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| ==''Mycobacterium tuberculosis'' salicylate synthase (Mbt1)== | | {{Sandbox_Reserved_Butler_CH462_Sp2015_#}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --> |
| <StructureSection load='3LOG' size='450' side='right' caption='([[3LOG]]) is a 4 chain structure of MbtI with sequence from [http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis Mycobacterium tuberculosis]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3LOG OCA].'> | | ==Your Protein Name here== |
| | <StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''> |
| | This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs. |
| | You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue. |
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| ==Introduction== | | == Biological Function == |
| <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 <ref name="8a">PMID:22307014</ref> <ref name="1a">PMID:20512795</ref>. These enzymes are present in bacteria, fungi, plants and apicomplexan parasites and catalyze the initial reactions of menaquinone, siderophore, and tryptophan biosynthesis <ref name="4a">PMID:21823653</ref> <ref name="1a">PMID:20512795</ref> <ref name="7a">PMID:10655517</ref>. 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 <ref name="8a">PMID:22307014</ref>. IS, IPL, and SS activity are also modulated by the pH of the medium <ref name="8a"/>. Isochorismate is the primary product at pH values below 7.5 and salicylate is the primary product formed at pH 8 <ref name="8a"/> <ref name="6a">PMID:17240979</ref>.
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| 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 <ref name="1a">PMID:20512795</ref>. This complex secondary metabolite is essential for both virulence and survival of ''M. tuberculosis''. <ref name="8a"/> <ref name="7a">PMID:10655517</ref><ref name="3a">PMID:16923875</ref> 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">PMID:10655517</ref> <ref name= "5a"/> <ref name= "4a">DOI:10.1021/bi2009739</ref> <ref name= "9a">PMID:14982443</ref>
| | == Structural Overview == |
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| [[Image:Pathways.png|500 px|center|thumb|'''Figure 1:''' Pathways catalyzed by wild-type MbtI<ref name= "8a">PMID:22307014</ref>.]]
| | == Mechanism of Action == |
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| ==Structure== | | == Zinc Ligand(s) == |
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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)]]
| | == Other Ligands == |
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| 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 <scene name='69/694235/Alpha_helics/2'>one large single domain</scene> 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 β sheets </scene>folded into a twisted beta-sandwich. The protein's core is then surrounded by <scene name='69/694235/Beta_sheets/4'>10 α 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 <ref name="3a"/>. One side of the groove is formed by β21, C-terminal helix, and α11 while the other side of the groove is formed by β16-17 loop, helix α7, and β15-α6 loop (Figure 2)<ref name="3a"/>. The β19-20 and β12-13 loops make up the bottom of the active side cleft (Figure 2) <ref name="3a">PMID:16923875</ref>.
| | This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes. |
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| For further structural and sequence information see [http://www.uniprot.org/uniprot/P9WFX1].
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| == Structural highlights ==
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| [[Image:Screen Shot 2015-04-26 at 6.31.19 PM.png|200 px|left|thumb|'''Figure 3:''' Overlay of chain A in [[3ST6]] (green) and [[3RV6]] (teal). [[3ST6]] contains the inhibitor AMT and represents the closed form of MbtI while [[3RV6]] contains an enolpyruvyl modified inhibitor (phenyl-AMT) and shows the movement of the backbone away from the closed form to accommodate the modified inhibitor. <ref name= "8a"/> <ref name="5a">PMID:22607697</ref>.]]
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| [[Image:Screen Shot 2015-04-26 at 6.29.56 PM.png|200 px|left|thumb|'''Figure 4:''' Overlay of chain A in [[3ST6]] (green) and [[3RV6]] (teal) with inhibitors bound and magnesium. <ref name= "8a"/> <ref name="5a"/>.]]
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| [[Image:Screen Shot 2015-04-26 at 6.30.55 PM.png|200 px|left|thumb|'''Figure 5:''' Flexibility of peptide backbone in regions proximal to the active site see through the overlay of chain A residues 269-293 and 324-336 in [[3ST6]] (green) and [[3RV6]] (teal)<ref name= "8a"/> <ref name="5a"/>.]]
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| MbtI structure has a mobile element (residues 268-293 and 324-336) that can adopt a closed or open conformation depending on whether or not ligands are bound to the active site (Figure 3,4,5)<ref name="3a"/>. The closed conformation partially obstructs the active site <ref name= "5a"/>. Inhibition studies have also shown a switch in binding mode at the MbtI active site for inhibitors carrying a substituted enolpyruvyl group compared to the chorismate substrate <ref name="5a">PMID:22607697</ref> <ref name="2a">PMID:23108268</ref> <ref name="1a">PMID:20512795</ref>. Crystal structures and fluorescent-based thermal shift assays show that substituents larger than a methyl group are accommodated in the active site of MbtI through localized flexibility in the peptide backbone.<ref name="5a"/> Positioning of the <scene name='69/694235/3st6_structure_bindingpocket/3'>active site residues</scene> of MbtI in [[3ST6]] with the inhibitor AMT bound is highly similar to the positioning of the <scene name='69/694235/3log_bindingpocket/2'>active site residues</scene> in closed form of MbtI [[3log]] with succinic acid bound <ref name= "5a"/>. The AMT inhibitor contains an unmodified enolpyruvyl side chain and resembles the structure of the natural substrate, chorismate. [[3log]] and [[3ST6]] are shown to share a similar binding mode, termed binding mode 1<ref name="5a"/>. Isochorismate inhibitors with modified enolpyruvl side chains ([[3VEH]], [[3RV9]], [[3RV8]], [[3RV7]], [[3RV6]]) utilize a novel binding mode, termed mode 2, which involves the <scene name='69/694235/3veh_structure_bindingpocket2/2'>reorientation of the isochorismate analogue within the active site</scene> <ref name="5a"/>. Movement of the peptide backbone away from the closed form of MbtI is required to accommodate the enolpyruyl modified inhibitors<ref name="5a"/>.
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| ==Molecular Mechanism==
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| '''Magnesium cation effect'''
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| [[Image:Screen Shot 2015-04-25 at 11.08.58 PM.png |300 px|left|thumb|'''Table 1:''' pKa values of active site residues of MbtI with and without Magnesium. <ref name="8a"/> 2-11.]] The presence of the [http://en.wikipedia.org/wiki/Magnesium_in_biology magnesium ion] induces changes in the structure of the active site and in the substrate, as well as causes significant pKa shifts in some of the key residues involved in the catalytic activity (Table 1).The <scene name='69/694235/3rv6_mg_shell/4'>coordination shell</scene> of the magnesium cation in the active site of MbtI in [[3rv6]] with phenyl-AMT inhibitor bound is composed of two water molecules, Glu434, Glu294, and the two oxygen atoms of the C1 carboxylate group of chorismate<ref name="5a"/>. In the presence of the magnesium ion, the positively charged Lys295 is displaced from the active site and the negatively charged Glu297 is faced toward the active site<ref name="5a"/><ref name="2a"/>. Magnesium cation also orients the C1 carboxylate group coplanar to the ring of chorismate, reducing the electron density on the C2 center and favoring nucleophilic attack<ref name= "8a">PMID:22307014</ref><ref name="5a"/><ref name="2a"/>.
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| '''Isochorismate pyruvate lyase (IPL)'''
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| Isochorismate is converted to salicylate and pyruvate through abstraction of the C2 hydrogen followed by protonation of C9 atom and the breakage of the C3-O7 bond (Figure 6) <ref name="6a">PMID:17240979</ref><ref name="8a"/>. Histidine residue (His334) was proposed to act as a base, abstracting the C2 proton of isochorismate through a second order elimination mechanism <ref name="6a">PMID:17240979</ref><ref name="8a"/>. However, recent studies have shown that this residue lies more than 13 A away from C2 atom and no other water molecules appear close enough to the C2 atom to act as a base <ref name="6a"/><ref name="8a"/>. IPL reaction has been proposed to proceed through an intramolecular pericyclic mechanisms, involving a concerted hydrogen transfer from C2 to C9 and breakage of the C3-O7 bond <ref name="6a"/><ref name="8a"/>.
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| [[Image:IPL2.png|500 px|center|thumb|'''Figure 6''': Isochorismate pyruvate activity <ref name= "8a"/>.]]
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| '''Isochorismate synthase (IS)'''
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| Currently, isochorismate is believed to be formed from chorismate through a proposed Sn2 mechanism involving nucleophilic attack of an activated water molecule to the C2 center followed by either a concerted or stepwise elimination of the C4 hydroxyl group (Figure 7)<ref name="9a"/><ref name="6a"/><ref name="8a"/>. Lys205 has been proposed to act as the catalytic base, activating a water molecule in the active site by abstracting one of its protons<ref name="6a"/><ref name="8a"/>. However, mutational analysis of Lys205 suggested that the lysine reside is not the sole determinant in the activation of a water molecule for nucleophilic attack of the C2 center<ref name="6a"/><ref name="8a"/>. Studies have shown that Lys205 is protonated at neutral pH and therefore can't act as a base to activate the water molecule, agreeing with the mutational analysis data<ref name="1a"/><ref name="2a"/>. Instead of Lys205, Glu297 residue has been proposed to act as a base in the activation of the water molecule<ref name="6a"/><ref name="8a"/>. The magnesium ion forces the negatively charged Glu297 residue to face toward the active site and the pKa of Glu297 (3.9) suggest an unprotonated state<ref name="8a"/>. Furthermore, Glu297 forms a hydrogen bond with a water molecule within the active site as well as with Lys205, which is in turn hydrogen bonded to C1 carboxylate group of chorismate and the oxygen of the nucleophilic water molecule<ref name="6a"/><ref name="8a"/>. The glutamic residue (Gly252) could protonate the C4 leaving hydroxyl group. The pKa of Gly252 (7.7) suggest that is it is the only protonated glutamate residue in the active site at pH 7 and thus able to protonate the C4 leaving group<ref name="8a"/>. The pKa of Gly252 also accounts for the accumulation of isochorismate at pH values below 7.5<ref name="8a"/>.
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| [[Image:IS2.png|500 px|center|thumb|'''Figure 7''': Isochorismate synthase activity <ref name="8a"/>.]]
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| '''Chorismate mutase (CM)'''
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| A magnesium ion in the active site orients the C1 carboxyl group of chorismate (Figure 8)<ref name="6a"/><ref name="8a"/>. A lysine residue then serves as a general base for the activation of a water molecule to attack at C2<ref name="6a"/><ref name="8a"/>. The catalytic mechanism for conversion of isochorismate to salicylate by MbtI is a sigmatropic, pericyclic mechanism that is pH-dependent<ref name="8a"/>. 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<ref name="6a"/><ref name="8a"/><ref name="2a"/>. The active site of MbtI is altered by the removal of the magnesium cofactor causing chromate mutase activity<ref name="6a"/><ref name="8a"/><ref name="2a"/>. MbtI has differing binding modes for chromate that leads to different substrate conformations/transition states and resulting in different products<ref name="1a"/><ref name="3a"/><ref name="5a"/>.
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| [[Image:CM2.png|450 px|center|thumb|'''Figure 8''': Isochorismate synthase activity <ref name="8a"/>.]]
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| '''Salicylate synthase (SS)'''
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| Chromate is converted to salicylate synthase and pyruvate by MbtI through an intermediate isochromate. The pyruvate molecule is expelled after the intermediate step and salicylate is incorporated in the biosynthesis of mycobactin T (Figure 9,10)<ref name="2a">PMID:23108268</ref>. Inhibition studies revealed two binding modes of MbtI based on the structure of the substrate <ref name="5a"/>. Mimics of isochromate inhibitors with modified enolpyruvly side chains showed the greatest inhibition capability and reoriented the substrate within the active side of the enzyme causing the backbone of the enzyme to shift away from the closed conformation (Figure 3,4,5)<ref name="5a"/>. A clear mechanism for the salicylate synthase activity of MbtI is currently unknown<ref name="5a"/>.
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| [[Image:Salicylate synthase chem draw.png|500 px|center|thumb|'''Figure 9''':salicylate synthase activity <ref name="5a"/><ref name="2a"/>]]
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| == Disease ==
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| [http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis Mycobacterium tuberculosis] is the causative agent of [http://www.cdc.gov/tb/ Tuberculosis] (TB), an infectious disease that affects one-third of the worlds population<ref name="CDC">Tuberculosis (TB). Ed. Sam Posner. Centers for Disease Control and Prevention, n.d. Web. 9 Apr. 2015.</ref>. Two TB-related conditions exist: latent TB infection and active TB disease<ref name="CDC"/>. Currently, there are four regimens that are approved for the treatment of latent TB infection through the use of the antibiotics isoniazid, rifampin, and rifapentine<ref name="CDC"/>.TB disease can also be treated through various antibiotic regimens<ref name="CDC"/>. There are 10 drugs currently approved by the FDA for treating TB disease<ref name="CDC"/>. The first-line anti-TB agents are the antibiotics isoniazid, rifampin, ethambutol, and pyrazinamide <ref name="CDC"/>. Although various treatments for TB infection and TB disease exist, the emergence of [http://www.cdc.gov/tb/publications/factsheets/drtb/mdrtb.htm multi-drug] and [http://www.cdc.gov/tb/topic/drtb/xdrtb.htm extensively-drug] resistant strains of ''M. tuberculosis'' has increased the need for anti-tubercular agents with novel modes of action <ref name="2a"/><ref name="5a"/><ref name="8a"/><ref name="CDC"/>.
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| [http://en.wikipedia.org/wiki/Iron#Biological_role Iron] is essential for mycobacterial growth and pathogenesis, therefore the pathways for iron acquisition are potential targets for antibacterial therapies<ref name="1a"/>.''M. tuberculosis'' obtains iron through two different pathways: chelating iron from the host through the siderophore mycobactin and the degradation of heme released from damaged red blood cells<ref name="1a"/>. MbtI catalyses the first committed step in the biosynthesis of the siderophore mycobactin and is a potential target for inhibition (Figure 10)<ref name="1a"/>. The salicylate synthase activity of MbtI produces salicylate and pyruvate from chorismate through an isochorismate intermediate<ref name="1a"/>. Inhibition of MbtI activity would decrease the production of salicylate and therefore the synthesis of mycobactin; leading to a decrease in iron acquisition and pathogenesis of ''M. tuberculosis''<ref name= "7a"/><ref name="1a"/><ref name="5a"/>.
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| [[Image:Screen Shot 2015-04-10 at 1.27.15 PM.png|500 px|center|thumb|'''Figure 10:''' Reaction catalyzed by MbtI in the mycobactin biosynthesis pathway. MbtI catalyses the conversion of chorismate to salicylate and pyruvate. Salicylate (red) is then involved in the biosynthesis of mycobactin T <ref name= "2a"/>.]]
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| ==Inhibition Studies==
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| MbtI Inhibition studies aid in the future design of [http://psychology.wikia.com/wiki/Antitubercular_drugs anti-tubercular agents] and [http://en.wikipedia.org/wiki/Broad-spectrum_antibiotic broad-spectrum antibiotics] with a novel mode of action. Mimics of the enzyme-bound intermediate of MbtI, <scene name='69/694235/3sr6_inhibitor/3'>isochorismate</scene>, prove to be significantly more potent inhibitors than mimics of the substrate, chorismate <ref name= "1a"/><ref name="2a"/><ref name="5a"/>. The isochorismate mimic based on a 2,3-dihydroxybenzoate scaffold showed low-micromolar inhibition constants against MbtI that were an order of magnitude more potents than the natural substrates<ref name="2a"/>. The most potent inhibitors contained hydrophobic enol ether side chains at C3 instead of the enol-pyruvyl side chains seen in chorismate and isochorismate. <ref name="1a">PMID:20512795</ref> Increased potency of <scene name='69/694235/3rv6_with_vae1/3'>inhibitors with a substituted enolpyruvyl group</scene> as seen in [[3RV6]] has been attributed to a change in the binding mode through localized flexibility of the peptide backbone<ref name="2a"/><ref name="5a"/>.
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| | </StructureSection> |
| == References == | | == References == |
| {{reflist}}
| | <references/> |
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| ==Student contributors==
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| Stephanie Raynor and Robin Gagnon
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| ==Related pdb files and proteopedia pages==
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| '''3D structures of isochorismate pyruvate lyase'''
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| [[3log]] – MtIPL/isochorismate synthase - ''Mycobacterium tuberculosis''<br />
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| [[3rv6]], [[3rv7]], [[3rv8]], [[3rv9]], [[3st6]], [[3veh]] - MtIPL/isochorismate synthase + inhibitor<br />
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| [[2h9c]] – PaIPL residues 1-99 – ''Pseudomonas aeruginosa''<br />
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| [[2h9d]] - PaIPL + pyruvate
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| [[3LOG]]
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| '''3D structure of isochorismate synthase'''
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| [[2eua]], [[3bzm]], [[3bzn]] - MenF from ''E. coli'' <br />
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| [[3os6]] - DhbC from ''Bacillus anthracis'' <br />
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| [[3gse]] - MenF from ''Yersinia pestis'' <br />
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| [[3hwo]] - EntC <br />
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| '''3D structure of salicylate synthase'''
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| [[3veh]] - MbtI with inhibitor methylAMT <br />
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| [[3st6]] - MbtI with isochorismate analogue inhibitor <br />
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| [[3rv6]] (Phenyl R-group), [[3rv7]] (Isopropyl R-group), [[3rv8]] (Cyclopropyl R-group), [[3rv9]] (Ethyl R-group) - MbtI with inhibitor <br />
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| [[2fn0]], [[2fn1]] (with products salicylate and pyruvate) - Irp9 from ''Yersinia enterocolitica'' <br />
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| [[2i6y]] - MbtI <br />
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