Sandbox Reserved 1068: Difference between revisions
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==Introduction== | ==Introduction== | ||
<scene name='69/694235/3log/12'>Salicylate synthase</scene> from [http://en.wikipedia.org/wiki/''Mycobacterium_tuberculosis''] (MtbI) is a highly promiscuous enzyme that has four distinct activities ''in vivo'': [http://en.wikipedia.org/wiki/Isochorismate_synthase isochorismate synthase] (IS), [http://www.proteopedia.org/wiki/index.php/Isochorismate_pyruvate_lyase isochorismate pyruvate lyase] (IPL), [http://www.rcsb.org/pdb/results/results.do?outformat=&qrid=8A8773E9&tabtoshow=Current salicylate synthase] (SS) and [http://en.wikipedia.org/wiki/Chorismate_mutase chorismate mutate] (CM) | <scene name='69/694235/3log/12'>Salicylate synthase</scene> from [http://en.wikipedia.org/wiki/''Mycobacterium_tuberculosis''] (MtbI) is a highly promiscuous enzyme that has four distinct activities ''in vivo'': [http://en.wikipedia.org/wiki/Isochorismate_synthase isochorismate synthase] (IS), [http://www.proteopedia.org/wiki/index.php/Isochorismate_pyruvate_lyase isochorismate pyruvate lyase] (IPL), [http://www.rcsb.org/pdb/results/results.do?outformat=&qrid=8A8773E9&tabtoshow=Current salicylate synthase] (SS) and [http://en.wikipedia.org/wiki/Chorismate_mutase chorismate mutate] (CM)<ref name="8a">PMID:22307014</ref>. MtbI belongs to the chorismate-utilizing enzyme family, which consists of structural homologues (<scene name='69/694235/Irp9/5'>Ipr9</scene>, <scene name='69/694235/Menf/3'>MenF</scene>, <scene name='69/694235/Entc/3'>EntC</scene>, and <scene name='69/694235/Mbti/3'>MbtI</scene>) that isomerize chromate to isochorismate and share a fold of two α/β subdomains, each comprising of a antiparallel β-sheet with helices packed against it(Ferrer 2012, Lamb 2011). These enzymes are present in bacteria, fungi, plants and apicomplexan parasites and catalyze the initial reactions of menaquinone, siderophore, and tryptophan biosynthesis(ferrer 2012, Lamb 2011, Voss 1999). The IS, IPL, and SS activity of MbtI require the presence of a magnesium ion within the active site, while CM activity is only observed in absence of the magnesium cation(ferrer 2012). IS, IPL, and SS activity are also modulated by the pH of the medium(ferrer 2012). Isochorismate is the primary product at pH values below 7.5 and salicylate is the primary product formed at pH 8(ferrer 2012, Zwahlen 2006). | ||
The salicylate synthase activity of MbtI catalyzes the first committed step in the synthesis of the iron chelating [http://en.wikipedia.org/wiki/Siderophore siderophore], mycobactin T, in ''Mycobacterium tuberculosis'' (Figure 1)<ref name= "5a">PMID:22607697</ref>. Mycobactin T is synthesized by the proteins encoded by the ''mbt'' and ''mbt2'' gene clusters <ref name="5a"/>. The gene Rv2386c is essential for the in vitro growth of ''M. tuberculosis'' and codes the enzyme MbtI (turvey, 2010). This complex secondary metabolite is essential for both virulence and survival of ''M. tuberculosis''(ferrer 2012, Voss 1999, Harrison 2006). Therefore, inhibitors of salicylate synthase may serve as potential TB therapies with a novel mode of action <ref name= "1a"> PMID:20512795</ref> <ref name= "2a">PMID:23108268</ref> <ref name= "7a">Voss, James J., Kerry Rutter, Benjamin G. Schroedor, Hua Su, and YaQi Zhu. "The salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages." Proceedings of the National Academy of Sciences 97.3 (2000): 1252-57. Web. 14 Mar. 2015.</ref> <ref name= "5a"/> <ref name= "4a">DOI:10.1021/bi2009739</ref> <ref name= "9a">PMID:14982443</ref> | The salicylate synthase activity of MbtI catalyzes the first committed step in the synthesis of the iron chelating [http://en.wikipedia.org/wiki/Siderophore siderophore], mycobactin T, in ''Mycobacterium tuberculosis'' (Figure 1)<ref name= "5a">PMID:22607697</ref>. Mycobactin T is synthesized by the proteins encoded by the ''mbt'' and ''mbt2'' gene clusters <ref name="5a"/>. The gene Rv2386c is essential for the in vitro growth of ''M. tuberculosis'' and codes the enzyme MbtI (turvey, 2010). This complex secondary metabolite is essential for both virulence and survival of ''M. tuberculosis''(ferrer 2012, Voss 1999, Harrison 2006). Therefore, inhibitors of salicylate synthase may serve as potential TB therapies with a novel mode of action <ref name= "1a"> PMID:20512795</ref> <ref name= "2a">PMID:23108268</ref> <ref name= "7a">Voss, James J., Kerry Rutter, Benjamin G. Schroedor, Hua Su, and YaQi Zhu. "The salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages." Proceedings of the National Academy of Sciences 97.3 (2000): 1252-57. Web. 14 Mar. 2015.</ref> <ref name= "5a"/> <ref name= "4a">DOI:10.1021/bi2009739</ref> <ref name= "9a">PMID:14982443</ref> | ||
[[Image:Pathways.png|500 px|center|thumb|'''Figure 1:''' Pathways catalyzed by wild-type MbtI<ref>PMID:22307014</ref>.]] | [[Image:Pathways.png|500 px|center|thumb|'''Figure 1:''' Pathways catalyzed by wild-type MbtI<ref name= "8a">PMID:22307014</ref>.]] | ||
==Structure== | ==Structure== | ||
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[[Image:Capture.PNG|300 px|left|thumb|'''Figure 2''': Monomeric ribbon diagram of MbtI with active site cleft highlighted with a white circle. Generated from [[3log]] (3a)]] | [[Image:Capture.PNG|300 px|left|thumb|'''Figure 2''': Monomeric ribbon diagram of MbtI with active site cleft highlighted with a white circle. Generated from [[3log]] (3a)]] | ||
The crystal asymmetric unit was found to contain <scene name='69/694235/3log/11'> four MbtI molecules</scene>, however crystal packing and size exclusion chromatography data suggest a monomeric enzyme <ref name= "3a">PMID | The crystal asymmetric unit was found to contain <scene name='69/694235/3log/11'> four MbtI molecules</scene>, however crystal packing and size exclusion chromatography data suggest a monomeric enzyme <ref name= "3a">PMID 16923875</ref>. There are no significant structural changes between the four monomers excepts from the localized differences in the active site <ref name= "3a"/>. The overall molecular structure consist of a polypeptide of 450 residues that forms one large single domain with a similar fold to other chromate-utilizing enzymes <ref name="3a"/>. The core of the protein is formed by <scene name='69/694234/Beta_sheets/1'>21 Beta sheets </scene>folded into a twisted beta-sandwich. The protein's core is then surrounded by <scene name='69/694235/Beta_sheets/4'>10 alpha helices</scene><ref name="3a"/>. The active site was identified by comparison to the product bound forms of Irp9 and TrpE and is situated in a cleft that is about 12Å in length, 10Å deep, and 7Å wide. One side of the groove is formed by β21, C-terminal helix, and α11. The other side of the groove is formed by β16-17 loop, helix α7, and β15-α6 loop. The β19-20 and β12-13 loops make up the bottom of the active side cleft (Figure 2)(Harrison 2006) | ||
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[[Image:IS2.png|500 px|center|thumb|Figure 3: Isochorismate synthase activity | [[Image:IS2.png|500 px|center|thumb|Figure 3: Isochorismate synthase activity <ref name="8a"/>.]] | ||
'''chorismate mutase (CM)''' | '''chorismate mutase (CM)''' | ||
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A magnesium ion in the active site orients the C1 carboxyl group of chorismate. A lysine residue then serves as a general base for the activation of a water molecule to attack at C2. The catalytic mechanism for conversion of isochorismate to salicylate by MbtI is a sigmatropic, pericyclic mechanism that is pH-dependent. Chromate mutase activity is only observed in the absence of magnesium ion in the active site while salicylate synthase activity is depended on magnesium ion. The active site of MbtI is altered by the removal of the magnesium cofactor causing chromate mutase activity. MbtI has differing binding modes for chromate that leads to different substrate conformations/transition states and resulting in different products. | A magnesium ion in the active site orients the C1 carboxyl group of chorismate. A lysine residue then serves as a general base for the activation of a water molecule to attack at C2. The catalytic mechanism for conversion of isochorismate to salicylate by MbtI is a sigmatropic, pericyclic mechanism that is pH-dependent. Chromate mutase activity is only observed in the absence of magnesium ion in the active site while salicylate synthase activity is depended on magnesium ion. The active site of MbtI is altered by the removal of the magnesium cofactor causing chromate mutase activity. MbtI has differing binding modes for chromate that leads to different substrate conformations/transition states and resulting in different products. | ||
[[Image:CM2.png|450 px|center|thumb|Figure 3: Isochorismate synthase activity | [[Image:CM2.png|450 px|center|thumb|Figure 3: Isochorismate synthase activity <ref name="8a"/>.]] | ||
