Sandbox Reserved 763: Difference between revisions

Line 92:

===Isoforms===

In '' Escherichia coli'' the shikimate kinase reaction is catalyzed by two different isoforms: SKI and SKII (Km= 20 μM Km= 200 μM, respectively) with 30% sequence identify. This is very unusual for two isoforms to occur in the middle of a biosynthetic pathway. Therefore, what may be occurring is that shikimate is at a branch point for two pathways. It is believed that SKII is the isoform participating in chorismate biosynthesis, while the role of the SKI isoform is unclear.<ref name="3dstructure">The three-dimensional structure of shikimate kinase. Journal of Molecular Biology http://www.sciencedirect.com.prox.lib.ncsu.edu/science/article/pii/S0022283698917557</ref>

In '' Escherichia coli'' the shikimate kinase reaction is catalyzed by two different isoforms: SKI and SKII (Km= 20 μM Km= 200 μM, respectively) with 30% sequence identify. This is very unusual for two isoforms to occur in the middle of a biosynthetic pathway. Therefore, what may be occurring is that shikimate is at a branch point for two pathways. It is believed that SKII is the isoform participating in chorismate biosynthesis, while the role of the SKI isoform is unclear.<ref name="3dstructure">Tino Krell et al. The three-dimensional structure of shikimate kinase. Journal of Molecular Biology http://www.sciencedirect.com.prox.lib.ncsu.edu/science/article/pii/S0022283698917557</ref>

===Oligomeric State===

Line 98:

Through experiments involving size exclusion liquid chromatography and gel filtration chromatography, the oligomeric state of homogeneous MtSK was found. The molecular mass was found to be 20.7 kDa and when compared to the suggested value of 18.5 kDa, it was found that MtSK is a monomer in solution.<ref>The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061918</ref>

Through experiments involving size exclusion liquid chromatography and gel filtration chromatography, the oligomeric state of homogeneous MtSK was found. The molecular mass was found to be 20.7 kDa and when compared to the suggested value of 18.5 kDa, it was found that MtSK is a monomer in solution.<ref>Leonardo Astolfi Rosado et al. The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061918</ref>

===Domains and Active Residues===

There are three domains present in Shikimate kinase: the CORE domain, and substrate-binding (SB) domain, and LID domain. Each domain plays a role in binding and is related to the active residues. The binding site for nucleotides is in the CORE, shikimate binds in the substrate-binding domain, and once ATP or shikimate binds, the LID domain closes over the active site.<ref name="UniProt">UniProt http://www.uniprot.org/uniprot/P0A4Z2</ref>

The CORE domain spans from residues 9-17 (phosphate binding loop), 148-155 (AB-loop), and 101-110 (segment which includes alpha6 from 104-110). The SB domain is comprised of residues 32-93 and consists of a sub-domain from residues 32-61 which relates to the NMP-binding domain in NMP kinases. Finally, the LID domain consists of residues 112-124. A global motion leads to LID flapping over the active site upon binding the first substrate (whether shikimate or nucleotide). This causes a change from the open to closed conformation. <ref name="miscinfo">Mechanism of Phosphoryl Transfer Catalyzed by Shikimate Kinase from Mycobacterium tuberculosis. Journal of Molecular Biology http://www.sciencedirect.com.prox.lib.ncsu.edu/science/article/pii/S0022283606011685#</ref>

The CORE domain spans from residues 9-17 (phosphate binding loop), 148-155 (AB-loop), and 101-110 (segment which includes alpha6 from 104-110). The SB domain is comprised of residues 32-93 and consists of a sub-domain from residues 32-61 which relates to the NMP-binding domain in NMP kinases. Finally, the LID domain consists of residues 112-124. A global motion leads to LID flapping over the active site upon binding the first substrate (whether shikimate or nucleotide). This causes a change from the open to closed conformation. <ref name="miscinfo">Marcus D. Hartmann et al. Mechanism of Phosphoryl Transfer Catalyzed by Shikimate Kinase from Mycobacterium tuberculosis. Journal of Molecular Biology http://www.sciencedirect.com.prox.lib.ncsu.edu/science/article/pii/S0022283606011685#</ref>

Understanding the active residues involved in ligand binding is essential to design inhibitors. The P-loop of SK is often targeted by inhibitors as the ATP-binding site catalyzes the reaction (the transfer of a phosphate group). SK binds shikimate at R57, R116, and R132. Shikimate binding occurs with helices α2, α3, and α4 (N-terminal region).<ref name="3dstructure" /> The hydroxyl groups present on shikimate form contact with M10, D33, G79-81, E114, V44, F48, and R116. The most critical residues for SK catalysis include D33, F48, R57, R116, and R132. It is believed that R57, R132, and F48 form a favorable environment to interact with shikimate, thus triggering a series of conformational changes. Catalyzing the phosphorylation of the 3-hydroxyl group of shikimate is initiated as R116 contacts shikimate during a conformational change.<ref name="compinhibitor"> Structures of Helicobacter pylori Shikimate Kinase Reveal a Selective Inhibitor-Induced-Fit Mechanism. PLoS One.

Understanding the active residues involved in ligand binding is essential to design inhibitors. The P-loop of SK is often targeted by inhibitors as the ATP-binding site catalyzes the reaction (the transfer of a phosphate group). SK binds shikimate at R57, R116, and R132. Shikimate binding occurs with helices α2, α3, and α4 (N-terminal region).<ref name="3dstructure" /> The hydroxyl groups present on shikimate form contact with M10, D33, G79-81, E114, V44, F48, and R116. The most critical residues for SK catalysis include D33, F48, R57, R116, and R132. It is believed that R57, R132, and F48 form a favorable environment to interact with shikimate, thus triggering a series of conformational changes. Catalyzing the phosphorylation of the 3-hydroxyl group of shikimate is initiated as R116 contacts shikimate during a conformational change.<ref name="compinhibitor"> Wen-Chi Cheng et al. Structures of Helicobacter pylori Shikimate Kinase Reveal a Selective Inhibitor-Induced-Fit Mechanism. PLoS One.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306394/#!po=19.5652</ref>

Line 111:

Protein folding from a random coil to native state with correct 3D structure is essential for proper protein function. The primary sequence of amino acids determines the folded structure. The topology of this enzyme is that of an α/β protein. An α/β/α fold exists consisting of a 5-stranded central parallel β-sheet (strand order 23145) and is flanked by 8 surrounding α-helices (α1 and α8 are to one side while α4, α5, and α7 are on the other side).<ref name="miscinfo" /><ref name="3dstructure" />

Shikimate kinase is a convenient protein to use in protein folding studies. This is because it is one of the smallest kinases and is a monomeric enzyme. It has been shown that the proposed refolding model includes a rapid hydrophobic collapse and then a slower secondary structure formation.<ref name="fold">The refolding of type II shikimate kinase from Erwinia chrysanthemi

Shikimate kinase is a convenient protein to use in protein folding studies. This is because it is one of the smallest kinases and is a monomeric enzyme. It has been shown that the proposed refolding model includes a rapid hydrophobic collapse and then a slower secondary structure formation.<ref name="fold">Eleonora Cerasoli et al. The refolding of type II shikimate kinase from Erwinia chrysanthemi

after denaturation in urea. European Journal of Biochemistry http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1033.2002.ejb.02862.x/pdf</ref>

Line 155:

<scene name='56/564039/Skm3po4ligand/1'>~~shikimate~~ 3-phosphate</scene>

<scene name='56/564039/Skm3po4ligand/1'>Shikimate 3-phosphate</scene>

Line 172:

Magnesium ions bind in the active site and hold influence over the position of shikimate hydroxy groups. This ion has a role in the transfer of the γ-phosphate of ATP to the 3-hydroxy group on shikimate. Chloride ions increase enzyme affinity for ADP and ATP and help to bind the nucleotide substrate in correct orientation.<ref name="mgcl"> Effects of the magnesium and chloride ions and shikimate on the structure of shikimate kinase from Mycobacterium tuberculosis Structural Biology and Crystallization Communications

Magnesium ions bind in the active site and hold influence over the position of shikimate hydroxy groups. This ion has a role in the transfer of the γ-phosphate of ATP to the 3-hydroxy group on shikimate. Chloride ions increase enzyme affinity for ADP and ATP and help to bind the nucleotide substrate in correct orientation.<ref name="mgcl"> Marcio Vinicius Bertacine Dias et al. Effects of the magnesium and chloride ions and shikimate on the structure of shikimate kinase from Mycobacterium tuberculosis Structural Biology and Crystallization Communications

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2330112/</ref> Sulfate is replaced with MgATP in the ternary complex of SK to allow for the reaction to occur.<ref name="sulfate"> Mechanism of Phosphoryl Transfer Catalyzed by Shikimate Kinase from Mycobacterium tuberculosis. Journal of Molecular Biology. http://www.sciencedirect.com/science/article/pii/S0022283606011685 </ref> Sulfate interacts with Arg117 and distorts the nucleotide binding site<ref name="miscinfo" /> Sulfate binds and distorts the nucleotide binding site, leading to a large decrease of motion.<ref name="miscinfo" />

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2330112/</ref> Sulfate is replaced with MgATP in the ternary complex of SK to allow for the reaction to occur.<ref name="sulfate"> Marcus D. Hartmann et all. Mechanism of Phosphoryl Transfer Catalyzed by Shikimate Kinase from Mycobacterium tuberculosis. Journal of Molecular Biology. http://www.sciencedirect.com/science/article/pii/S0022283606011685 </ref> Sulfate interacts with Arg117 and distorts the nucleotide binding site<ref name="miscinfo" /> Sulfate binds and distorts the nucleotide binding site, leading to a large decrease of motion.<ref name="miscinfo" />

===Inhibitors===

Line 180:

==Implications or Possible Applications==

Enzymes present in the shikimate pathway are important in microorganism survival yet absent in mammals. This means the enzymes of the SKM pathway are good candidates for antimicrobial agents, herbicides, inhibitors, and anti-parasitic drug target.

<ref name="miscinfo" /><ref name="inhibitors"> Identification of new potential Mycobacterium tuberculosis shikimate kinase inhibitors through molecular docking simulations. Journal of Molecular Modeling http://link.springer.com.prox.lib.ncsu.edu/article/10.1007%2Fs00894-011-1113-5

<ref name="miscinfo" /><ref name="inhibitors"> Carolina Pasa Vianna et al. Identification of new potential Mycobacterium tuberculosis shikimate kinase inhibitors through molecular docking simulations. Journal of Molecular Modeling http://link.springer.com.prox.lib.ncsu.edu/article/10.1007%2Fs00894-011-1113-5

</ref>

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@@ Line 92: / Line 92: @@
 ===Isoforms===
-In '' Escherichia coli'' the shikimate kinase reaction is catalyzed by two different isoforms: SKI and SKII (Km= 20 μM Km= 200 μM, respectively) with 30% sequence identify. This is very unusual for two isoforms to occur in the middle of a biosynthetic pathway. Therefore, what may be occurring is that shikimate is at a branch point for two pathways. It is believed that SKII is the isoform participating in chorismate biosynthesis, while the role of the SKI isoform is unclear.<ref name="3dstructure">The three-dimensional structure of shikimate kinase. Journal of Molecular Biology http://www.sciencedirect.com.prox.lib.ncsu.edu/science/article/pii/S0022283698917557</ref>
+In '' Escherichia coli'' the shikimate kinase reaction is catalyzed by two different isoforms: SKI and SKII (Km= 20 μM Km= 200 μM, respectively) with 30% sequence identify. This is very unusual for two isoforms to occur in the middle of a biosynthetic pathway. Therefore, what may be occurring is that shikimate is at a branch point for two pathways. It is believed that SKII is the isoform participating in chorismate biosynthesis, while the role of the SKI isoform is unclear.<ref name="3dstructure">Tino Krell et al. The three-dimensional structure of shikimate kinase. Journal of Molecular Biology http://www.sciencedirect.com.prox.lib.ncsu.edu/science/article/pii/S0022283698917557</ref>
 ===Oligomeric State===
@@ Line 98: / Line 98: @@
 <ref name="3dstructure" />
-Through experiments involving size exclusion liquid chromatography and gel filtration chromatography, the oligomeric state of homogeneous MtSK was found. The molecular mass was found to be 20.7 kDa and when compared to the suggested value of 18.5 kDa, it was found that MtSK is a monomer in solution.<ref>The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061918</ref>
+Through experiments involving size exclusion liquid chromatography and gel filtration chromatography, the oligomeric state of homogeneous MtSK was found. The molecular mass was found to be 20.7 kDa and when compared to the suggested value of 18.5 kDa, it was found that MtSK is a monomer in solution.<ref>Leonardo Astolfi Rosado et al. The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061918</ref>
 ===Domains and Active Residues===
 There are three domains present in Shikimate kinase: the CORE domain, and substrate-binding (SB) domain, and LID domain. Each domain plays a role in binding and is related to the active residues. The binding site for nucleotides is in the CORE, shikimate binds in the substrate-binding domain, and once ATP or shikimate binds, the LID domain closes over the active site.<ref name="UniProt">UniProt http://www.uniprot.org/uniprot/P0A4Z2</ref>
-The CORE domain spans from residues 9-17 (phosphate binding loop), 148-155 (AB-loop), and 101-110 (segment which includes alpha6 from 104-110). The SB domain is comprised of residues 32-93 and consists of a sub-domain from residues 32-61 which relates to the NMP-binding domain in NMP kinases. Finally, the LID domain consists of residues 112-124. A global motion leads to LID flapping over the active site upon binding the first substrate (whether shikimate or nucleotide). This causes a change from the open to closed conformation. <ref name="miscinfo">Mechanism of Phosphoryl Transfer Catalyzed by Shikimate Kinase from Mycobacterium tuberculosis. Journal of Molecular Biology http://www.sciencedirect.com.prox.lib.ncsu.edu/science/article/pii/S0022283606011685#</ref>
+The CORE domain spans from residues 9-17 (phosphate binding loop), 148-155 (AB-loop), and 101-110 (segment which includes alpha6 from 104-110). The SB domain is comprised of residues 32-93 and consists of a sub-domain from residues 32-61 which relates to the NMP-binding domain in NMP kinases. Finally, the LID domain consists of residues 112-124. A global motion leads to LID flapping over the active site upon binding the first substrate (whether shikimate or nucleotide). This causes a change from the open to closed conformation. <ref name="miscinfo">Marcus D. Hartmann et al. Mechanism of Phosphoryl Transfer Catalyzed by Shikimate Kinase from Mycobacterium tuberculosis. Journal of Molecular Biology http://www.sciencedirect.com.prox.lib.ncsu.edu/science/article/pii/S0022283606011685#</ref>
-Understanding the active residues involved in ligand binding is essential to design inhibitors. The P-loop of SK is often targeted by inhibitors as the ATP-binding site catalyzes the reaction (the transfer of a phosphate group). SK binds shikimate at R57, R116, and R132. Shikimate binding occurs with helices α2, α3, and α4 (N-terminal region).<ref name="3dstructure" /> The hydroxyl groups present on shikimate form contact with M10, D33, G79-81, E114, V44, F48, and R116.  The most critical residues for SK catalysis include D33, F48, R57, R116, and R132. It is believed that R57, R132, and F48 form a favorable environment to interact with shikimate, thus triggering a series of  conformational changes. Catalyzing the phosphorylation of the 3-hydroxyl group of shikimate is initiated as R116 contacts shikimate during a conformational change.<ref name="compinhibitor"> Structures of Helicobacter pylori Shikimate Kinase Reveal a Selective Inhibitor-Induced-Fit Mechanism. PLoS One.
+Understanding the active residues involved in ligand binding is essential to design inhibitors. The P-loop of SK is often targeted by inhibitors as the ATP-binding site catalyzes the reaction (the transfer of a phosphate group). SK binds shikimate at R57, R116, and R132. Shikimate binding occurs with helices α2, α3, and α4 (N-terminal region).<ref name="3dstructure" /> The hydroxyl groups present on shikimate form contact with M10, D33, G79-81, E114, V44, F48, and R116.  The most critical residues for SK catalysis include D33, F48, R57, R116, and R132. It is believed that R57, R132, and F48 form a favorable environment to interact with shikimate, thus triggering a series of  conformational changes. Catalyzing the phosphorylation of the 3-hydroxyl group of shikimate is initiated as R116 contacts shikimate during a conformational change.<ref name="compinhibitor"> Wen-Chi Cheng et al. Structures of Helicobacter pylori Shikimate Kinase Reveal a Selective Inhibitor-Induced-Fit Mechanism. PLoS One.
 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306394/#!po=19.5652</ref>
@@ Line 111: / Line 111: @@
 Protein folding from a random coil to native state with correct 3D structure is essential for proper protein function. The primary sequence of amino acids determines the folded structure. The topology of this enzyme is that of an α/β protein. An α/β/α fold exists consisting of a 5-stranded central parallel β-sheet (strand order 23145) and is flanked by 8 surrounding α-helices (α1 and α8 are to one side while α4, α5, and α7 are on the other side).<ref name="miscinfo" /><ref name="3dstructure" />
-Shikimate kinase is a convenient protein to use in protein folding studies. This is because it is one of the smallest kinases and is a monomeric enzyme. It has been shown that the proposed refolding model includes a rapid hydrophobic collapse and then a slower secondary structure formation.<ref name="fold">The refolding of type II shikimate kinase from Erwinia chrysanthemi
+Shikimate kinase is a convenient protein to use in protein folding studies. This is because it is one of the smallest kinases and is a monomeric enzyme. It has been shown that the proposed refolding model includes a rapid hydrophobic collapse and then a slower secondary structure formation.<ref name="fold">Eleonora Cerasoli et al. The refolding of type II shikimate kinase from Erwinia chrysanthemi
 after denaturation in urea. European Journal of Biochemistry http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1033.2002.ejb.02862.x/pdf</ref>
@@ Line 155: / Line 155: @@
 <scene name='56/564039/Skmligand/1'>SKM</scene>
-<scene name='56/564039/Skm3po4ligand/1'>shikimate 3-phosphate</scene>
+<scene name='56/564039/Skm3po4ligand/1'>Shikimate 3-phosphate</scene>
 <scene name='56/564039/Adpligand/1'>ADP</scene>
@@ Line 172: / Line 172: @@
-Magnesium ions bind in the active site and hold influence over the position of shikimate hydroxy groups. This ion has a role in the transfer of the γ-phosphate of ATP to the 3-hydroxy group on shikimate. Chloride ions increase enzyme affinity for ADP and ATP and help to bind the nucleotide substrate in correct orientation.<ref name="mgcl"> Effects of the magnesium and chloride ions and shikimate on the structure of shikimate kinase from Mycobacterium tuberculosis Structural Biology and Crystallization Communications
+Magnesium ions bind in the active site and hold influence over the position of shikimate hydroxy groups. This ion has a role in the transfer of the γ-phosphate of ATP to the 3-hydroxy group on shikimate. Chloride ions increase enzyme affinity for ADP and ATP and help to bind the nucleotide substrate in correct orientation.<ref name="mgcl"> Marcio Vinicius Bertacine Dias et al. Effects of the magnesium and chloride ions and shikimate on the structure of shikimate kinase from Mycobacterium tuberculosis Structural Biology and Crystallization Communications
-http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2330112/</ref> Sulfate is replaced with MgATP in the ternary complex of SK to allow for the reaction to occur.<ref name="sulfate"> Mechanism of Phosphoryl Transfer Catalyzed by Shikimate Kinase from Mycobacterium tuberculosis. Journal of Molecular Biology. http://www.sciencedirect.com/science/article/pii/S0022283606011685 </ref> Sulfate interacts with Arg117 and distorts the nucleotide binding site<ref name="miscinfo" /> Sulfate binds and distorts the nucleotide binding site, leading to a large decrease of motion.<ref name="miscinfo" />
+http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2330112/</ref> Sulfate is replaced with MgATP in the ternary complex of SK to allow for the reaction to occur.<ref name="sulfate"> Marcus D. Hartmann et all. Mechanism of Phosphoryl Transfer Catalyzed by Shikimate Kinase from Mycobacterium tuberculosis. Journal of Molecular Biology. http://www.sciencedirect.com/science/article/pii/S0022283606011685 </ref> Sulfate interacts with Arg117 and distorts the nucleotide binding site<ref name="miscinfo" /> Sulfate binds and distorts the nucleotide binding site, leading to a large decrease of motion.<ref name="miscinfo" />
 ===Inhibitors===
@@ Line 180: / Line 180: @@
 ==Implications or Possible Applications==
 Enzymes present in the shikimate pathway are important in microorganism survival yet absent in mammals. This means the enzymes of the SKM pathway are good candidates for antimicrobial agents, herbicides, inhibitors, and anti-parasitic drug target.
-<ref name="miscinfo" /><ref name="inhibitors"> Identification of new potential Mycobacterium tuberculosis shikimate kinase inhibitors through molecular docking simulations. Journal of Molecular Modeling http://link.springer.com.prox.lib.ncsu.edu/article/10.1007%2Fs00894-011-1113-5
+<ref name="miscinfo" /><ref name="inhibitors"> Carolina Pasa Vianna et al. Identification of new potential Mycobacterium tuberculosis shikimate kinase inhibitors through molecular docking simulations. Journal of Molecular Modeling http://link.springer.com.prox.lib.ncsu.edu/article/10.1007%2Fs00894-011-1113-5
 </ref>