Proteopedia - User contributions [en]

https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Michael+O%27Shaughnessy Proteopedia - User contributions [en] 2026-10-05T00:59:21Z User contributions MediaWiki 1.43.8 https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3558313 Thymidylate synthase 2022-05-04T18:43:01Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>[[Thymidylate synthase]] (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref name="Kholodar" >DOI:10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg|1000px]]<br /> <br /> The above image depicts the overall reaction catalyzed by TS. The section of the molecules contained in boxes highlights the differences between the substrates and products as a result of this reaction. <br /> == Function ==<br /> Thymidylate synthase catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|1000px]]<br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Dump_binding/4'>active sites</scene> can each bind the substrate dUMP<ref>PMID:9053905</ref>. The sketch below shows some of the ionic and hydrogen bonding interactions between the protein and the substrate, most of which are also shown in the 3D scene. You can measure distances and angles after turning off the spinning (for detailed instructions, see viewing guide, [https://proteopedia.org/wiki/index.php/Help:Viewing_pages]).<br /> <br /> [[Image:DUMP binding.PNG|400px]]<br /> <br /> Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> To distinguish between path A (in which cysteine remains covalently bound during methyl and hydride transfer) and path B (in which cysteine shows two separated nucleophilic attacks on carbon C6) in the mechanistic hypotheses depicted above, Kholodar et al co-crystallized the enzyme with an analog of the <scene name='49/493689/Ts_7jxf_percentb/5'>bisubstrate intermediate</scene>.<ref name="Kholodar" /><br /> [[Image:IntB.jpg|300px]]<br /> <br /> While the natural intermediate contains a carbonyl oxygen (circled in yellow), the cocrystallized intermediate has an amine (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> define current selected;<br /> select [VNM]301:B.NA4%A #9177;<br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in that position instead. In the crystal structure determination, one active site binds to the bisubstrate analog (as shown) while the other active site binds to the products. This fits with the half-the-sites mechanism mentioned above.<br /> <br /> The active site cysteine initially covalently binds to carbon 6 of the dUMP-ring. As seen here in the analog of intermediate conformations, it is not covalently bound here. The active site is disordered in the structure, showing <jmol><br /> <jmolLink><br /> <script>moveto 1.0 { 897 150 415 177.22} 615.28 0.0 0.0 {-7.028727272727273 50.28431818181818 5.7841818181818185} 49.47386078304503 {0 0 0} 0 0 0 3.0 0.0 0.0;<br /> </script><br /> <text>two conformations</text><br /> </jmolLink><br /> </jmol>. One conformation shows the active site cysteine 4.21 Angstroms away from the C6 carbon of the dMP ring and the other 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to <scene name='49/493689/Inhibited/2'>bind to the active site</scene> of TS and inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction.<br /> <br /> The image below shows folic acid on the top and the inhibitor methotrexate on the bottom. Compared the the substrate CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F, methotrexate has an additional amino group, lacks four hydrogens in the ring (i.e. has more double bonds), and is N-methylated in a different position.<br /> <br /> [[Image:Methotrexate.png|400px]]<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> ==Acknowledgements==<br /> [[User:Michael O'Shaughnessy|Michael O'Shaughnessy]] would like to thank [[User:Karsten Theis|Dr. Karsten Theis]] as well his my classmates from Biochemistry II ([[User:Shaylie Albright|Shaylie Albright]], [[User:Anna Postnikova|Anna Postnikova]], and [[User:Kia Yang| Kia Yang]])from the Spring 2022 semester for their contributions to this page, and would also like to thank Dr. Craig Martin from University of Massachusetts Amherst for his feedback on this page. <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3558312 Thymidylate synthase 2022-05-04T18:42:16Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>[[Thymidylate synthase]] (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref name="Kholodar" >DOI:10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg|1000px]]<br /> <br /> The above image depicts the overall reaction catalyzed by TS. The section of the molecules contained in boxes highlights the differences between the substrates and products as a result of this reaction. <br /> == Function ==<br /> Thymidylate synthase catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|1000px]]<br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Dump_binding/4'>active sites</scene> can each bind the substrate dUMP<ref>PMID:9053905</ref>. The sketch below shows some of the ionic and hydrogen bonding interactions between the protein and the substrate, most of which are also shown in the 3D scene. You can measure distances and angles after turning off the spinning (for detailed instructions, see viewing guide, [https://proteopedia.org/wiki/index.php/Help:Viewing_pages]).<br /> <br /> [[Image:DUMP binding.PNG|400px]]<br /> <br /> Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> To distinguish between path A (in which cysteine remains covalently bound during methyl and hydride transfer) and path B (in which cysteine shows two separated nucleophilic attacks on carbon C6) in the mechanistic hypotheses depicted above, Kholodar et al co-crystallized the enzyme with an analog of the <scene name='49/493689/Ts_7jxf_percentb/5'>bisubstrate intermediate</scene>.<ref name="Kholodar" /><br /> [[Image:IntB.jpg|300px]]<br /> <br /> While the natural intermediate contains a carbonyl oxygen (circled in yellow), the cocrystallized intermediate has an amine (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> define current selected;<br /> select [VNM]301:B.NA4%A #9177;<br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in that position instead. In the crystal structure determination, one active site binds to the bisubstrate analog (as shown) while the other active site binds to the products. This fits with the half-the-sites mechanism mentioned above.<br /> <br /> The active site cysteine initially covalently binds to carbon 6 of the dUMP-ring. As seen here in the analog of intermediate conformations, it is not covalently bound here. The active site is disordered in the structure, showing <jmol><br /> <jmolLink><br /> <script>moveto 1.0 { 897 150 415 177.22} 615.28 0.0 0.0 {-7.028727272727273 50.28431818181818 5.7841818181818185} 49.47386078304503 {0 0 0} 0 0 0 3.0 0.0 0.0;<br /> </script><br /> <text>two conformations</text><br /> </jmolLink><br /> </jmol>. One conformation shows the active site cysteine 4.21 Angstroms away from the C6 carbon of the dMP ring and the other 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to <scene name='49/493689/Inhibited/2'>bind to the active site</scene> of TS and inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction.<br /> <br /> The image below shows folic acid on the top and the inhibitor methotrexate on the bottom. Compared the the substrate CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F, methotrexate has an additional amino group, lacks four hydrogens in the ring (i.e. has more double bonds), and is N-methylated in a different position.<br /> <br /> [[Image:Methotrexate.png|400px]]<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> ==Acknowledgements==<br /> [[User:Michael O'Shaughnessy|Michael O'Shaughnessy]] would like to thank Dr. Karsten Theis as well his my classmates from Biochemistry II ([[User:Shaylie Albright|Shaylie Albright]], [[User:Anna Postnikova|Anna Postnikova]], and [[User:Kia Yang| Kia Yang]])from the Spring 2022 semester for their contributions to this page, and would also like to thank Dr. Craig Martin from University of Massachusetts Amherst for his feedback on this page. <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3558309 Thymidylate synthase 2022-05-04T18:36:30Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>[[Thymidylate synthase]] (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref name="Kholodar" >DOI:10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg|1000px]]<br /> <br /> The above image depicts the overall reaction catalyzed by TS. The section of the molecules contained in boxes highlights the differences between the substrates and products as a result of this reaction. <br /> == Function ==<br /> Thymidylate synthase catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|1000px]]<br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Dump_binding/4'>active sites</scene> can each bind the substrate dUMP<ref>PMID:9053905</ref>. The sketch below shows some of the ionic and hydrogen bonding interactions between the protein and the substrate, most of which are also shown in the 3D scene. You can measure distances and angles after turning off the spinning (for detailed instructions, see viewing guide, [https://proteopedia.org/wiki/index.php/Help:Viewing_pages]).<br /> <br /> [[Image:DUMP binding.PNG|400px]]<br /> <br /> Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> To distinguish between path A (in which cysteine remains covalently bound during methyl and hydride transfer) and path B (in which cysteine shows two separated nucleophilic attacks on carbon C6) in the mechanistic hypotheses depicted above, Kholodar et al co-crystallized the enzyme with an analog of the <scene name='49/493689/Ts_7jxf_percentb/5'>bisubstrate intermediate</scene>.<ref name="Kholodar" /><br /> [[Image:IntB.jpg|300px]]<br /> <br /> While the natural intermediate contains a carbonyl oxygen (circled in yellow), the cocrystallized intermediate has an amine (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> define current selected;<br /> select [VNM]301:B.NA4%A #9177;<br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in that position instead. In the crystal structure determination, one active site binds to the bisubstrate analog (as shown) while the other active site binds to the products. This fits with the half-the-sites mechanism mentioned above.<br /> <br /> The active site cysteine initially covalently binds to carbon 6 of the dUMP-ring. As seen here in the analog of intermediate conformations, it is not covalently bound here. The active site is disordered in the structure, showing <jmol><br /> <jmolLink><br /> <script>moveto 1.0 { 897 150 415 177.22} 615.28 0.0 0.0 {-7.028727272727273 50.28431818181818 5.7841818181818185} 49.47386078304503 {0 0 0} 0 0 0 3.0 0.0 0.0;<br /> </script><br /> <text>two conformations</text><br /> </jmolLink><br /> </jmol>. One conformation shows the active site cysteine 4.21 Angstroms away from the C6 carbon of the dMP ring and the other 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to <scene name='49/493689/Inhibited/2'>bind to the active site</scene> of TS and inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction.<br /> <br /> The image below shows folic acid on the top and the inhibitor methotrexate on the bottom. Compared the the substrate CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F, methotrexate has an additional amino group, lacks four hydrogens in the ring (i.e. has more double bonds), and is N-methylated in a different position.<br /> <br /> [[Image:Methotrexate.png|400px]]<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> ==Acknowledgements==<br /> [[User:Michael O'Shaughnessy|Michael O'Shaughnessy]] would like to thank Dr. Karsten Theis as well his my classmates from Biochemistry II from the Spring 2022 semester for their contributions to this page, and would also like to thank Dr. Craig Martin from University of Massachusetts Amherst for his feedback on this page. <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Methylenetetrahydrofolate_reductase&diff=3558307 Methylenetetrahydrofolate reductase 2022-05-04T18:35:26Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>[[Methylenetetrahydrofolate reductase]] (MTHFR) is an enzyme that is a regulatory agent of folate [[one-carbon metabolism]]. The enzyme is present in both eukaryotes and prokaryotes however, the structure is unique in eukaryotes as it has a SAM binding domain. The enzyme has an essential role in S-Adenosyl Methionine (SAM) regulation in order to promote homeostasis within the folate cycle. This shows the importance of MTHFR within the human body, and identifies the issues that may arise if MTHFR dysfunction occurs<ref>https://doi.org/10.1038/s41467-018-04735-2</ref>.<br /> <br /> == Function ==<br /> Methylenetetrahydrofolate reductase (MTHFR) enzyme catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate to be recycled back into the folate cycle, and for aiding folate uptake in the body. This reduction reaction requires the cofactor molecule flavin adenine dinucleotide (FAD) and the second substrate nicotinamide adenine dinucleotide phosphate (NADPH) as the electron donor in the reaction. MTHFR has a unique folding structure. Its N-terminal is abundant in serine and acts as a phosphorylation site, its situated in close proximity to it's C-terminal S-adenosyl methionine (SAM) binding site. A linker joins the catalytic domain (N-terminal) to the regulatory domain (C-terminal) for interaction and increases the sensitivity to SAM binding and feedback properties.<ref>DOI: 10.1038/s41467-018-04735-2</ref> <br /> <br /> ==Mechanism==<br /> This reaction mechanism is characterized by a hydride transfer (highlighted in yellow) from the FADH to the partially positive 5,10-methyl group. This causes the 5-membered ring to open and Nitrogen 10 to receive the electrons that were involved in the C-N10 bond. This allows Nitrogen 10 to act as a Bronsted-Lowry base and accept the proton highlighted in green. <br /> <br /> 5,10-methylenetetrahydrofolate + NADPH + H+ → 5-methyltetrahydrofolate + NADP+<br /> <br /> [[Image:MTHFR Reaction.jpg|800px]]<br /> <br /> <br /> == Disease ==<br /> In addition to the folate cycle, MTHFR is also a major component of the homeostasis of homocysteine in the blood stream. When this homeostasis is disrupted, mutations are created that result in hyperhomocysteinemia with homocystinuria, or mild hyperhomocysteinemia. Hyperhomocysteinemia is an excess of the amino acid circulating in the body, and is a direct correlation of cardiovascular disease, Alzheimer's disease, depression, and neural tube defects within the fetus. Furthermore, homocystinuria is clinically described as the body's inability to adequately process homocysteine and the amino acid methionine. This dysfunction can be clinically presented with skeletal, vision, and blood clotting abnormalities coupled with learning disorders<ref>Leclerc D, Sibani S, Rozen R. Molecular biology of methylenetetrahydrofolate reductase (MTHFR) and overview of mutations/polymorphisms. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013. Available from: https://www.ncbi.nlm.nih.gov/books/NBK6561/</ref>.<br /> <br /> == Structure ==<br /> <StructureSection load='' size='400' side='right' scene='90/907473/Mthfr_protein/2'><br /> MTHFR occurs as a <scene name='90/907473/Mthfr_protein/3'>dimer</scene>. The <scene name='90/907473/Mthfr_protein/4'>N- and C-terminal domains of MTHFR</scene> are joined together by a linker (red). The N-terminal domain (silver) functions as the catalytic domain to convert 5,10-MTHF to 5-MTHF (this product will be used with [[Methionine synthase]]). This domain contains FAD as a cofactor and binds to NADPH as an electron donor<ref>PMID:16114881</ref>. The C-terminal (yellow) functions as the regulatory domain and binds to SAH, to recognize the need for inhibition or activation.<br /> <br /> MTHFR catalyzes the reaction using a <scene name='90/907473/Superposition/7'>ping pong mechanism</scene>, where a substrate binds and forms an intermediate before releasing the product, followed by the binding of a second substrate and later release of a second product <ref>DOI:10.1021/bi9007325</ref>.<br /> {{Template:Button Toggle Animation2}}<jmol><br /> <jmolCheckbox><br /> <scriptWhenUnChecked>select NAI or C2F or FAD; spacefill off;<br /> </scriptWhenUnChecked><br /> <scriptWhenchecked>select NAI or C2F or FAD; spacefill on;<br /> </scriptWhenchecked><br /> <checked>false</checked> <br /> <text>Ligands as spacefilling</text><br /> </jmolCheckbox><br /> </jmol><br /> <br /> [[Image:MTHFR ping pong.gif]]<br /> <br /> </StructureSection><br /> ==Acknowledgements==<br /> <br /> == References ==<br /> <references/><br /> [[Category:One-carbon metabolism]]<br /> [[Category:5-methyltetrahydrofolate]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3555036 Thymidylate synthase 2022-05-02T14:24:20Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>[[Thymidylate synthase]] (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref name="Kholodar" >DOI:10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg|1000px]]<br /> <br /> The above image depicts the overall reaction catalyzed by TS. The section of the molecules contained in boxes highlights the differences between the substrates and products as a result of this reaction. <br /> == Function ==<br /> Thymidylate synthase catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|1000px]]<br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Dump_binding/4'>active sites</scene> can each bind the substrate dUMP<ref>PMID:9053905</ref>. The sketch below shows some of the ionic and hydrogen bonding interactions between the protein and the substrate, most of which are also shown in the 3D scene. You can measure distances and angles after turning off the spinning (for detailed instructions, see viewing guide, [https://proteopedia.org/wiki/index.php/Help:Viewing_pages]).<br /> <br /> [[Image:DUMP binding.PNG|400px]]<br /> <br /> Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> To distinguish between path A (in which cysteine remains covalently bound during methyl and hydride transfer) and path B (in which cysteine shows two separated nucleophilic attacks on carbon C6) in the mechanistic hypotheses depicted above, Kholodar et al co-crystallized the enzyme with an analog of the <scene name='49/493689/Ts_7jxf_percentb/5'>bisubstrate intermediate</scene>.<ref name="Kholodar" /><br /> [[Image:IntB.jpg|300px]]<br /> <br /> While the natural intermediate contains a carbonyl oxygen (circled in yellow), the cocrystallized intermediate has an amine (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> define current selected;<br /> select [VNM]301:B.NA4%A #9177;<br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in that position instead. In the crystal structure determination, one active site binds to the bisubstrate analog (as shown) while the other active site binds to the products. This fits with the half-the-sites mechanism mentioned above.<br /> <br /> The active site cysteine initially covalently binds to carbon 6 of the dUMP-ring. As seen here in the analog of intermediate conformations, it is not covalently bound here. The active site is disordered in the structure, showing <jmol><br /> <jmolLink><br /> <script>moveto 1.0 { 897 150 415 177.22} 615.28 0.0 0.0 {-7.028727272727273 50.28431818181818 5.7841818181818185} 49.47386078304503 {0 0 0} 0 0 0 3.0 0.0 0.0;<br /> </script><br /> <text>two conformations</text><br /> </jmolLink><br /> </jmol>. One conformation shows the active site cysteine 4.21 Angstroms away from the C6 carbon of the dMP ring and the other 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to <scene name='49/493689/Inhibited/2'>bind to the active site</scene> of TS and inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction.<br /> <br /> The image below shows folic acid on the top and the inhibitor methotrexate on the bottom. Compared the the substrate CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F, methotrexate has an additional amino group, lacks four hydrogens in the ring (i.e. has more double bonds), and is N-methylated in a different position.<br /> <br /> [[Image:Methotrexate.png|400px]]<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> ==Acknowledgements==<br /> I would like to thank Doctor Karsten Theis as well as my classmates from Biochemistry II from the Spring 2022 semester for their contributions to this page. I would also like to thank Doctor Craig Martin from University of Massachusetts Amherst for his feedback on this page. <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3554888 Thymidylate synthase 2022-05-01T17:32:02Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>[[Thymidylate synthase]] (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref name="Kholodar" >DOI:10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg|1000px]]<br /> <br /> The above image depicts the overall reaction catalyzed by TS. The section of the molecules contained in boxes highlights the differences between the substrates and products as a result of this reaction. <br /> == Function ==<br /> Thymidylate synthase catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|1000px]]<br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Dump_binding/4'>active sites</scene> can each bind the substrate dUMP<ref>PMID:9053905</ref>. The sketch below shows some of the ionic and hydrogen bonding interactions between the protein and the substrate, most of which are also shown in the 3D scene. You can measure distances and angles after turning off the spinning (for detailed instructions, see viewing guide, [https://proteopedia.org/wiki/index.php/Help:Viewing_pages]).<br /> <br /> [[Image:DUMP binding.PNG|400px]]<br /> <br /> Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> To distinguish between path A (in which cysteine remains covalently bound during methyl and hydride transfer) and path B (in which cysteine shows two separated nucleophilic attacks on carbon C6) in the mechanistic hypotheses depicted above, Kholodar et al co-crystallized the enzyme with an analog of the <scene name='49/493689/Ts_7jxf_percentb/5'>bisubstrate intermediate</scene>.<ref name="Kholodar" /><br /> [[Image:IntB.jpg|300px]]<br /> <br /> While the natural intermediate contains a carbonyl oxygen (circled in yellow), the cocrystallized intermediate has an amine (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> define current selected;<br /> select [VNM]301:B.NA4%A #9177;<br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in that position instead. In the crystal structure determination, one active site binds to the bisubstrate analog (as shown) while the other active site binds to the products. This fits with the half-the-sites mechanism mentioned above.<br /> <br /> The active site cysteine initially covalently binds to carbon 6 of the dUMP-ring. As seen here in the analog of intermediate conformations, it is not covalently bound here. The active site is disordered in the structure, showing <jmol><br /> <jmolLink><br /> <script>moveto 1.0 { 897 150 415 177.22} 615.28 0.0 0.0 {-7.028727272727273 50.28431818181818 5.7841818181818185} 49.47386078304503 {0 0 0} 0 0 0 3.0 0.0 0.0;<br /> </script><br /> <text>two conformations</text><br /> </jmolLink><br /> </jmol>. One conformation shows the active site cysteine 4.21 Angstroms away from the C6 carbon of the dMP ring and the other 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to <scene name='49/493689/Inhibited/2'>bind to the active site</scene> of TS and inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction.<br /> <br /> The image below shows folic acid on the top and the inhibitor methotrexate on the bottom. Compared the the substrate CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F, methotrexate has an additional amino group, lacks four hydrogens in the ring (i.e. has more double bonds), and is N-methylated in a different position.<br /> <br /> [[Image:Methotrexate.png|400px]]<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> ==Acknowledgements==<br /> I would like to thank Doctor Karsten Theis as well as my classmates from Biochemistry II for their contributions to this page. I would also like to thank Doctor Craig Martin from University of Massachusetts Amherst for his feedback on this page. <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3554885 Thymidylate synthase 2022-05-01T17:27:34Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>[[Thymidylate synthase]] (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref name="Kholodar" >DOI:10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg|1000px]]<br /> <br /> The above image depicts the overall reaction catalyzed by TS. The section of the molecules contained in boxes highlights the differences between the substrates and products as a result of this reaction. <br /> == Function ==<br /> Thymidylate synthase catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|1000px]]<br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Dump_binding/4'>active sites</scene> can each bind the substrate dUMP<ref>PMID:9053905</ref>. The sketch below shows some of the ionic and hydrogen bonding interactions between the protein and the substrate, most of which are also shown in the 3D scene. You can measure distances and angles after turning off the spinning (for detailed instructions, see viewing guide, [https://proteopedia.org/wiki/index.php/Help:Viewing_pages]).<br /> <br /> [[Image:DUMP binding.PNG|400px]]<br /> <br /> Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> To distinguish between path A (in which cysteine remains covalently bound during methyl and hydride transfer) and path B (in which cysteine shows two separated nucleophilic attacks on carbon C6) in the mechanistic hypotheses depicted above, Kholodar et al co-crystallized the enzyme with an analog of the <scene name='49/493689/Ts_7jxf_percentb/5'>bisubstrate intermediate</scene>.<ref name="Kholodar" /><br /> [[Image:IntB.jpg|300px]]<br /> <br /> While the natural intermediate contains a carbonyl oxygen (circled in yellow), the cocrystallized intermediate has an amine (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> define current selected;<br /> select [VNM]301:B.NA4%A #9177;<br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in that position instead. In the crystal structure determination, one active site binds to the bisubstrate analog (as shown) while the other active site binds to the products. This fits with the half-the-sites mechanism mentioned above.<br /> <br /> The active site cysteine initially covalently binds to carbon 6 of the dUMP-ring. As seen here in the analog of intermediate conformations, it is not covalently bound here. The active site is disordered in the structure, showing <jmol><br /> <jmolLink><br /> <script>moveto 1.0 { 897 150 415 177.22} 615.28 0.0 0.0 {-7.028727272727273 50.28431818181818 5.7841818181818185} 49.47386078304503 {0 0 0} 0 0 0 3.0 0.0 0.0;<br /> </script><br /> <text>two conformations</text><br /> </jmolLink><br /> </jmol>. One conformation shows the active site cysteine 4.21 Angstroms away from the C6 carbon of the dMP ring and the other 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to <scene name='49/493689/Inhibited/2'>bind to the active site</scene> of TS and inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction.<br /> <br /> The image below shows folic acid on the top and the inhibitor methotrexate on the bottom. Compared the the substrate CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F, methotrexate has an additional amino group, lacks four hydrogens in the ring (i.e. has more double bonds), and is N-methylated in a different position.<br /> <br /> [[Image:Methotrexate.png|400px]]<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> ==Acknowledgements==<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3553678 Thymidylate synthase 2022-04-27T15:04:57Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref name="Kholodar" >DOI:10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg|1000px]]<br /> The above image depicts the overall reaction catalyzed by TS. The section of the molecules contained in boxes highlights the differences between the substrates and products as a result of this reaction. <br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|1000px]]<br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Dump_binding/4'>active sites</scene> can each bind the substrate dUMP<ref>PMID:9053905</ref>. The sketch below shows some of the ionic and hydrogen bonding interactions between the protein and the substrate, most of which are also shown in the 3D scene. You can measure distances and angles after turning off the spinning (for detailed instructions, see viewing guide, [https://proteopedia.org/wiki/index.php/Help:Viewing_pages]).<br /> <br /> [[Image:DUMP binding.PNG|400px]]<br /> <br /> Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> To distinguish between path A (in which cysteine remains covalently bound during methyl and hydride transfer) and path B (in which cysteine shows two separated nucleophilic attacks on carbon C6) in the mechanistic hypotheses depicted above, Kholodar et al co-crystallized the enzyme with an analog of the <scene name='49/493689/Ts_7jxf_percentb/5'>bisubstrate intermediate</scene>.<ref name="Kholodar" /><br /> [[Image:IntB.jpg|300px]]<br /> <br /> While the natural intermediate contains a carbonyl oxygen (circled in yellow), the cocrystallized intermediate has an amine (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> define current selected;<br /> select [VNM]301:B.NA4%A #9177;<br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in that position instead. In the crystal structure determination, one active site binds to the bisubstrate analog (as shown) while the other active site binds to the products. This fits with the half-the-sites mechanism mentioned above.<br /> <br /> The active site cysteine initially covalently binds to carbon 6 of the dUMP-ring. As seen here in the analog of intermediate conformations, it is not covalently bound here. The active site is disordered in the structure, showing <jmol><br /> <jmolLink><br /> <script>moveto 1.0 { 897 150 415 177.22} 615.28 0.0 0.0 {-7.028727272727273 50.28431818181818 5.7841818181818185} 49.47386078304503 {0 0 0} 0 0 0 3.0 0.0 0.0;<br /> </script><br /> <text>two conformations</text><br /> </jmolLink><br /> </jmol>. One conformation shows the active site cysteine 4.21 Angstroms away from the C6 carbon of the dMP ring and the other 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/2'>inhibits</scene> TS.<br /> <br /> The image below shows folic acid on the top and the inhibitor methotrexate on the bottom<br /> <br /> [[Image:Methotrexate.png|400px]]<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> ==Acknowledgements==<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3553676 Thymidylate synthase 2022-04-27T15:01:31Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref name="Kholodar" >DOI:10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg|1000px]]<br /> The above image depicts the overall reaction catalyzed by TS. The section of the substrates and products contained in boxes are the relevant sites for this reaction. <br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|1000px]]<br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Dump_binding/4'>active sites</scene> can each bind the substrate dUMP<ref>PMID:9053905</ref>. The sketch below shows some of the ionic and hydrogen bonding interactions between the protein and the substrate, most of which are also shown in the 3D scene. You can measure distances and angles after turning off the spinning (for detailed instructions, see viewing guide, [https://proteopedia.org/wiki/index.php/Help:Viewing_pages]).<br /> <br /> [[Image:DUMP binding.PNG|400px]]<br /> <br /> Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> To distinguish between path A (in which cysteine remains covalently bound during methyl and hydride transfer) and path B (in which cysteine shows two separated nucleophilic attacks on carbon C6) in the mechanistic hypotheses depicted above, Kholodar et al co-crystallized the enzyme with an analog of the <scene name='49/493689/Ts_7jxf_percentb/5'>bisubstrate intermediate</scene>.<ref name="Kholodar" /><br /> [[Image:IntB.jpg|300px]]<br /> <br /> While the natural intermediate contains a carbonyl oxygen (circled in yellow), the cocrystallized intermediate has an amine (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> define current selected;<br /> select [VNM]301:B.NA4%A #9177;<br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in that position instead. In the crystal structure determination, one active site binds to the bisubstrate analog (as shown) while the other active site binds to the products. This fits with the half-the-sites mechanism mentioned above.<br /> <br /> The active site cysteine initially covalently binds to carbon 6 of the dUMP-ring. As seen here in the analog of intermediate conformations, it is not covalently bound here. The active site is disordered in the structure, showing <jmol><br /> <jmolLink><br /> <script>moveto 1.0 { 897 150 415 177.22} 615.28 0.0 0.0 {-7.028727272727273 50.28431818181818 5.7841818181818185} 49.47386078304503 {0 0 0} 0 0 0 3.0 0.0 0.0;<br /> </script><br /> <text>two conformations</text><br /> </jmolLink><br /> </jmol>. One conformation shows the active site cysteine 4.21 Angstroms away from the C6 carbon of the dMP ring and the other 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/2'>inhibits</scene> TS.<br /> <br /> The image below shows folic acid on the top and the inhibitor methotrexate on the bottom<br /> <br /> [[Image:Methotrexate.png|400px]]<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> ==Acknowledgements==<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3553673 Thymidylate synthase 2022-04-27T14:56:41Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref name="Kholodar" >DOI:10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg|1000px]]<br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|1000px]]<br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Dump_binding/4'>active sites</scene> can each bind the substrate dUMP<ref>PMID:9053905</ref>. The sketch below shows some of the ionic and hydrogen bonding interactions between the protein and the substrate, most of which are also shown in the 3D scene. You can measure distances and angles after turning off the spinning (for detailed instructions, see viewing guide, [https://proteopedia.org/wiki/index.php/Help:Viewing_pages]).<br /> <br /> [[Image:DUMP binding.PNG|400px]]<br /> <br /> Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> To distinguish between path A (in which cysteine remains covalently bound during methyl and hydride transfer) and path B (in which cysteine shows two separated nucleophilic attacks on carbon C6) in the mechanistic hypotheses depicted above, Kholodar et al co-crystallized the enzyme with an analog of the <scene name='49/493689/Ts_7jxf_percentb/5'>bisubstrate intermediate</scene>.<ref name="Kholodar" /><br /> [[Image:IntB.jpg|300px]]<br /> <br /> While the natural intermediate contains a carbonyl oxygen (circled in yellow), the cocrystallized intermediate has an amine (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> define current selected;<br /> select [VNM]301:B.NA4%A #9177;<br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in that position instead. In the crystal structure determination, one active site binds to the bisubstrate analog (as shown) while the other active site binds to the products. This fits with the half-the-sites mechanism mentioned above.<br /> <br /> The active site cysteine initially covalently binds to carbon 6 of the dUMP-ring. As seen here in the analog of intermediate conformations, it is not covalently bound here. The active site is disordered in the structure, showing <jmol><br /> <jmolLink><br /> <script>moveto 1.0 { 897 150 415 177.22} 615.28 0.0 0.0 {-7.028727272727273 50.28431818181818 5.7841818181818185} 49.47386078304503 {0 0 0} 0 0 0 3.0 0.0 0.0;<br /> </script><br /> <text>two conformations</text><br /> </jmolLink><br /> </jmol>. One conformation shows the active site cysteine 4.21 Angstroms away from the C6 carbon of the dMP ring and the other 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/2'>inhibits</scene> TS.<br /> <br /> The image below shows folic acid on the top and the inhibitor methotrexate on the bottom<br /> <br /> [[Image:Methotrexate.png|400px]]<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> ==Acknowledgements==<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=File:IntB.jpg&diff=3550776 File:IntB.jpg 2022-04-25T16:51:17Z

<p>Michael O'Shaughnessy: uploaded a new version of "Image:IntB.jpg": Redesigned IntB in Professional ChemDraw</p> <hr /> <div>== Summary ==<br /> A complexed intermediate of the substrates of TS<br /> == Licensing ==<br /> {{self|cc-by-sa-3.0|GFDL}}</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=File:IntB.jpg&diff=3550775 File:IntB.jpg 2022-04-25T16:31:37Z

<p>Michael O'Shaughnessy: uploaded a new version of "Image:IntB.jpg": Reverted to version as of 17:59, 20 April 2022</p> <hr /> <div>== Summary ==<br /> A complexed intermediate of the substrates of TS<br /> == Licensing ==<br /> {{self|cc-by-sa-3.0|GFDL}}</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Methionine_adenosyltransferase&diff=3550772 Methionine adenosyltransferase 2022-04-25T15:12:01Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>[[Methionine adenosyltransferase]] (MAT) or S-adenosylmethionine synthetase (SAM synthetase) synthesizes S-adenosylmethionine (SAM or AdoMet) from the substrates adenosine triphosphate (ATP) and methionine. ATP isn’t used only as a source of energy like it is in other reactions but gets a methionine added onto the 5' carbon while the three phosphate groups are broken down and released from the active site. This enzyme is conserved and found in many organisms, so it is essential for life. Importantly, the production of SAM by MAT provides methyl for methylation of nucleic acids, allowing for epigenetic modification. Problems with this enzyme have been shown to cause diseases including various cancers.<br /> <br /> == Relevance ==<br /> The product of this enzymatic reaction, SAM, is the universal methyl donor of metabolism. DNA methyltransferases can transfer a methyl group from SAM to the 5th carbon of cytosine residues <ref>DOI:10.1038/npp.2012.112</ref>. In this way, MAT is indirectly important for regulation of gene expression by providing methyl through SAM. SAM is also involved in N-methylation, O-methylation and C-methylation, yielding S-adenosyl homocysteine as a product that gets recycled by the [[one-carbon metabolism]]. Radical SAM enzymes break down SAM into an adenosyl radical and methionine, enabling a host of otherwise difficult to achieve reactions, e.g. in molybdenum cofactors biosynthesis<ref>DOI:10.1073/pnas.0404624101</ref>. Accumulation of S-adenosyl homocysteine (or homocysteine itself) indicates an imbalance in supply and demand for SAM in the organism. Methionine metabolism impairment in liver diseases is related in alteration in MAT<ref>PMID:7741002</ref>.<br /> <br /> <br /> == Function and reaction mechanism ==<br /> '''S-adenosylmethionine synthetase''' or '''S-adenosylmethionine synthase''' or '''S-adenosylmethionine transferase''' or '''methionine adenosyltransferase''' (MAT) catalyzes the conversion of methionine and ATP to S-adenosylmethionine (AdoMet), pyrophosphate (PPi) and orthophosphate (Pi). The catalytic entity of MAT is a dimer. MAT cofactors are Mg+2 (or Co+2) and K+ ions<ref>PMID:8611562</ref>. <br /> <br /> [[Image:Sam rxn 2.jpg|800px]]<br /> <br /> The nucleophilic sulfur atom of methionine attacks the slightly positive 5' carbon of the adenosine sugar unit. Following this, the bond from the 5' carbon to the oxygen breaks, separating the tripolyphosphate from the newly formed S-adenosylmethionine (SAM) <ref name="Murray et al.">Murray B, Antonyuk SV, Marina A, Lu SC, Mato JM, Hasnain SS, Rojas Al. Crystallography captures catalytic steps in human methionine adenosyltransferase enzymes. PNAS. 2016 Feb 8;113 (8) 2104-2109. doi: https://doi.org/10.1073/pnas.1510959113</ref>. This is an example of an SN2 reaction, where the substrates move through a transition state to then form the products. The products are only released after the methionine binds and the C-O bond breaks.<br /> <br /> == Structure ==<br /> <StructureSection load='5a1i' size='400' side='right' caption='MAT dimer in complex with methionine, PPNP, and adenosine (PBD 5a1i)' scene=''><br /> MAT consists of α and β subunits. The MATα1 and <scene name='90/907472/Mat2a/4'>MATα2</scene> subunits are catalytic subunits while MATβ is a regulatory subunit. MATα2 subunits can form <scene name='49/493038/Tetramer/1'>tetramers</scene>, where the active site is found <scene name='49/493038/Tetramer/2'>between two of the subunits</scene>. The subunits are encoded on different genes in humans, so they are created separately and can then come together to form various complexes, such as MATαβ or MATα2 dimers <ref name="Murray et al." />. Not much is currently known about the function of this regulatory subunit and how it regulates the function of the enzyme<ref>DOI:10.1107/S2052252514012585</ref>. However, Murray et al.<ref name="Murray et al." /> show that even in the absence of the regulatory subunit, the active site found in the catalytic subunit remains functional. <br /> <br /> The biological assembly of rat S-adenosylmethionine synthetase is a <scene name='49/493038/Cv/11'>homotetramer</scene>. The <scene name='49/493038/Cv/10'>active site of MAT is located between adjacent subunits</scene><ref>PMID:12888348</ref>. The <scene name='49/493038/Substrates/4'>substrates</scene> used by the enzyme are methionine and ATP. Notably, ATP is not used as a source of energy in this reaction like it is for many other processes. Instead, it is used as a substrate in the synthesis reaction. Methionine and ATP enter the active site and are stabilized by residues present there, including lysine and histidine. Once the reaction begins to take place, methionine flips toward the 5' carbon of the adenosine sugar<ref>doi:10.1042/BJ20121580</ref>. Following nucleophilic attack of the sulfur on the carbon, the C-O bond between the phosphates and the carbon breaks, and the <scene name='49/493038/Product/4'>products</scene> are formed (tripolyphosphate not pictured). SAM is released from the active site first. MAT also catalyzes hydrolysis of the tripolyphosphate into pyrophosphate and orthophosphate, which are then released from the active site <ref>Niland CN, Ghosh A, Cahill SM, Schramm VL. Mechanism and Inhibition of Human Methionine Adenosyltransferase 2A. ACS Biochemistry. 2021 Mar 3;60 (10) 791-801. doi: https://doi.org/10.1021/acs.biochem.0c00998</ref>.<br /> <br /> == Gating Loop ==<br /> MAT has been shown to have a <scene name='49/493038/Loop/6'>gating loop</scene> next to the active site <ref name="Murray et al." />. <br /> <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>model 1</script><br /> <text>ordered with SAM</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>model 2</script><br /> <text>disordered apo</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>model 0</script><br /> <text>both</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>model 1; delay 0.5;model 2; delay 0.5;model 1; delay 0.5;model 2; delay 0.5;model 1; delay 0.5;model 2;</script><br /> <text>animate 3 cycles</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> This structure is thought to allow access to the active site, becoming ordered or disordered. When the loop is ordered, the active site is closed, and it is opened again when the loop is disordered. Murray et al. <ref name="Murray et al." /> found that when SAM or adenosine is bound to the active site the gate is closed, and when PPNP (tripolyphosphate in the body) is bound to the active site the gate is open. It is still unknown what causes the gating loop to open and what triggers the release of products, but it is not caused by hydrolysis of tripolyphosphate <ref name="Murray et al." />. This finding shows that hydrolysis of tripolyphosphate does not provide energy for the opening of the gating loop, nor does ATP provide energy for any part of this reaction.<br /> </StructureSection><br /> <br /> ==3D structures of S-adenosylmethionine synthetase==<br /> <br /> [[S-adenosylmethionine synthetase 3D structures]]<br /> <br /> {{REVISIONDAY2}}-{{MONTHNAME|{{REVISIONMONTH}}}}-{{REVISIONYEAR}}<br /> {{#tree:id=OrganizedByTopic|openlevels=0|<br /> <br /> *S-adenosylmethionine synthetase<br /> <br /> **[[1fug]] – EcMAT – ''Escherichia coli''<br /><br /> **[[3iml]] – MAT – ''Burkholderia pseudomallei''<br /><br /> **[[2ydy]] – hMAT II subunit β – human<br /><br /> **[[4le5]] – MAT- ''Campylobacter jejuni''<br /><br /> **[[4hpv]] - SsMAT – ''Sulfolobus solfataricus''<br /><br /> **[[3tde]] – MAT- ''Mycobacterium tuberculosis''<br /><br /> **[[3s82]] – MAT- ''Mycobacterium avium''<br /><br /> **[[3rv2]] – MAT- ''Mycobacterium marinum''<br /><br /> <br /> <br /> *S-adenosylmethionine synthetase complexes<br /> <br /> **[[1xra]], [[1xrb]] – EcMAT + Pi + K + Mg <br /><br /> **[[1xrc]] - EcMAT + Pi + Co + K<br /><br /> **[[1mxa]] - EcMAT + Pi + PPi + Mg + K<br /><br /> **[[1mxb]] - EcMAT + Pi + ADP + Mg + K<br /><br /> **[[1mxc]] - EcMAT + Pi + Br-ADP + Mg + K<br /><br /> **[[1p7l]] – EcMAT + methionine + AdoMet + AMPPNP + Mg + K<br /><br /> **[[1rg9]] - EcMAT + AdoMet + AMPPNP + Mg + K<br /><br /> **[[1qm4]] - rMAT+ methionine analog + K + Mg – rat<br /><br /> **[[1o90]] - rMAT + Pi + Mg + K<br /><br /> **[[1o92]] – rMAT + methionine analog + ADP + Mg + K<br /><br /> **[[1o93]] – rMAT + methionine analog + ATP + Mg + K<br /><br /> **[[1o9t]] – rMAT + methionine + ADP + Mg + K<br /><br /> **[[2obv]] – hMAT I + AdoMet + Na <br /><br /> **[[2p02]] - hMAT II + AdoMet<br /><br /> **[[2ydx]] – MAT + resveratrol + NADP + Ca + ADP derivative<br /><br /> **[[4l2z]], [[4k0b]] - SsMAT + Pi + PPi + Mg + SAM derivative <br /><br /> }}<br /> == References ==<br /> <references/><br /> [[Category:Topic Page]][[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Methionine_adenosyltransferase&diff=3550771 Methionine adenosyltransferase 2022-04-25T15:09:44Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>[[Methionine adenosyltransferase]] (MAT) or S-adenosylmethionine synthetase (SAM synthetase) synthesizes S-adenosylmethionine (SAM or AdoMet) from the substrates adenosine triphosphate (ATP) and methionine. ATP isn’t used only as a source of energy like it is in other reactions but gets a methionine added onto the 5' carbon while the three phosphate groups are broken down and released from the active site. This enzyme is conserved and found in many organisms, so it is essential for life. Importantly, the production of SAM by MAT provides methyl for methylation of nucleic acids, allowing for epigenetic modification. Problems with this enzyme have been shown to cause diseases including various cancers.<br /> <br /> == Relevance ==<br /> The product of this enzymatic reaction, SAM, is the universal methyl donor of metabolism. DNA methyltransferases can transfer a methyl group from SAM to the 5th carbon of cytosine residues <ref>DOI:10.1038/npp.2012.112</ref>. In this way, MAT is indirectly important for regulation of gene expression by providing methyl through SAM. SAM is also involved in N-methylation, O-methylation and C-methylation, yielding S-adenosyl homocysteine as a product that gets recycled by the [[one-carbon metabolism]]. Radical SAM enzymes break down SAM into an adenosyl radical and methionine, enabling a host of otherwise difficult to achieve reactions, e.g. in molybdenum cofactors biosynthesis<ref>DOI:10.1073/pnas.0404624101</ref>. Accumulation of S-adenosyl homocysteine (or homocysteine itself) indicates an imbalance in supply and demand for SAM in the organism. Methionine metabolism impairment in liver diseases is related in alteration in MAT<ref>PMID:7741002</ref>.<br /> <br /> <br /> == Function and reaction mechanism ==<br /> '''S-adenosylmethionine synthetase''' or '''S-adenosylmethionine synthase''' or '''S-adenosylmethionine transferase''' or '''methionine adenosyltransferase''' (MAT) catalyzes the conversion of methionine and ATP to S-adenosylmethionine (AdoMet), pyrophosphate (PPi) and orthophosphate (Pi). The catalytic entity of MAT is a dimer. MAT cofactors are Mg+2 (or Co+2) and K+ ions<ref>PMID:8611562</ref>. <br /> <br /> [[Image:Sam rxn 2.jpg|800px]]<br /> <br /> The nucleophilic sulfur atom of methionine attacks the slightly positive 5' carbon of the adenosine sugar unit. Following this, the bond from the 5' carbon to the oxygen breaks, separating the tripolyphosphate from the newly formed S-adenosylmethionine (SAM) <ref name="Murray et al.">Murray B, Antonyuk SV, Marina A, Lu SC, Mato JM, Hasnain SS, Rojas Al. Crystallography captures catalytic steps in human methionine adenosyltransferase enzymes. PNAS. 2016 Feb 8;113 (8) 2104-2109. doi: https://doi.org/10.1073/pnas.1510959113</ref>. This is an example of an SN2 reaction, where the substrates move through a transition state to then form the products. The products are only released after the methionine binds and the C-O bond breaks.<br /> <br /> == Structure ==<br /> <StructureSection load='5a1i' size='400' side='right' caption='MAT dimer in complex with methionine, PPNP, and adenosine (PBD 5a1i)' scene=''><br /> MAT consists of α and β subunits. The MATα1 and <scene name='90/907472/Mat2a/4'>MATα2</scene> subunits are catalytic subunits while MATβ is a regulatory subunit. MATα2 subunits can form <scene name='49/493038/Tetramer/1'>tetramers</scene>, where the active site is found <scene name='49/493038/Tetramer/2'>between two of the subunits</scene>. The subunits are encoded on different genes in humans, so they are created separately and can then come together to form various complexes, such as MATαβ or MATα2 dimers <ref name="Murray et al." />. Not much is currently known about the function of this regulatory subunit and how it regulates the function of the enzyme<ref>DOI:10.1107/S2052252514012585</ref>. However, Murray et al.<ref name="Murray et al." /> show that even in the absence of the regulatory subunit, the active site found in the catalytic subunit remains functional. <br /> <br /> The biological assembly of rat S-adenosylmethionine synthetase is a <scene name='49/493038/Cv/7'>homotetramer</scene>. The <scene name='49/493038/Cv/10'>active site of MAT is located between adjacent subunits</scene><ref>PMID:12888348</ref>. The <scene name='49/493038/Substrates/4'>substrates</scene> used by the enzyme are methionine and ATP. Notably, ATP is not used as a source of energy in this reaction like it is for many other processes. Instead, it is used as a substrate in the synthesis reaction. Methionine and ATP enter the active site and are stabilized by residues present there, including lysine and histidine. Once the reaction begins to take place, methionine flips toward the 5' carbon of the adenosine sugar<ref>doi:10.1042/BJ20121580</ref>. Following nucleophilic attack of the sulfur on the carbon, the C-O bond between the phosphates and the carbon breaks, and the <scene name='49/493038/Product/4'>products</scene> are formed (tripolyphosphate not pictured). SAM is released from the active site first. MAT also catalyzes hydrolysis of the tripolyphosphate into pyrophosphate and orthophosphate, which are then released from the active site <ref>Niland CN, Ghosh A, Cahill SM, Schramm VL. Mechanism and Inhibition of Human Methionine Adenosyltransferase 2A. ACS Biochemistry. 2021 Mar 3;60 (10) 791-801. doi: https://doi.org/10.1021/acs.biochem.0c00998</ref>.<br /> <br /> == Gating Loop ==<br /> MAT has been shown to have a <scene name='49/493038/Loop/6'>gating loop</scene> next to the active site <ref name="Murray et al." />. <br /> <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>model 1</script><br /> <text>ordered with SAM</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>model 2</script><br /> <text>disordered apo</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>model 0</script><br /> <text>both</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>model 1; delay 0.5;model 2; delay 0.5;model 1; delay 0.5;model 2; delay 0.5;model 1; delay 0.5;model 2;</script><br /> <text>animate 3 cycles</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> This structure is thought to allow access to the active site, becoming ordered or disordered. When the loop is ordered, the active site is closed, and it is opened again when the loop is disordered. Murray et al. <ref name="Murray et al." /> found that when SAM or adenosine is bound to the active site the gate is closed, and when PPNP (tripolyphosphate in the body) is bound to the active site the gate is open. It is still unknown what causes the gating loop to open and what triggers the release of products, but it is not caused by hydrolysis of tripolyphosphate <ref name="Murray et al." />. This finding shows that hydrolysis of tripolyphosphate does not provide energy for the opening of the gating loop, nor does ATP provide energy for any part of this reaction.<br /> </StructureSection><br /> <br /> ==3D structures of S-adenosylmethionine synthetase==<br /> <br /> [[S-adenosylmethionine synthetase 3D structures]]<br /> <br /> {{REVISIONDAY2}}-{{MONTHNAME|{{REVISIONMONTH}}}}-{{REVISIONYEAR}}<br /> {{#tree:id=OrganizedByTopic|openlevels=0|<br /> <br /> *S-adenosylmethionine synthetase<br /> <br /> **[[1fug]] – EcMAT – ''Escherichia coli''<br /><br /> **[[3iml]] – MAT – ''Burkholderia pseudomallei''<br /><br /> **[[2ydy]] – hMAT II subunit β – human<br /><br /> **[[4le5]] – MAT- ''Campylobacter jejuni''<br /><br /> **[[4hpv]] - SsMAT – ''Sulfolobus solfataricus''<br /><br /> **[[3tde]] – MAT- ''Mycobacterium tuberculosis''<br /><br /> **[[3s82]] – MAT- ''Mycobacterium avium''<br /><br /> **[[3rv2]] – MAT- ''Mycobacterium marinum''<br /><br /> <br /> <br /> *S-adenosylmethionine synthetase complexes<br /> <br /> **[[1xra]], [[1xrb]] – EcMAT + Pi + K + Mg <br /><br /> **[[1xrc]] - EcMAT + Pi + Co + K<br /><br /> **[[1mxa]] - EcMAT + Pi + PPi + Mg + K<br /><br /> **[[1mxb]] - EcMAT + Pi + ADP + Mg + K<br /><br /> **[[1mxc]] - EcMAT + Pi + Br-ADP + Mg + K<br /><br /> **[[1p7l]] – EcMAT + methionine + AdoMet + AMPPNP + Mg + K<br /><br /> **[[1rg9]] - EcMAT + AdoMet + AMPPNP + Mg + K<br /><br /> **[[1qm4]] - rMAT+ methionine analog + K + Mg – rat<br /><br /> **[[1o90]] - rMAT + Pi + Mg + K<br /><br /> **[[1o92]] – rMAT + methionine analog + ADP + Mg + K<br /><br /> **[[1o93]] – rMAT + methionine analog + ATP + Mg + K<br /><br /> **[[1o9t]] – rMAT + methionine + ADP + Mg + K<br /><br /> **[[2obv]] – hMAT I + AdoMet + Na <br /><br /> **[[2p02]] - hMAT II + AdoMet<br /><br /> **[[2ydx]] – MAT + resveratrol + NADP + Ca + ADP derivative<br /><br /> **[[4l2z]], [[4k0b]] - SsMAT + Pi + PPi + Mg + SAM derivative <br /><br /> }}<br /> == References ==<br /> <references/><br /> [[Category:Topic Page]][[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Methionine_adenosyltransferase&diff=3550769 Methionine adenosyltransferase 2022-04-25T15:06:18Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>[[Methionine adenosyltransferase]] (MAT) or S-adenosylmethionine synthetase (SAM synthetase) synthesizes S-adenosylmethionine (SAM or AdoMet) from the substrates adenosine triphosphate (ATP) and methionine. ATP isn’t used only as a source of energy like it is in other reactions but gets a methionine added onto the 5' carbon while the three phosphate groups are broken down and released from the active site. This enzyme is conserved and found in many organisms, so it is essential for life. Importantly, the production of SAM by MAT provides methyl for methylation of nucleic acids, allowing for epigenetic modification. Problems with this enzyme have been shown to cause diseases including various cancers.<br /> <br /> == Relevance ==<br /> The product of this enzymatic reaction, SAM, is the universal methyl donor of metabolism. DNA methyltransferases can transfer a methyl group from SAM to the 5th carbon of cytosine residues <ref>DOI:10.1038/npp.2012.112</ref>. In this way, MAT is indirectly important for regulation of gene expression by providing methyl through SAM. SAM is also involved in N-methylation, O-methylation and C-methylation, yielding S-adenosyl homocysteine as a product that gets recycled by the [[one-carbon metabolism]]. Radical SAM enzymes break down SAM into an adenosyl radical and methionine, enabling a host of otherwise difficult to achieve reactions, e.g. in molybdenum cofactors biosynthesis<ref>DOI:10.1073/pnas.0404624101</ref>. Accumulation of S-adenosyl homocysteine (or homocysteine itself) indicates an imbalance in supply and demand for SAM in the organism. Methionine metabolism impairment in liver diseases is related in alteration in MAT<ref>PMID:7741002</ref>.<br /> <br /> <br /> == Function and reaction mechanism ==<br /> '''S-adenosylmethionine synthetase''' or '''S-adenosylmethionine synthase''' or '''S-adenosylmethionine transferase''' or '''methionine adenosyltransferase''' (MAT) catalyzes the conversion of methionine and ATP to S-adenosylmethionine (AdoMet), pyrophosphate (PPi) and orthophosphate (Pi). The catalytic entity of MAT is a dimer. MAT cofactors are Mg+2 (or Co+2) and K+ ions<ref>PMID:8611562</ref>. <br /> <br /> [[Image:Sam rxn 2.jpg|800px]]<br /> <br /> The nucleophilic sulfur atom of methionine attacks the slightly positive 5' carbon of the adenosine sugar unit. Following this, the bond from the 5' carbon to the oxygen breaks, separating the tripolyphosphate from the newly formed S-adenosylmethionine (SAM) <ref name="Murray et al.">Murray B, Antonyuk SV, Marina A, Lu SC, Mato JM, Hasnain SS, Rojas Al. Crystallography captures catalytic steps in human methionine adenosyltransferase enzymes. PNAS. 2016 Feb 8;113 (8) 2104-2109. doi: https://doi.org/10.1073/pnas.1510959113</ref>. This is an example of an SN2 reaction, where the substrates move through a transition state to then form the products. The products are only released after the methionine binds and the C-O bond breaks.<br /> <br /> == Structure ==<br /> <StructureSection load='5a1i' size='400' side='right' caption='MAT dimer in complex with methionine, PPNP, and adenosine (PBD 5a1i)' scene=''><br /> MAT consists of α and β subunits. The MATα1 and <scene name='90/907472/Mat2a/4'>MATα2</scene> subunits are catalytic subunits while MATβ is a regulatory subunit. MATα2 subunits can form <scene name='49/493038/Tetramer/1'>tetramers</scene>, where the active site is found <scene name='49/493038/Tetramer/2'>between two of the subunits</scene>. The subunits are encoded on different genes in humans, so they are created separately and can then come together to form various complexes, such as MATαβ or MATα2 dimers <ref name="Murray et al." />. Not much is currently known about the function of this regulatory subunit and how it regulates the function of the enzyme<ref>DOI:10.1107/S2052252514012585</ref>. However, Murray et al.<ref name="Murray et al." /> show that even in the absence of the regulatory subunit, the active site found in the catalytic subunit remains functional. <br /> <br /> The biological assembly of rat S-adenosylmethionine synthetase is a <scene name='49/493038/Cv/7'>homotetramer</scene>. The <scene name='49/493038/Cv/9'>active site of MAT is located between adjacent subunits</scene><ref>PMID:12888348</ref>. The <scene name='49/493038/Substrates/2'>substrates</scene> used by the enzyme are methionine and ATP. Notably, ATP is not used as a source of energy in this reaction like it is for many other processes. Instead, it is used as a substrate in the synthesis reaction. Methionine and ATP enter the active site and are stabilized by residues present there, including lysine and histidine. Once the reaction begins to take place, methionine flips toward the 5' carbon of the adenosine sugar<ref>doi:10.1042/BJ20121580</ref>. Following nucleophilic attack of the sulfur on the carbon, the C-O bond between the phosphates and the carbon breaks, and the <scene name='49/493038/Product/3'>products</scene> are formed (tripolyphosphate not pictured). SAM is released from the active site first. MAT also catalyzes hydrolysis of the tripolyphosphate into pyrophosphate and orthophosphate, which are then released from the active site <ref>Niland CN, Ghosh A, Cahill SM, Schramm VL. Mechanism and Inhibition of Human Methionine Adenosyltransferase 2A. ACS Biochemistry. 2021 Mar 3;60 (10) 791-801. doi: https://doi.org/10.1021/acs.biochem.0c00998</ref>.<br /> <br /> == Gating Loop ==<br /> MAT has been shown to have a <scene name='49/493038/Loop/2'>gating loop</scene> next to the active site <ref name="Murray et al." />. <br /> <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>model 1</script><br /> <text>ordered with SAM</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>model 2</script><br /> <text>disordered apo</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>model 0</script><br /> <text>both</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>model 1; delay 0.5;model 2; delay 0.5;model 1; delay 0.5;model 2; delay 0.5;model 1; delay 0.5;model 2;</script><br /> <text>animate 3 cycles</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> This structure is thought to allow access to the active site, becoming ordered or disordered. When the loop is ordered, the active site is closed, and it is opened again when the loop is disordered. Murray et al. <ref name="Murray et al." /> found that when SAM or adenosine is bound to the active site the gate is closed, and when PPNP (tripolyphosphate in the body) is bound to the active site the gate is open. It is still unknown what causes the gating loop to open and what triggers the release of products, but it is not caused by hydrolysis of tripolyphosphate <ref name="Murray et al." />. This finding shows that hydrolysis of tripolyphosphate does not provide energy for the opening of the gating loop, nor does ATP provide energy for any part of this reaction.<br /> </StructureSection><br /> <br /> ==3D structures of S-adenosylmethionine synthetase==<br /> <br /> [[S-adenosylmethionine synthetase 3D structures]]<br /> <br /> {{REVISIONDAY2}}-{{MONTHNAME|{{REVISIONMONTH}}}}-{{REVISIONYEAR}}<br /> {{#tree:id=OrganizedByTopic|openlevels=0|<br /> <br /> *S-adenosylmethionine synthetase<br /> <br /> **[[1fug]] – EcMAT – ''Escherichia coli''<br /><br /> **[[3iml]] – MAT – ''Burkholderia pseudomallei''<br /><br /> **[[2ydy]] – hMAT II subunit β – human<br /><br /> **[[4le5]] – MAT- ''Campylobacter jejuni''<br /><br /> **[[4hpv]] - SsMAT – ''Sulfolobus solfataricus''<br /><br /> **[[3tde]] – MAT- ''Mycobacterium tuberculosis''<br /><br /> **[[3s82]] – MAT- ''Mycobacterium avium''<br /><br /> **[[3rv2]] – MAT- ''Mycobacterium marinum''<br /><br /> <br /> <br /> *S-adenosylmethionine synthetase complexes<br /> <br /> **[[1xra]], [[1xrb]] – EcMAT + Pi + K + Mg <br /><br /> **[[1xrc]] - EcMAT + Pi + Co + K<br /><br /> **[[1mxa]] - EcMAT + Pi + PPi + Mg + K<br /><br /> **[[1mxb]] - EcMAT + Pi + ADP + Mg + K<br /><br /> **[[1mxc]] - EcMAT + Pi + Br-ADP + Mg + K<br /><br /> **[[1p7l]] – EcMAT + methionine + AdoMet + AMPPNP + Mg + K<br /><br /> **[[1rg9]] - EcMAT + AdoMet + AMPPNP + Mg + K<br /><br /> **[[1qm4]] - rMAT+ methionine analog + K + Mg – rat<br /><br /> **[[1o90]] - rMAT + Pi + Mg + K<br /><br /> **[[1o92]] – rMAT + methionine analog + ADP + Mg + K<br /><br /> **[[1o93]] – rMAT + methionine analog + ATP + Mg + K<br /><br /> **[[1o9t]] – rMAT + methionine + ADP + Mg + K<br /><br /> **[[2obv]] – hMAT I + AdoMet + Na <br /><br /> **[[2p02]] - hMAT II + AdoMet<br /><br /> **[[2ydx]] – MAT + resveratrol + NADP + Ca + ADP derivative<br /><br /> **[[4l2z]], [[4k0b]] - SsMAT + Pi + PPi + Mg + SAM derivative <br /><br /> }}<br /> == References ==<br /> <references/><br /> [[Category:Topic Page]][[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Methionine_adenosyltransferase&diff=3550766 Methionine adenosyltransferase 2022-04-25T14:57:29Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>[[Methionine adenosyltransferase]] (MAT) or S-adenosylmethionine synthetase (SAM synthetase) synthesizes S-adenosylmethionine (SAM or AdoMet) from the substrates adenosine triphosphate (ATP) and methionine. ATP isn’t used only as a source of energy like it is in other reactions but gets a methionine added onto the 5' carbon while the three phosphate groups are broken down and released from the active site. This enzyme is conserved and found in many organisms, so it is essential for life. Importantly, the production of SAM by MAT provides methyl for methylation of nucleic acids, allowing for epigenetic modification. Problems with this enzyme have been shown to cause diseases including various cancers.<br /> <br /> == Relevance ==<br /> The product of this enzymatic reaction, SAM, is the universal methyl donor of metabolism. DNA methyltransferases can transfer a methyl group from SAM to the 5th carbon of cytosine residues <ref>DOI:10.1038/npp.2012.112</ref>. In this way, MAT is indirectly important for regulation of gene expression by providing methyl through SAM. SAM is also involved in N-methylation, O-methylation and C-methylation, yielding S-adenosyl homocysteine as a product that gets recycled by the [[one-carbon metabolism]]. Radical SAM enzymes break down SAM into an adenosyl radical and methionine, enabling a host of otherwise difficult to achieve reactions, e.g. in molybdenum cofactors biosynthesis<ref>DOI:10.1073/pnas.0404624101</ref>. Accumulation of S-adenosyl homocysteine (or homocysteine itself) indicates an imbalance in supply and demand for SAM in the organism. Methionine metabolism impairment in liver diseases is related in alteration in MAT<ref>PMID:7741002</ref>.<br /> <br /> <br /> == Function and reaction mechanism ==<br /> '''S-adenosylmethionine synthetase''' or '''S-adenosylmethionine synthase''' or '''S-adenosylmethionine transferase''' or '''methionine adenosyltransferase''' (MAT) catalyzes the conversion of methionine and ATP to S-adenosylmethionine (AdoMet), pyrophosphate (PPi) and orthophosphate (Pi). The catalytic entity of MAT is a dimer. MAT cofactors are Mg+2 (or Co+2) and K+ ions<ref>PMID:8611562</ref>. <br /> <br /> [[Image:Sam rxn 2.jpg|800px]]<br /> <br /> The nucleophilic sulfur atom of methionine attacks the slightly positive 5' carbon of the adenosine sugar unit. Following this, the bond from the 5' carbon to the oxygen breaks, separating the tripolyphosphate from the newly formed S-adenosylmethionine (SAM) <ref name="Murray et al.">Murray B, Antonyuk SV, Marina A, Lu SC, Mato JM, Hasnain SS, Rojas Al. Crystallography captures catalytic steps in human methionine adenosyltransferase enzymes. PNAS. 2016 Feb 8;113 (8) 2104-2109. doi: https://doi.org/10.1073/pnas.1510959113</ref>. This is an example of an SN2 reaction, where the substrates move through a transition state to then form the products. The products are only released after the methionine binds and the C-O bond breaks.<br /> <br /> == Structure ==<br /> <StructureSection load='5a1i' size='400' side='right' caption='MAT dimer in complex with methionine, PPNP, and adenosine (PBD 5a1i)' scene=''><br /> MAT consists of α and β subunits. The MATα1 and <scene name='90/907472/Mat2a/4'>MATα2</scene> subunits are catalytic subunits while MATβ is a regulatory subunit. The subunits are encoded on different genes in humans, so they are created separately and can then come together to form various complexes, such as MATαβ or MATα2 dimers <ref name="Murray et al." />. Not much is currently known about the function of this regulatory subunit and how it regulates the function of the enzyme<ref>DOI:10.1107/S2052252514012585</ref>. However, Murray et al.<ref name="Murray et al." /> show that even in the absence of the regulatory subunit, the active site found in the catalytic subunit remains functional. <br /> <br /> The biological assembly of rat S-adenosylmethionine synthetase is a <scene name='49/493038/Cv/7'>homotetramer</scene>. The <scene name='49/493038/Cv/8'>active site of MAT is located between adjacent subunits</scene><ref>PMID:12888348</ref>. The <scene name='49/493038/Substrates/2'>substrates</scene> used by the enzyme are methionine and ATP. Notably, ATP is not used as a source of energy in this reaction like it is for many other processes. Instead, it is used as a substrate in the synthesis reaction. Methionine and ATP enter the active site and are stabilized by residues present there, including lysine and histidine. Once the reaction begins to take place, methionine flips toward the 5' carbon of the adenosine sugar<ref>doi:10.1042/BJ20121580</ref>. Following nucleophilic attack of the sulfur on the carbon, the C-O bond between the phosphates and the carbon breaks, and the <scene name='49/493038/Product/3'>products</scene> are formed (tripolyphosphate not pictured). SAM is released from the active site first. MAT also catalyzes hydrolysis of the tripolyphosphate into pyrophosphate and orthophosphate, which are then released from the active site <ref>Niland CN, Ghosh A, Cahill SM, Schramm VL. Mechanism and Inhibition of Human Methionine Adenosyltransferase 2A. ACS Biochemistry. 2021 Mar 3;60 (10) 791-801. doi: https://doi.org/10.1021/acs.biochem.0c00998</ref>.<br /> <br /> == Gating Loop ==<br /> MAT has been shown to have a <scene name='49/493038/Loop/2'>gating loop</scene> next to the active site <ref name="Murray et al." />. <br /> <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>model 1</script><br /> <text>ordered with SAM</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>model 2</script><br /> <text>disordered apo</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>model 0</script><br /> <text>both</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>model 1; delay 0.5;model 2; delay 0.5;model 1; delay 0.5;model 2; delay 0.5;model 1; delay 0.5;model 2;</script><br /> <text>animate 3 cycles</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> This structure is thought to allow access to the active site, becoming ordered or disordered. When the loop is ordered, the active site is closed, and it is opened again when the loop is disordered. Murray et al. <ref name="Murray et al." /> found that when SAM or adenosine is bound to the active site the gate is closed, and when PPNP (tripolyphosphate in the body) is bound to the active site the gate is open. It is still unknown what causes the gating loop to open and what triggers the release of products, but it is not caused by hydrolysis of tripolyphosphate <ref name="Murray et al." />. This finding shows that hydrolysis of tripolyphosphate does not provide energy for the opening of the gating loop, nor does ATP provide energy for any part of this reaction.<br /> </StructureSection><br /> <br /> ==3D structures of S-adenosylmethionine synthetase==<br /> Scene 1: <scene name='49/493038/Tetramer/1'>tetramer</scene><br /> Scene 2:<scene name='49/493038/Tetramer/2'>binding site detail</scene><br /> <br /> [[S-adenosylmethionine synthetase 3D structures]]<br /> <br /> {{REVISIONDAY2}}-{{MONTHNAME|{{REVISIONMONTH}}}}-{{REVISIONYEAR}}<br /> {{#tree:id=OrganizedByTopic|openlevels=0|<br /> <br /> *S-adenosylmethionine synthetase<br /> <br /> **[[1fug]] – EcMAT – ''Escherichia coli''<br /><br /> **[[3iml]] – MAT – ''Burkholderia pseudomallei''<br /><br /> **[[2ydy]] – hMAT II subunit β – human<br /><br /> **[[4le5]] – MAT- ''Campylobacter jejuni''<br /><br /> **[[4hpv]] - SsMAT – ''Sulfolobus solfataricus''<br /><br /> **[[3tde]] – MAT- ''Mycobacterium tuberculosis''<br /><br /> **[[3s82]] – MAT- ''Mycobacterium avium''<br /><br /> **[[3rv2]] – MAT- ''Mycobacterium marinum''<br /><br /> <br /> <br /> *S-adenosylmethionine synthetase complexes<br /> <br /> **[[1xra]], [[1xrb]] – EcMAT + Pi + K + Mg <br /><br /> **[[1xrc]] - EcMAT + Pi + Co + K<br /><br /> **[[1mxa]] - EcMAT + Pi + PPi + Mg + K<br /><br /> **[[1mxb]] - EcMAT + Pi + ADP + Mg + K<br /><br /> **[[1mxc]] - EcMAT + Pi + Br-ADP + Mg + K<br /><br /> **[[1p7l]] – EcMAT + methionine + AdoMet + AMPPNP + Mg + K<br /><br /> **[[1rg9]] - EcMAT + AdoMet + AMPPNP + Mg + K<br /><br /> **[[1qm4]] - rMAT+ methionine analog + K + Mg – rat<br /><br /> **[[1o90]] - rMAT + Pi + Mg + K<br /><br /> **[[1o92]] – rMAT + methionine analog + ADP + Mg + K<br /><br /> **[[1o93]] – rMAT + methionine analog + ATP + Mg + K<br /><br /> **[[1o9t]] – rMAT + methionine + ADP + Mg + K<br /><br /> **[[2obv]] – hMAT I + AdoMet + Na <br /><br /> **[[2p02]] - hMAT II + AdoMet<br /><br /> **[[2ydx]] – MAT + resveratrol + NADP + Ca + ADP derivative<br /><br /> **[[4l2z]], [[4k0b]] - SsMAT + Pi + PPi + Mg + SAM derivative <br /><br /> }}<br /> == References ==<br /> <references/><br /> [[Category:Topic Page]][[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Methionine_adenosyltransferase&diff=3550760 Methionine adenosyltransferase 2022-04-25T14:53:20Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>[[Methionine adenosyltransferase]] (MAT) or S-adenosylmethionine synthetase (SAM synthetase) synthesizes S-adenosylmethionine (SAM or AdoMet) from the substrates adenosine triphosphate (ATP) and methionine. ATP isn’t used only as a source of energy like it is in other reactions but gets a methionine added onto the 5' carbon while the three phosphate groups are broken down and released from the active site. This enzyme is conserved and found in many organisms, so it is essential for life. Importantly, the production of SAM by MAT provides methyl for methylation of nucleic acids, allowing for epigenetic modification. Problems with this enzyme have been shown to cause diseases including various cancers.<br /> <br /> == Relevance ==<br /> The product of this enzymatic reaction, SAM, is the universal methyl donor of metabolism. DNA methyltransferases can transfer a methyl group from SAM to the 5th carbon of cytosine residues <ref>DOI:10.1038/npp.2012.112</ref>. In this way, MAT is indirectly important for regulation of gene expression by providing methyl through SAM. SAM is also involved in N-methylation, O-methylation and C-methylation, yielding S-adenosyl homocysteine as a product that gets recycled by the [[one-carbon metabolism]]. Radical SAM enzymes break down SAM into an adenosyl radical and methionine, enabling a host of otherwise difficult to achieve reactions, e.g. in molybdenum cofactors biosynthesis<ref>DOI:10.1073/pnas.0404624101</ref>. Accumulation of S-adenosyl homocysteine (or homocysteine itself) indicates an imbalance in supply and demand for SAM in the organism. Methionine metabolism impairment in liver diseases is related in alteration in MAT<ref>PMID:7741002</ref>.<br /> <br /> <br /> == Function and reaction mechanism ==<br /> '''S-adenosylmethionine synthetase''' or '''S-adenosylmethionine synthase''' or '''S-adenosylmethionine transferase''' or '''methionine adenosyltransferase''' (MAT) catalyzes the conversion of methionine and ATP to S-adenosylmethionine (AdoMet), pyrophosphate (PPi) and orthophosphate (Pi). The catalytic entity of MAT is a dimer. MAT cofactors are Mg+2 (or Co+2) and K+ ions<ref>PMID:8611562</ref>. <br /> <br /> [[Image:Sam rxn 2.jpg|800px]]<br /> <br /> The nucleophilic sulfur atom of methionine attacks the slightly positive 5' carbon of the adenosine sugar unit. Following this, the bond from the 5' carbon to the oxygen breaks, separating the tripolyphosphate from the newly formed S-adenosylmethionine (SAM) <ref name="Murray et al.">Murray B, Antonyuk SV, Marina A, Lu SC, Mato JM, Hasnain SS, Rojas Al. Crystallography captures catalytic steps in human methionine adenosyltransferase enzymes. PNAS. 2016 Feb 8;113 (8) 2104-2109. doi: https://doi.org/10.1073/pnas.1510959113</ref>. This is an example of an SN2 reaction, where the substrates move through a transition state to then form the products. The products are only released after the methionine binds and the C-O bond breaks.<br /> <br /> == Structure ==<br /> <StructureSection load='5a1i' size='400' side='right' caption='MAT dimer in complex with methionine, PPNP, and adenosine' scene=''><br /> MAT consists of α and β subunits. The MATα1 and <scene name='90/907472/Mat2a/4'>MATα2</scene> subunits are catalytic subunits while MATβ is a regulatory subunit. The subunits are encoded on different genes in humans, so they are created separately and can then come together to form various complexes, such as MATαβ or MATα2 dimers <ref name="Murray et al." />. Not much is currently known about the function of this regulatory subunit and how it regulates the function of the enzyme<ref>DOI:10.1107/S2052252514012585</ref>. However, Murray et al.<ref name="Murray et al." /> show that even in the absence of the regulatory subunit, the active site found in the catalytic subunit remains functional. <br /> <br /> The biological assembly of rat S-adenosylmethionine synthetase is a <scene name='49/493038/Cv/7'>homotetramer</scene>. The <scene name='49/493038/Cv/8'>active site of MAT is located between adjacent subunits</scene><ref>PMID:12888348</ref>. The <scene name='90/907472/Substrates/2'>substrates</scene> used by the enzyme are methionine and ATP. Notably, ATP is not used as a source of energy in this reaction like it is for many other processes. Instead, it is used as a substrate in the synthesis reaction. Methionine and ATP enter the active site and are stabilized by residues present there, including lysine and histidine. Once the reaction begins to take place, methionine flips toward the 5' carbon of the adenosine sugar<ref>doi:10.1042/BJ20121580</ref>. Following nucleophilic attack of the sulfur on the carbon, the C-O bond between the phosphates and the carbon breaks, and the <scene name='90/907472/Product/3'>products</scene> are formed (tripolyphosphate not pictured). SAM is released from the active site first. MAT also catalyzes hydrolysis of the tripolyphosphate into pyrophosphate and orthophosphate, which are then released from the active site <ref>Niland CN, Ghosh A, Cahill SM, Schramm VL. Mechanism and Inhibition of Human Methionine Adenosyltransferase 2A. ACS Biochemistry. 2021 Mar 3;60 (10) 791-801. doi: https://doi.org/10.1021/acs.biochem.0c00998</ref>.<br /> <br /> == Gating Loop ==<br /> MAT has been shown to have a <scene name='49/493038/Loop/2'>gating loop</scene> next to the active site <ref name="Murray et al." />. <br /> <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>model 1</script><br /> <text>ordered with SAM</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>model 2</script><br /> <text>disordered apo</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>model 0</script><br /> <text>both</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>model 1; delay 0.5;model 2; delay 0.5;model 1; delay 0.5;model 2; delay 0.5;model 1; delay 0.5;model 2;</script><br /> <text>animate 3 cycles</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> This structure is thought to allow access to the active site, becoming ordered or disordered. When the loop is ordered, the active site is closed, and it is opened again when the loop is disordered. Murray et al. <ref name="Murray et al." /> found that when SAM or adenosine is bound to the active site the gate is closed, and when PPNP (tripolyphosphate in the body) is bound to the active site the gate is open. It is still unknown what causes the gating loop to open and what triggers the release of products, but it is not caused by hydrolysis of tripolyphosphate <ref name="Murray et al." />. This finding shows that hydrolysis of tripolyphosphate does not provide energy for the opening of the gating loop, nor does ATP provide energy for any part of this reaction.<br /> </StructureSection><br /> <br /> ==3D structures of S-adenosylmethionine synthetase==<br /> <br /> [[S-adenosylmethionine synthetase 3D structures]]<br /> <br /> {{REVISIONDAY2}}-{{MONTHNAME|{{REVISIONMONTH}}}}-{{REVISIONYEAR}}<br /> {{#tree:id=OrganizedByTopic|openlevels=0|<br /> <br /> *S-adenosylmethionine synthetase<br /> <br /> **[[1fug]] – EcMAT – ''Escherichia coli''<br /><br /> **[[3iml]] – MAT – ''Burkholderia pseudomallei''<br /><br /> **[[2ydy]] – hMAT II subunit β – human<br /><br /> **[[4le5]] – MAT- ''Campylobacter jejuni''<br /><br /> **[[4hpv]] - SsMAT – ''Sulfolobus solfataricus''<br /><br /> **[[3tde]] – MAT- ''Mycobacterium tuberculosis''<br /><br /> **[[3s82]] – MAT- ''Mycobacterium avium''<br /><br /> **[[3rv2]] – MAT- ''Mycobacterium marinum''<br /><br /> <br /> <br /> *S-adenosylmethionine synthetase complexes<br /> <br /> **[[1xra]], [[1xrb]] – EcMAT + Pi + K + Mg <br /><br /> **[[1xrc]] - EcMAT + Pi + Co + K<br /><br /> **[[1mxa]] - EcMAT + Pi + PPi + Mg + K<br /><br /> **[[1mxb]] - EcMAT + Pi + ADP + Mg + K<br /><br /> **[[1mxc]] - EcMAT + Pi + Br-ADP + Mg + K<br /><br /> **[[1p7l]] – EcMAT + methionine + AdoMet + AMPPNP + Mg + K<br /><br /> **[[1rg9]] - EcMAT + AdoMet + AMPPNP + Mg + K<br /><br /> **[[1qm4]] - rMAT+ methionine analog + K + Mg – rat<br /><br /> **[[1o90]] - rMAT + Pi + Mg + K<br /><br /> **[[1o92]] – rMAT + methionine analog + ADP + Mg + K<br /><br /> **[[1o93]] – rMAT + methionine analog + ATP + Mg + K<br /><br /> **[[1o9t]] – rMAT + methionine + ADP + Mg + K<br /><br /> **[[2obv]] – hMAT I + AdoMet + Na <br /><br /> **[[2p02]] - hMAT II + AdoMet<br /><br /> **[[2ydx]] – MAT + resveratrol + NADP + Ca + ADP derivative<br /><br /> **[[4l2z]], [[4k0b]] - SsMAT + Pi + PPi + Mg + SAM derivative <br /><br /> }}<br /> == References ==<br /> <references/><br /> [[Category:Topic Page]][[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3550750 Thymidylate synthase 2022-04-25T14:32:11Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref name="Kholodar" >DOI:10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg|1000px]]<br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|1000px]]<br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Dump_binding/4'>active sites</scene> can each bind the substrate dUMP<ref>PMID:9053905</ref>. The sketch below shows some of the ionic and hydrogen bonding interactions between the protein and the substrate, most of which are also shown in the 3D scene. You can measure distances and angles after turning off the spinning (for detailed instructions, see viewing guide, [https://proteopedia.org/wiki/index.php/Help:Viewing_pages]).<br /> <br /> [[Image:DUMP binding.PNG|400px]]<br /> <br /> Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> To distinguish between path A (in which cysteine remains covalently bound during methyl and hydride transfer) and path B (in which cysteine shows two separated nucleophilic attacks on carbon C6) in the mechanistic hypotheses depicted above, Kholodar et al co-crystallized the enzyme with an analog of the <scene name='49/493689/Ts_7jxf_percentb/5'>bisubstrate intermediate</scene>.<ref name="Kholodar" /><br /> [[Image:IntB.jpg|300px]]<br /> <br /> While the natural intermediate contains a carbonyl oxygen (circled in yellow), the cocrystallized intermediate has an amine (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> define current selected;<br /> select [VNM]301:B.NA4%A #9177;<br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in that position instead. This prevents the reaction from progressing to its products, allowing to capture the intermediate in the crystal structure determination.<br /> <br /> The active site cysteine initially covalently binds to carbon 6 of the dUMP-ring. As seen here in the analog of intermediate conformations, it is not covalently bound here. The active site is disordered in the structure, showing <jmol><br /> <jmolLink><br /> <script>moveto 1.0 { 897 150 415 177.22} 615.28 0.0 0.0 {-7.028727272727273 50.28431818181818 5.7841818181818185} 49.47386078304503 {0 0 0} 0 0 0 3.0 0.0 0.0;<br /> </script><br /> <text>two conformations</text><br /> </jmolLink><br /> </jmol>. One conformation shows the active site cysteine 4.21 Angstroms away from the C6 carbon of the dMP ring and the other 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/1'>inhibits</scene> TS.<br /> <br /> The image below shows folic acid on the top and the inhibitor methotrexate on the bottom<br /> <br /> [[Image:Methotrexate.png|400px]]<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=File:TS_Mechanism.jpg&diff=3550688 File:TS Mechanism.jpg 2022-04-24T16:59:06Z

<p>Michael O'Shaughnessy: uploaded a new version of "Image:TS Mechanism.jpg": Notation fix</p> <hr /> <div>== Summary ==<br /> Overview of two proposed mechanisms for the reaction catalyzed by the enzyme Thymidylate Synthase. This image is a modification of the image used in "Caught in Action: X‐ray Structure of Thymidylate Synthase with Noncovalent Intermediate Analog" by Kholodar et. al. (https://doi.org/10.1021/acs.biochem.1c00063) Image was modified for clarity. <br /> == Licensing ==<br /> {{self|cc-by-sa-3.0|GFDL}}</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=File:TS_Mechanism.jpg&diff=3550687 File:TS Mechanism.jpg 2022-04-24T16:57:36Z

<p>Michael O'Shaughnessy: uploaded a new version of "Image:TS Mechanism.jpg": Fixed notation for general base</p> <hr /> <div>== Summary ==<br /> Overview of two proposed mechanisms for the reaction catalyzed by the enzyme Thymidylate Synthase. This image is a modification of the image used in "Caught in Action: X‐ray Structure of Thymidylate Synthase with Noncovalent Intermediate Analog" by Kholodar et. al. (https://doi.org/10.1021/acs.biochem.1c00063) Image was modified for clarity. <br /> == Licensing ==<br /> {{self|cc-by-sa-3.0|GFDL}}</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=File:IntB.jpg&diff=3550686 File:IntB.jpg 2022-04-24T16:55:33Z

<p>Michael O'Shaughnessy: uploaded a new version of "Image:IntB.jpg": Reverted to version as of 22:42, 22 April 2022</p> <hr /> <div>== Summary ==<br /> A complexed intermediate of the substrates of TS<br /> == Licensing ==<br /> {{self|cc-by-sa-3.0|GFDL}}</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Methionine_synthase&diff=3550504 Methionine synthase 2022-04-22T15:21:16Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>This page is being worked on during the Spring 2022 semester.<br /> <br /> Addlt ref 1<ref>DOI: 10.1128/JB.00208-06</ref><br /> <br /> <br /> Addlt ref 2<ref>DOI:10.1073/pnas.1133218100</ref><br /> <br /> '''Methionine synthase''' (MS; EC: 2.1.1.13) is an important enzyme in [[one-carbon metabolism]]. MS catalyzes the transfer of a methyl group from methyltetrahydrofolate (MTHF) to homocysteine, resulting in the formation of methionine. Methionine is an essential amino acid required by our bodies for healthy cell and tissue growth. It is essential as it is not naturally derived in our bodies, thus requiring the conversion of homocysteine to methionine as needed.<br /> <br /> ==Function==<br /> <br /> [[Image:Overall.jpeg]]<br /> <br /> MS is a B12-dependent enzyme responsible for regenerating methionine from homocysteine and uses vitamin B12 Cobalamin as a cofactor. The change from homocysteine to methionine is an SN2 reaction, as seen above, where the methyl group on N-5 from methyltetrahydrofolate (MTHF), is donated. MTHF is a product of methylenetetrahydrofolate reductase [[MTHFR]] from the folate cycle. This is a complex reaction as tetrahydrofolate (THF), the product, is a poor leaving group and requires a "super nucleophile", vitamin B12 Cob(I)alamin, to carry out the reaction<ref>DOI:10.1146/annurev.biochem.72.121801.161828</ref><ref name="Kung et al">DOI: 10.1038/nature10916</ref>; the methyl carrier. <br /> <br /> === Oxidation states ===<br /> == Oxidation States of Cobalamin == <br /> <br /> Cobalamin exists in three different oxidation states during the MS cycle.<br /> <br /> '''Cob(I)alamin''': Cobalt in the +1 oxidation state is nicknamed the "super nucleophile" as its high energy is required to carry out the complex SN2 reaction of breaking the bond between THF and the methyl group, in the catalytic cycle.<br /> <br /> '''Co(III)alamin''': Cobalt in +3 oxidation state occurs when His 759 replaces the dimethylbenzimidazole (DMB) ligand to allow for the methyl to be accepted by Cob(I)alamin, forming Me-Cob(III)alamin.<br /> <br /> '''Cob(II)alamin''': Cob(I)alamin is highlight reactive towards oxygen so occasionally under aerobic conditions, Cob(I)alamin will occasionally undergo oxidation leading to an inactive Cob(II)alamin enzyme in the +2 oxidation state. This is regulated by reductive methylation by using Flavodoxin as an electron donor to reactivate Cob(I)alamin, and subsequently regenerates Me-Cob(III)alamin with a methyl being donated from SAM.<br /> <br /> == Relevance ==<br /> <br /> MS is an important enzyme responsible for generating methionine, required by our bodies for healthy cell and tissue growth, and protein synthesis. Any MS and/or B12 deficiencies can result in diseases such as abnormal birth defects or anemia<ref name="Kung et al"/>.<br /> <br /> Uses in treating cancer.<br /> <br /> <br /> == Structural highlights ==<br /> <br /> === 1. Domain organization ===<br /> <StructureSection load='1bmt' size='310' side='right' caption='Homodimer of B12 binding domain of MS. Cobalt in pink.' scene=''><br /> <br /> The full structure of MS has yet to be determined but studies have found it contains four domains, each with a unique function that bind to Cob(I)alamin as the methyl carrier, MTHF as the methyl donor in the catalytic cycle, homocysteine as the methyl acceptor, and S-adenosylmethionine or SAM, as the methyl donor in the reactivation cycle<ref name="Bandarian et al">DOI: 10.1038/nsb738</ref>. The orientation of the domains changes during the catalytic cycle.<br /> <br /> Shown here is the <scene name='90/907471/Superposition/7'>theoretical prediction</scene> of the structure by the alphafold algorithm, with experimental structures of the N-terminal 2 domains as well as of the C-terminal 2 domins superposed.<br /> <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide 1.1</script><br /> <text>experimental fragments</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>display 1.1</script><br /> <text>theoretical</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>display all</script><br /> <text>both</text><br /> <checked>true</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> As the graph below shows, the prediction has high confidence in the internal structure of individual domains but not the relative orientation.<br /> <br /> [[Image:Position error alphafold P13009.jpg|400px]]<br /> <br /> During each cycle, the domains must be positioned close enough to Cobalamin in order for methyl transfers to be successful. Conformations of MS allows substrates to be presented to Cobalamin for reactions to occur.<br /> <br /> === 2. Cobalamin binding ===<br /> <br /> [[Image:cob_1_alamin.jpeg|500px]]<br /> <br /> The vitamin B12 Cobalamin binding domain has a special characteristic in that, it is most naturally found in a protective conformation to prevent unwanted chemistry from occurring (PDB: 1BMT). This is referred to as a 'capping' mechanism.<br /> <br /> <scene name='90/907471/Bindingdomain2/1'>scene 1</scene><br /> <br /> <scene name='90/907471/Bindingdomain3/2'>scene 2</scene><br /> <br /> <br /> === 3. Cobalamin activation ===<br /> <br /> === 4. Cap domain ===<br /> <br /> When B12 is not engaged with one of the other three substrate binding domains, it is protected by a <scene name='90/907471/Cap/1'>cap</scene>.<br /> <br /> In the Cob(I)alamin binding domain, the imidazole side chain containing His 759 replaces the dimethylbenzimidazole (DMB) ligand. His 759 bonds to Asp 757 and Ser 810 via hydrogen bonds to create a ligand trifecta that increases the efficiency of the methyl transfer during the catalytic cycle. With His on, the cap is off of Cobalamin to allow for it to hold onto the methyl from MTHF. With His off, the cap is on thus no reaction.<ref name="Bandarian et al"/>.<br /> <br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <br /> <references/><br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3550502 Thymidylate synthase 2022-04-22T14:32:12Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref name="Kholodar" >DOI:10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg|1000px]]<br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|1000px]]<br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Dump_binding/3'>active sites</scene> can each bind the substrate dUMP<ref>PMID:9053905</ref>. The sketch below shows some of the ionic and hydrogen bonding interactions between the protein and the substrate, most of which are also shown in the 3D scene. You can measure distances and angles after turning off the spinning (for detailed instructions, see viewing guide, [https://proteopedia.org/wiki/index.php/Help:Viewing_pages]).<br /> <br /> [[Image:DUMP binding.PNG|400px]]<br /> <br /> Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> To distinguish between path A (in which cysteine remains covalently bound during methyl and hydride transfer) and path B (in which cysteine shows two separated nucleophilic attacks on carbon C6) in the mechanistic hypotheses depicted above, Kholodar et al co-crystallized the enzyme with an analog of the <scene name='49/493689/Ts_7jxf_percentb/5'>bisubstrate intermediate</scene>.<ref name="Kholodar" /><br /> [[Image:IntB.jpg|300px]]<br /> <br /> While the natural intermediate contains a carbonyl oxygen (circled in yellow), the cocrystallized intermediate has an amine (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> define current selected;<br /> select [VNM]301:B.NA4%A #9177;<br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in that position instead. This prevents the reaction from progressing to its products, allowing to capture the intermediate in the crystal structure determination.<br /> <br /> The active site cysteine initially covalently binds to carbon 6 of the dUMP-ring. As seen here in the analog of intermediate conformations, it is not covalently bound here. The active site is disordered in the structure, showing <jmol><br /> <jmolLink><br /> <script>moveto 1.0 { 897 150 415 177.22} 615.28 0.0 0.0 {-7.028727272727273 50.28431818181818 5.7841818181818185} 49.47386078304503 {0 0 0} 0 0 0 3.0 0.0 0.0;<br /> </script><br /> <text>two conformations</text><br /> </jmolLink><br /> </jmol>. One conformation shows the active site cysteine 4.21 Angstroms away from the C6 carbon of the dMP ring and the other 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/1'>inhibits</scene> TS.<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> The image below shows tetrahydrofolic acid on the top and the inhibitor methotrexate on the bottom<br /> <br /> [[Image:Methotrexate.png|400px]]<br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3550501 Thymidylate synthase 2022-04-22T14:29:20Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref name="Kholodar" >DOI:10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|1000px]]<br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Dump_binding/3'>active sites</scene> can each bind the substrate dUMP<ref>PMID:9053905</ref>. The sketch below shows some of the ionic and hydrogen bonding interactions between the protein and the substrate, most of which are also shown in the 3D scene. You can measure distances and angles after turning off the spinning (for detailed instructions, see viewing guide, [https://proteopedia.org/wiki/index.php/Help:Viewing_pages]).<br /> <br /> [[Image:DUMP binding.PNG|400px]]<br /> <br /> Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> To distinguish between path A (in which cysteine remains covalently bound during methyl and hydride transfer) and path B (in which cysteine shows two separated nucleophilic attacks on carbon C6) in the mechanistic hypotheses depicted above, Kholodar et al co-crystallized the enzyme with an analog of the <scene name='49/493689/Ts_7jxf_percentb/5'>bisubstrate intermediate</scene>.<ref name="Kholodar" /><br /> [[Image:IntB.jpg|300px]]<br /> <br /> While the natural intermediate contains a carbonyl oxygen (circled in yellow), the cocrystallized intermediate has an amine (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> define current selected;<br /> select [VNM]301:B.NA4%A #9177;<br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in that position instead. This prevents the reaction from progressing to its products, allowing to capture the intermediate in the crystal structure determination.<br /> <br /> The active site cysteine initially covalently binds to carbon 6 of the dUMP-ring. As seen here in the analog of intermediate conformations, it is not covalently bound here. The active site is disordered in the structure, showing <jmol><br /> <jmolLink><br /> <script>moveto 1.0 { 897 150 415 177.22} 615.28 0.0 0.0 {-7.028727272727273 50.28431818181818 5.7841818181818185} 49.47386078304503 {0 0 0} 0 0 0 3.0 0.0 0.0;<br /> </script><br /> <text>two conformations</text><br /> </jmolLink><br /> </jmol>. One conformation shows the active site cysteine 4.21 Angstroms away from the C6 carbon of the dMP ring and the other 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/1'>inhibits</scene> TS.<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> The image below shows tetrahydrofolic acid on the top and the inhibitor methotrexate on the bottom<br /> <br /> [[Image:Methotrexate.png|400px]]<br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3547547 Thymidylate synthase 2022-04-20T19:35:16Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|800px]]<br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Cv/4'>active sites can each bind the substrate dUMP</scene><ref>PMID:9053905</ref>. Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> The active site Cysteine has two <scene name='49/493689/Ts_7jxf_percentb/4'>conformations</scene>. Conformation 1 shows the active site cysteine unbound to the intermediate 4.21 Angstroms away from the C6 carbon of the dMP ring. Conformation 2 shows the active site cysteine bound to the intermediate 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> The active site cysteine can covalently bind to and unbind from the appropriate carbon on the dUMP-ring, as seen here in the analog of intermediate conformations. The conversion of the carboxylic oxygen (circled in yellow) of the folate to nitrogen (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> define current selected;<br /> select [VNM]301:B.NA4%A #9177;<br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in the intermediate prevents the reaction from progressing to its products.<br /> <br /> [[Image:IntB.jpg|400px]]<br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/1'>inhibits</scene> TS.<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> The image below shows tetrahydrofolic acid on the top and the inhibitor methotrexate on the bottom<br /> [[Image:Methotrexate.png|400px]]<br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3547542 Thymidylate synthase 2022-04-20T19:12:13Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|800px]]<br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Cv/4'>active sites can each bind the substrate dUMP</scene><ref>PMID:9053905</ref>. Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> The active site Cysteine has two <scene name='49/493689/Ts_7jxf_percentb/4'>conformations</scene>. Conformation 1 shows the active site cysteine unbound to the intermediate 4.21 Angstroms away from the C6 carbon of the dMP ring. Conformation 2 shows the active site cysteine bound to the intermediate 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> The active site cysteine can covalently bind to and unbind from the appropriate carbon on the dUMP-ring, as seen here in the analog of intermediate conformations. The conversion of the carboxylic oxygen (circled in yellow) of the folate to nitrogen (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%A #9177;<br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in the intermediate prevents the reaction from progressing to its products.<br /> <br /> [[Image:IntB.jpg|400px]]<br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/1'>inhibits</scene> TS.<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> The image below shows tetrahydrofolic acid on the top and the inhibitor methotrexate on the bottom<br /> [[Image:Methotrexate.png|400px]]<br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3547541 Thymidylate synthase 2022-04-20T19:10:35Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|800px]]<br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Cv/4'>active sites can each bind the substrate dUMP</scene><ref>PMID:9053905</ref>. Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> The active site Cysteine has two <scene name='49/493689/Ts_7jxf_percentb/4'>conformations</scene>. Conformation 1 shows the active site cysteine unbound to the intermediate 4.21 Angstroms away from the C6 carbon of the dMP ring. Conformation 2 shows the active site cysteine bound to the intermediate 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> The active site cysteine can covalently bind to and unbind from the appropriate carbon on the dUMP-ring, as seen here in the analog of intermediate conformations. The conversion of the carboxylic oxygen (circled in yellow) of the folate to nitrogen (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> select [VNM]301:B.NA4%A #9177;<br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in the intermediate prevents the reaction from progressing to its products.<br /> <br /> [[Image:IntB.jpg|400px]]<br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/1'>inhibits</scene> TS.<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> The image below shows tetrahydrofolic acid on the top and the inhibitor methotrexate on the bottom<br /> [[Image:Methotrexate.png|400px]]<br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3547540 Thymidylate synthase 2022-04-20T19:09:06Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|800px]]<br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Cv/4'>active sites can each bind the substrate dUMP</scene><ref>PMID:9053905</ref>. Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> The active site Cysteine has two <scene name='49/493689/Ts_7jxf_percentb/4'>conformations</scene>. Conformation 1 shows the active site cysteine unbound to the intermediate 4.21 Angstroms away from the C6 carbon of the dMP ring. Conformation 2 shows the active site cysteine bound to the intermediate 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> The active site cysteine can covalently bind to and unbind from the appropriate carbon on the dUMP-ring, as seen here in the analog of intermediate conformations. The conversion of the carboxylic oxygen (circled in yellow) of the folate to nitrogen (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178 or [VNM]301:B.NA4%A #9177; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in the intermediate prevents the reaction from progressing to its products.<br /> <br /> [[Image:IntB.jpg|400px]]<br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/1'>inhibits</scene> TS.<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> The image below shows tetrahydrofolic acid on the top and the inhibitor methotrexate on the bottom<br /> [[Image:Methotrexate.png|400px]]<br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3547539 Thymidylate synthase 2022-04-20T19:05:29Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|800px]]<br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Cv/4'>active sites can each bind the substrate dUMP</scene><ref>PMID:9053905</ref>. Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> The active site Cysteine has two <scene name='49/493689/Ts_7jxf_percentb/4'>conformations</scene>. Conformation 1 shows the active site cysteine unbound to the intermediate 4.21 Angstroms away from the C6 carbon of the dMP ring. Conformation 2 shows the active site cysteine bound to the intermediate 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> The active site cysteine can covalently bind to and unbind from the appropriate carbon on the dUMP-ring, as seen here in the analog of intermediate conformations. The conversion of the carboxylic oxygen (circled in yellow) of the folate to nitrogen (<jmol><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> </jmol>) in the intermediate prevents the reaction from progressing to its products.<br /> <br /> [[Image:IntB.jpg|400px]]<br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/1'>inhibits</scene> TS.<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> The image below shows tetrahydrofolic acid on the top and the inhibitor methotrexate on the bottom<br /> [[Image:Methotrexate.png|400px]]<br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3547538 Thymidylate synthase 2022-04-20T19:04:46Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|800px]]<br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Cv/4'>active sites can each bind the substrate dUMP</scene><ref>PMID:9053905</ref>. Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> The active site Cysteine has two <scene name='49/493689/Ts_7jxf_percentb/4'>conformations</scene>. Conformation 1 shows the active site cysteine unbound to the intermediate 4.21 Angstroms away from the C6 carbon of the dMP ring. Conformation 2 shows the active site cysteine bound to the intermediate 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> The active site cysteine can covalently bind to and unbind from the appropriate carbon on the dUMP-ring, as seen here in the analog of intermediate conformations. The conversion of the carboxylic oxygen (circled in yellow) of the folate to nitrogen (<br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> ) in the intermediate prevents the reaction from progressing to its products.<br /> <br /> [[Image:IntB.jpg|400px]]<br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/1'>inhibits</scene> TS.<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> The image below shows tetrahydrofolic acid on the top and the inhibitor methotrexate on the bottom<br /> [[Image:Methotrexate.png|400px]]<br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=User:Michael_O%27Shaughnessy/_TS&diff=3547536 User:Michael O'Shaughnessy/ TS 2022-04-20T19:03:05Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>== Overview ==<br /> This page is a work in progress during the spring 2022 semester. <br /> <br /> <br /> The enzyme Thymidylate Synthase (TS) is an important enzyme in one-carbon metabolism[https://proteopedia.org/wiki/index.php/One-carbon_metabolism]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> <StructureSection load= '7jxf' size='340' side='right' caption='Caption for this structure' scene=''><br /> <br /> <br /> <br /> <scene name='90/907470/Ts/1'>Text To Be Displayed</scene><br /> <br /> <scene name='90/907470/Ts/2'>TS with no DDT or UMP</scene><br /> <br /> <ref>PMID:11590022</ref><br /> <br /> <br /> <br /> <br /> ==Small Molecules==<br /> 2-deoxyuridine-5'-monophosphate(dUMP)<br /> <br /> [[Image:dUMP-full.jpg]]<br /> 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F)<br /> <br /> [[Image:CH2H4F-full.jpg]]<br /> thymidine 5'-monophosphate(dTMP)<br /> <br /> [[Image:DTMP-full.jpg]]<br /> <br /> Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image: H2F-small.jpg]]<br /> <br /> ==Mechanism==<br /> <br /> [[Image:TS Mechanism.jpg|800px]]<br /> <br /> <br /> == Structural highlights ==<br /> <br /> The active site Cysteine has two <scene name='90/907470/Ts_7jxf_percentb/3'>conformations</scene>. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 1</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both confirmations</text><br /> <checked>true</checked><br /> </item><br /> </jmolRadioGroup><br /> <jmolLink><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmolLink><br /> <br /> </jmol><br /> <br /> <br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=User:Michael_O%27Shaughnessy/_TS&diff=3547535 User:Michael O'Shaughnessy/ TS 2022-04-20T19:01:17Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>== Overview ==<br /> This page is a work in progress during the spring 2022 semester. <br /> <br /> <br /> The enzyme Thymidylate Synthase (TS) is an important enzyme in one-carbon metabolism[https://proteopedia.org/wiki/index.php/One-carbon_metabolism]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> <StructureSection load= '7jxf' size='340' side='right' caption='Caption for this structure' scene=''><br /> <br /> <br /> <br /> <scene name='90/907470/Ts/1'>Text To Be Displayed</scene><br /> <br /> <scene name='90/907470/Ts/2'>TS with no DDT or UMP</scene><br /> <br /> <ref>PMID:11590022</ref><br /> <br /> <br /> <br /> <br /> ==Small Molecules==<br /> 2-deoxyuridine-5'-monophosphate(dUMP)<br /> <br /> [[Image:dUMP-full.jpg]]<br /> 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F)<br /> <br /> [[Image:CH2H4F-full.jpg]]<br /> thymidine 5'-monophosphate(dTMP)<br /> <br /> [[Image:DTMP-full.jpg]]<br /> <br /> Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image: H2F-small.jpg]]<br /> <br /> ==Mechanism==<br /> <br /> [[Image:TS Mechanism.jpg|800px]]<br /> <br /> <br /> == Structural highlights ==<br /> <br /> The active site Cysteine has two <scene name='90/907470/Ts_7jxf_percentb/3'>conformations</scene>. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 1</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both confirmations</text><br /> <checked>true</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> <br /> <br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=User:Michael_O%27Shaughnessy/_TS&diff=3547534 User:Michael O'Shaughnessy/ TS 2022-04-20T19:00:45Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>== Overview ==<br /> This page is a work in progress during the spring 2022 semester. <br /> <br /> <br /> The enzyme Thymidylate Synthase (TS) is an important enzyme in one-carbon metabolism[https://proteopedia.org/wiki/index.php/One-carbon_metabolism]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> <StructureSection load= '7jxf' size='340' side='right' caption='Caption for this structure' scene=''><br /> <br /> <br /> <br /> <scene name='90/907470/Ts/1'>Text To Be Displayed</scene><br /> <br /> <scene name='90/907470/Ts/2'>TS with no DDT or UMP</scene><br /> <br /> <ref>PMID:11590022</ref><br /> <br /> <br /> <br /> <br /> ==Small Molecules==<br /> 2-deoxyuridine-5'-monophosphate(dUMP)<br /> <br /> [[Image:dUMP-full.jpg]]<br /> 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F)<br /> <br /> [[Image:CH2H4F-full.jpg]]<br /> thymidine 5'-monophosphate(dTMP)<br /> <br /> [[Image:DTMP-full.jpg]]<br /> <br /> Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image: H2F-small.jpg]]<br /> <br /> ==Mechanism==<br /> <br /> [[Image:TS Mechanism.jpg|800px]]<br /> <br /> <br /> == Structural highlights ==<br /> <br /> The active site Cysteine has two <scene name='90/907470/Ts_7jxf_percentb/3'>conformations</scene>. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 1</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both confirmations</text><br /> <checked>true</checked><br /> </item><br /> </jmolRadioGroup><br /> <script><br /> define current selected;<br /> select [VNM]301:B.NA4%B #9178; <br /> selectionHalos on; <br /> delay 0.5; <br /> selectionHalos off;<br /> select current;<br /> </script><br /> <text>☼</text><br /> </jmol><br /> <br /> <br /> <br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3547533 Thymidylate synthase 2022-04-20T18:52:13Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer from the folate substrate to the dMP substrate. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|800px]]<br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Cv/4'>active sites can each bind the substrate dUMP</scene><ref>PMID:9053905</ref>. Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> The active site Cysteine has two <scene name='49/493689/Ts_7jxf_percentb/4'>conformations</scene>. Conformation 1 shows the active site cysteine unbound to the intermediate 4.21 Angstroms away from the C6 carbon of the dMP ring. Conformation 2 shows the active site cysteine bound to the intermediate 3.84 Angstroms away from the C6 carbon of the dMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> The active site cysteine can covalently bind to and unbind from the appropriate carbon on the dUMP-ring, as seen here in the analog of intermediate conformations. The conversion of the carboxylic oxygen (circled in yellow) of the folate to nitrogen (*) in the intermediate prevents the reaction from progressing to its products.<br /> <br /> [[Image:IntB.jpg|400px]]<br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/1'>inhibits</scene> TS.<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> The image below shows tetrahydrofolic acid on the top and the inhibitor methotrexate on the bottom<br /> [[Image:Methotrexate.png|400px]]<br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3547532 Thymidylate synthase 2022-04-20T18:50:17Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Mechanism==<br /> There are two potential pathways that the mechanism for TS could operate by, however it is unclear which pathway the mechanism follows. In both pathways, the substrates form a covalent ternary complex with the enzyme. This complex facilitates the transfer of the methyl group from CH<sub>2</sub>H<sub>4</sub>F to dUMP and is followed by a hydride transfer. During the A pathway (highlighted in red), the intermediate complex remains covalently bound to the enzyme. The B pathway (highlighted in blue) involves the decoupling of the intermediate from the active site cysteine. <br /> <br /> [[Image:TS Mechanism.jpg|800px]]<br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Cv/4'>active sites can each bind the substrate dUMP</scene><ref>PMID:9053905</ref>. Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> The active site Cysteine has two <scene name='49/493689/Ts_7jxf_percentb/4'>conformations</scene>. Conformation 1 shows the active site cysteine unbound to the intermediate 4.21 Angstroms away from the C6 carbon of the dNMP ring. Conformation 2 shows the active site cysteine bound to the intermediate 3.84 Angstroms away from the C6 carbon of the dNMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> The active site cysteine can covalently bind to and unbind from the appropriate carbon on the dUMP-ring, as seen here in the analog of intermediate conformations. The conversion of the carboxylic oxygen (circled in yellow) of the folate to nitrogen (*) in the intermediate prevents the reaction from progressing to its products.<br /> <br /> [[Image:IntB.jpg|400px]]<br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/1'>inhibits</scene> TS.<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> The image below shows tetrahydrofolic acid on the top and the inhibitor methotrexate on the bottom<br /> [[Image:Methotrexate.png|400px]]<br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3547519 Thymidylate synthase 2022-04-20T18:01:24Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Cv/4'>active sites can each bind the substrate dUMP</scene><ref>PMID:9053905</ref>. Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> The active site Cysteine has two <scene name='49/493689/Ts_7jxf_percentb/4'>conformations</scene>. Conformation 1 shows the active site cysteine unbound to the intermediate 4.21 Angstroms away from the C6 carbon of the dNMP ring. Conformation 2 shows the active site cysteine bound to the intermediate 3.84 Angstroms away from the C6 carbon of the dNMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> The active site cysteine can covalently bind to and unbind from the appropriate carbon on the dUMP-ring, as seen here in the analog of intermediate conformations. The conversion of the carboxylic oxygen (circled in yellow) of the folate to nitrogen (*) in the intermediate prevents the reaction from progressing to its products.<br /> <br /> [[Image:IntB.jpg|400px]]<br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/1'>inhibits</scene> TS.<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> The image below shows tetrahydrofolic acid on the top and the inhibitor methotrexate on the bottom<br /> [[Image:Methotrexate.png|400px]]<br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3547518 Thymidylate synthase 2022-04-20T18:00:41Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Cv/4'>active sites can each bind the substrate dUMP</scene><ref>PMID:9053905</ref>. Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction<ref>DOI:10.1021/bi00005a001</ref>. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> The active site Cysteine has two <scene name='49/493689/Ts_7jxf_percentb/4'>conformations</scene>. Conformation 1 shows the active site cysteine unbound to the intermediate 4.21 Angstroms away from the C6 carbon of the dNMP ring. Conformation 2 shows the active site cysteine bound to the intermediate 3.84 Angstroms away from the C6 carbon of the dNMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> The active site cysteine can covalently bind to and unbind from the appropriate carbon on the dUMP-ring, as seen here in the analog of intermediate conformations. The conversion of the carboxylic oxygen of the folate to nitrogen (*) in the intermediate prevents the reaction from progressing to its products.<br /> <br /> [[Image:IntB.jpg|400px]]<br /> <br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/1'>inhibits</scene> TS.<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> The image below shows tetrahydrofolic acid on the top and the inhibitor methotrexate on the bottom<br /> [[Image:Methotrexate.png|400px]]<br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=File:IntB.jpg&diff=3547517 File:IntB.jpg 2022-04-20T17:59:55Z

<p>Michael O'Shaughnessy: A complexed intermediate of the substrates of TS</p> <hr /> <div>== Summary ==<br /> A complexed intermediate of the substrates of TS<br /> == Licensing ==<br /> {{self|cc-by-sa-3.0|GFDL}}</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=User:Michael_O%27Shaughnessy/_TS&diff=3547512 User:Michael O'Shaughnessy/ TS 2022-04-20T16:27:10Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>== Overview ==<br /> This page is a work in progress during the spring 2022 semester. <br /> <br /> <br /> The enzyme Thymidylate Synthase (TS) is an important enzyme in one-carbon metabolism[https://proteopedia.org/wiki/index.php/One-carbon_metabolism]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> <StructureSection load= '7jxf' size='340' side='right' caption='Caption for this structure' scene=''><br /> <br /> <br /> <br /> <scene name='90/907470/Ts/1'>Text To Be Displayed</scene><br /> <br /> <scene name='90/907470/Ts/2'>TS with no DDT or UMP</scene><br /> <br /> <ref>PMID:11590022</ref><br /> <br /> <br /> <br /> <br /> ==Small Molecules==<br /> 2-deoxyuridine-5'-monophosphate(dUMP)<br /> <br /> [[Image:dUMP-full.jpg]]<br /> 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F)<br /> <br /> [[Image:CH2H4F-full.jpg]]<br /> thymidine 5'-monophosphate(dTMP)<br /> <br /> [[Image:DTMP-full.jpg]]<br /> <br /> Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image: H2F-small.jpg]]<br /> <br /> ==Mechanism==<br /> <br /> [[Image:TS Mechanism.jpg|800px]]<br /> <br /> <br /> == Structural highlights ==<br /> <br /> The active site Cysteine has two <scene name='90/907470/Ts_7jxf_percentb/3'>conformations</scene>. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 1</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both confirmations</text><br /> <checked>true</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> <br /> <br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=User:Michael_O%27Shaughnessy/_TS&diff=3547511 User:Michael O'Shaughnessy/ TS 2022-04-20T16:26:40Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>== Overview ==<br /> This page is a work in progress during the spring 2022 semester. <br /> <br /> <br /> The enzyme Thymidylate Synthase (TS) is an important enzyme in one-carbon metabolism[https://proteopedia.org/wiki/index.php/One-carbon_metabolism]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> <StructureSection load= '7jxf' size='340' side='right' caption='Caption for this structure' scene=''><br /> <br /> <br /> <br /> <scene name='90/907470/Ts/1'>Text To Be Displayed</scene><br /> <br /> <scene name='90/907470/Ts/2'>TS with no DDT or UMP</scene><br /> <br /> <ref>PMID:11590022</ref><br /> <br /> <br /> <br /> <br /> ==Small Molecules==<br /> 2-deoxyuridine-5'-monophosphate(dUMP)<br /> <br /> [[Image:dUMP-full.jpg]]<br /> 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F)<br /> <br /> [[Image:CH2H4F-full.jpg]]<br /> thymidine 5'-monophosphate(dTMP)<br /> <br /> [[Image:DTMP-full.jpg]]<br /> <br /> Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image: H2F-small.jpg]]<br /> <br /> ==Mechanism==<br /> <br /> [[Image:TS Mechanism.jpg|900px]]<br /> <br /> <br /> == Structural highlights ==<br /> <br /> The active site Cysteine has two <scene name='90/907470/Ts_7jxf_percentb/3'>conformations</scene>. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 1</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both confirmations</text><br /> <checked>true</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> <br /> <br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=User:Michael_O%27Shaughnessy/_TS&diff=3547510 User:Michael O'Shaughnessy/ TS 2022-04-20T16:26:16Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>== Overview ==<br /> This page is a work in progress during the spring 2022 semester. <br /> <br /> <br /> The enzyme Thymidylate Synthase (TS) is an important enzyme in one-carbon metabolism[https://proteopedia.org/wiki/index.php/One-carbon_metabolism]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> <StructureSection load= '7jxf' size='340' side='right' caption='Caption for this structure' scene=''><br /> <br /> <br /> <br /> <scene name='90/907470/Ts/1'>Text To Be Displayed</scene><br /> <br /> <scene name='90/907470/Ts/2'>TS with no DDT or UMP</scene><br /> <br /> <ref>PMID:11590022</ref><br /> <br /> <br /> <br /> <br /> ==Small Molecules==<br /> 2-deoxyuridine-5'-monophosphate(dUMP)<br /> <br /> [[Image:dUMP-full.jpg]]<br /> 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F)<br /> <br /> [[Image:CH2H4F-full.jpg]]<br /> thymidine 5'-monophosphate(dTMP)<br /> <br /> [[Image:DTMP-full.jpg]]<br /> <br /> Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image: H2F-small.jpg]]<br /> <br /> ==Mechanism==<br /> <br /> [[Image:TS Mechanism.jpg|800px]]<br /> <br /> <br /> == Structural highlights ==<br /> <br /> The active site Cysteine has two <scene name='90/907470/Ts_7jxf_percentb/3'>conformations</scene>. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 1</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both confirmations</text><br /> <checked>true</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> <br /> <br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=User:Michael_O%27Shaughnessy/_TS&diff=3547509 User:Michael O'Shaughnessy/ TS 2022-04-20T16:25:44Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>== Overview ==<br /> This page is a work in progress during the spring 2022 semester. <br /> <br /> <br /> The enzyme Thymidylate Synthase (TS) is an important enzyme in one-carbon metabolism[https://proteopedia.org/wiki/index.php/One-carbon_metabolism]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> <StructureSection load= '7jxf' size='340' side='right' caption='Caption for this structure' scene=''><br /> <br /> <br /> <br /> <scene name='90/907470/Ts/1'>Text To Be Displayed</scene><br /> <br /> <scene name='90/907470/Ts/2'>TS with no DDT or UMP</scene><br /> <br /> <ref>PMID:11590022</ref><br /> <br /> <br /> <br /> <br /> ==Small Molecules==<br /> 2-deoxyuridine-5'-monophosphate(dUMP)<br /> <br /> [[Image:dUMP-full.jpg]]<br /> 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F)<br /> <br /> [[Image:CH2H4F-full.jpg]]<br /> thymidine 5'-monophosphate(dTMP)<br /> <br /> [[Image:DTMP-full.jpg]]<br /> <br /> Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image: H2F-small.jpg]]<br /> <br /> ==Mechanism==<br /> <br /> [[Image:TS Mechanism.jpg|700px]]<br /> <br /> <br /> == Structural highlights ==<br /> <br /> The active site Cysteine has two <scene name='90/907470/Ts_7jxf_percentb/3'>conformations</scene>. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 1</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both confirmations</text><br /> <checked>true</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> <br /> <br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=User:Michael_O%27Shaughnessy/_TS&diff=3547508 User:Michael O'Shaughnessy/ TS 2022-04-20T16:25:17Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>== Overview ==<br /> This page is a work in progress during the spring 2022 semester. <br /> <br /> <br /> The enzyme Thymidylate Synthase (TS) is an important enzyme in one-carbon metabolism[https://proteopedia.org/wiki/index.php/One-carbon_metabolism]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> <StructureSection load= '7jxf' size='340' side='right' caption='Caption for this structure' scene=''><br /> <br /> <br /> <br /> <scene name='90/907470/Ts/1'>Text To Be Displayed</scene><br /> <br /> <scene name='90/907470/Ts/2'>TS with no DDT or UMP</scene><br /> <br /> <ref>PMID:11590022</ref><br /> <br /> <br /> <br /> <br /> ==Small Molecules==<br /> 2-deoxyuridine-5'-monophosphate(dUMP)<br /> <br /> [[Image:dUMP-full.jpg]]<br /> 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F)<br /> <br /> [[Image:CH2H4F-full.jpg]]<br /> thymidine 5'-monophosphate(dTMP)<br /> <br /> [[Image:DTMP-full.jpg]]<br /> <br /> Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image: H2F-small.jpg]]<br /> <br /> ==Mechanism==<br /> <br /> [[Image:TS Mechanism.jpg|500px]]<br /> <br /> <br /> == Structural highlights ==<br /> <br /> The active site Cysteine has two <scene name='90/907470/Ts_7jxf_percentb/3'>conformations</scene>. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 1</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both confirmations</text><br /> <checked>true</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> <br /> <br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=User:Michael_O%27Shaughnessy/_TS&diff=3547507 User:Michael O'Shaughnessy/ TS 2022-04-20T15:25:26Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>== Overview ==<br /> This page is a work in progress during the spring 2022 semester. <br /> <br /> <br /> The enzyme Thymidylate Synthase (TS) is an important enzyme in one-carbon metabolism[https://proteopedia.org/wiki/index.php/One-carbon_metabolism]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <br /> <StructureSection load= '7jxf' size='340' side='right' caption='Caption for this structure' scene=''><br /> <br /> <br /> <br /> <scene name='90/907470/Ts/1'>Text To Be Displayed</scene><br /> <br /> <scene name='90/907470/Ts/2'>TS with no DDT or UMP</scene><br /> <br /> <ref>PMID:11590022</ref><br /> <br /> <br /> <br /> <br /> ==Small Molecules==<br /> 2-deoxyuridine-5'-monophosphate(dUMP)<br /> <br /> [[Image:dUMP-full.jpg]]<br /> 5,10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F)<br /> <br /> [[Image:CH2H4F-full.jpg]]<br /> thymidine 5'-monophosphate(dTMP)<br /> <br /> [[Image:DTMP-full.jpg]]<br /> <br /> Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image: H2F-small.jpg]]<br /> <br /> ==Mechanism==<br /> <br /> [[Image:TS Mechanism.jpg|400px]]<br /> <br /> <br /> == Structural highlights ==<br /> <br /> The active site Cysteine has two <scene name='90/907470/Ts_7jxf_percentb/3'>conformations</scene>. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 1</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both confirmations</text><br /> <checked>true</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> <br /> <br /> <br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=File:TS_Mechanism.jpg&diff=3547505 File:TS Mechanism.jpg 2022-04-20T15:20:27Z

<p>Michael O'Shaughnessy: uploaded a new version of "Image:TS Mechanism.jpg": Actually has the numbers this time</p> <hr /> <div>== Summary ==<br /> Overview of two proposed mechanisms for the reaction catalyzed by the enzyme Thymidylate Synthase. This image is a modification of the image used in "Caught in Action: X‐ray Structure of Thymidylate Synthase with Noncovalent Intermediate Analog" by Kholodar et. al. (https://doi.org/10.1021/acs.biochem.1c00063) Image was modified for clarity. <br /> == Licensing ==<br /> {{self|cc-by-sa-3.0|GFDL}}</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3547500 Thymidylate synthase 2022-04-20T15:11:27Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Structure and ligand binding==<br /> TS forms a homodimer consisting of two domains (reload <scene name='49/493689/Cv/1'>initial scene</scene>). Their <scene name='49/493689/Cv/4'>active sites can each bind the substrate dUMP</scene><ref>PMID:9053905</ref>. Maley et al. show that E.coli TS uses a half-the-sites mechanism when catalyzing its reaction. One of the two active sites catalyzes the reaction while the other remains inactive. The way the two domains communicate with each other to coordinate this is still unknown.<br /> <br /> The active site Cysteine has two <scene name='49/493689/Ts_7jxf_percentb/4'>conformations</scene>. Conformation 1 shows the active site cysteine unbound to the intermediate 4.21 Angstroms away from the C6 carbon of the dNMP ring. Conformation 2 shows the active site cysteine bound to the intermediate 3.84 Angstroms away from the C6 carbon of the dNMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor competes with folate substrate to bind to the active site of TS. Inhibits the conversion of CH<sub>2</sub>H<sub>4</sub>F to H<sub>2</sub>F. This inhibition stops the conversion of products to reactants, stopping cellular reproduction. <scene name='49/493689/Inhibited/1'>inhibits</scene> TS.<br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> [[Image:Methotrexate.png|400px]]<br /> </StructureSection><br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3547493 Thymidylate synthase 2022-04-20T14:35:34Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> One of the nucleobase of DNA, cytosine, spontaneously deaminates to form uracil, changing the encoded message. Different from RNA, DNA does not contain uracil but instead contains the methylated form thymine, avoiding missense mutations. The [https://proteopedia.org/wiki/index.php/Category:Dna-repair DNA repair protein] [[Uracil DNA glycosylase]] will recognize uracil as damaged DNA, removing it and re-establishing the original message<ref>DOI:10.1038/sj.onc.1205996</ref>. Because thymine contains an additional methyl group, it is distinguishable from uracil, and is not removed through DNA repair. Thymidylate synthase helps to turn the abundant uridine (containing the nucleobase uracil) into deoxythymidine (containing the nucleobase thymine) in preparation for DNA synthesis prior to cell division. Thus, thymidylate synthase plays a role in making DNA a more reliable long-term storage of genetic information compared to RNA.<br /> <br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> ==Structure and ligand binding==<br /> Thymidylate synthase forms a dimer (reload <scene name='49/493689/Cv/1'>initial scene</scene>).<br /> TS <scene name='49/493689/Cv/4'>active site contains the substrate dUMP</scene><ref>PMID:9053905</ref>. Water molecules are shown as red spheres. <br /> <br /> The active site Cysteine has two <scene name='49/493689/Ts_7jxf_percentb/4'>conformations</scene>. Conformation 1 shows the active site cysteineunbound to the intermediate 4.21 Angstroms away from the C6 carbon of the dNMP ring. Conformation 2 shows the active site cysteine bound to the intermediate 3.84 Angstroms away from the C6 carbon of the dNMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 1</text><br /> <checked>true</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both conformations</text><br /> <checked>false</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> ==Inhibition==<br /> Methotrexate, a competitive inhibitor of folates, <scene name='49/493689/Inhibited/1'>inhibits</scene> TS.<br /> </StructureSection><br /> <br /> <br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=File:TS_Mechanism.jpg&diff=3547431 File:TS Mechanism.jpg 2022-04-19T22:34:42Z

<p>Michael O'Shaughnessy: uploaded a new version of "Image:TS Mechanism.jpg": modified to show numbers of relevant atoms</p> <hr /> <div>== Summary ==<br /> Overview of two proposed mechanisms for the reaction catalyzed by the enzyme Thymidylate Synthase. This image is a modification of the image used in "Caught in Action: X‐ray Structure of Thymidylate Synthase with Noncovalent Intermediate Analog" by Kholodar et. al. (https://doi.org/10.1021/acs.biochem.1c00063) Image was modified for clarity. <br /> == Licensing ==<br /> {{self|cc-by-sa-3.0|GFDL}}</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=Thymidylate_synthase&diff=3546294 Thymidylate synthase 2022-04-15T17:10:16Z

<p>Michael O'Shaughnessy: </p> <hr /> <div>Thymidylate Synthase (TS) is an important enzyme in [[one-carbon metabolism]]. TS catalyzes the transfer of a methyl group and a hydride from 5,10-methylenetetrahydrofolate to 2-deoxyuridine-5'-monophosphate, resulting in the formation of thymidine 5'-monophosphate and dihydrofolate. This is the only de novo source of dTMP (a precursor to Thymine) in humans. <ref>DOI 10.1021/acs.biochem.1c00063</ref><br /> <br /> 2-deoxyuridine-5'-monophosphate(dUMP) + 5, 10-methylenetetrahydrofolate(CH<sub>2</sub>H<sub>4</sub>F) ⇌ thymidine 5'-monophosphate(dTMP) + Dihydrofolate(H<sub>2</sub>F)<br /> <br /> [[Image:TS Overview.jpg]]<br /> <StructureSection load='' size='350' side='right' caption='Thymidylate synthase complex with dUMP (PDB entry [[1tsv]])' scene='49/493689/Cv/1'><br /> == Function ==<br /> '''Thymidylate synthase''' (TS) catalyzes the methylation of dUMP to dTMP using 5,10-methylenetetrahydrofolate as a cofactor. TS is essential for DNA replication and repair<ref>PMID:2243092</ref>. In protozoa, [[dihydrofolate reductase]] (DHFR) and TS are expressed as a bifunctional monomeric enzyme ('''DHFR-TS)''' with the DHFR entity at the N terminal. DHFR and TS catalyze consecutive reactions in the dTMP biosynthesis. There are two different types of TS – '''ThyA''' and '''ThyX'''. The types differ in their activity and structure. The TS ThyX are flavin-dependent enzymes.<br /> <br /> == Relevance ==<br /> TS inhibition at its folate-binding site is used in anticancer therapeutic drugs. DHFR-TS inhibitors are potential drug targets against parasite-transferred diseases. TS exhibits oncogene-like activity.<ref>PMID:15093541</ref><br /> <br /> Due to its role in cell division, thymidylate synthase has become a popular target for anticancer drugs. Indirect inhibition of thymidylate synthase by the drug 5-fluorouracil (5-FU) is one of the most used inhibitors for study of TS function. This drug indirectly inhibits TS as it it eventually converted to FdUMP, which forms a covalent complex with both the active site cysteine and CH<sub>2</sub>H<sub>4</sub>F. Inhibition of TS halts the production of dTMP and, indirectly, 2'-deoxythymidine-5'-triphosphate (dTTP). Both dTMP and dTTP are essential building blocks for DNA synthesis and their absence halts the ability of cells to replicate their genetic information. This is especially effective in cancer cells that rapidly divide and require large amounts of dTMP and dTTP. <ref>DOI 10.2174/0929867054864868</ref><br /> <br /> == Structural highlights ==<br /> TS <scene name='49/493689/Cv/4'>active site contains the substrate dUMP</scene><ref>PMID:9053905</ref>. Water molecules are shown as red spheres. <br /> <br /> The active site Cysteine has two <scene name='49/493689/Ts_7jxf_percentb/4'>conformations</scene>. Conformation 1 shows the active site cysteine bound to the intermediate 3.84 Angstroms away from the C6 carbon of the dNMP ring. Conformation 2 shows the active site cysteine unbound to the intermediate 4.21 Angstroms away from the C6 carbon of the dNMP ring. <br /> <jmol><br /> <jmolRadioGroup><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=B</script><br /> <text>Conformation 1</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide (VNM or 146:B) and altloc=A</script><br /> <text>Conformation 2</text><br /> <checked>false</checked><br /> </item><br /> <item><br /> <script>hide none</script><br /> <text>both confirmations</text><br /> <checked>true</checked><br /> </item><br /> </jmolRadioGroup><br /> </jmol><br /> ==3D structures of thymidylate synthase==<br /> [[Thymidylate synthase 3D structures]]<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/><br /> [[Category:Topic Page]]<br /> [[Category:Dihydrofolate]]<br /> [[Category:One-carbon metabolism]]</div>

Michael O'Shaughnessy https://proteopedia.org/index.php?title=File:TS_Overview.jpg&diff=3546293 File:TS Overview.jpg 2022-04-15T17:08:49Z

<p>Michael O'Shaughnessy: uploaded a new version of "Image:TS Overview.jpg": actual adjusted resolution</p> <hr /> <div>== Summary ==<br /> An overview of the reaction catalyzed by the enzyme thymidylate synthase<br /> == Licensing ==<br /> {{self|cc-by-sa-3.0|GFDL}}</div>

Michael O'Shaughnessy