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==''Mycobacterium tuberculosis'' salicylate synthase (Mbt1)==

==Introduction==

Salicylate synthase from ''Mycobacterim tuberculosis'' (MtbI) is a highly promiscuous enzyme that has four distinct activities ''in vivo'': isochorismate synthase (IS), isochorismate pyruvate lyase (IPL), salicylate synthase (SS) and chromate mutate (CM). MtbI belongs to the chorismate-utilising enzyme family, which consists of structural homologues (<scene name='69/694235/Irp9/2'>Ipr9</scene>, <scene name='69/694235/Menf/2'>MenF</scene>, <scene name='69/694235/Entc/2'>EntC</scene>, and <scene name='69/694235/Mbti/2'>MbtI</scene>) that isomerize chromate to isochorismate. These enzymes are present in bacteria, fungi, plants and apicomplexan parasites and catalyze the initial reactions of menaquinone, siderophore, and tryptophan biosynthesis. 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. IS, IPL, and SS activity are also modulated by the pH of the medium. Isochorismate is the primary product at pH values below 7.5 and salicylate is the primary product formed at pH 8.

[[Image:Pathways.png|500 px|center|thumb|Figure 1: This is the pathway of reactions catilized by wild-type MbtI <ref>PMID:22307014</ref>.]]

==Structure==

[[Image:Active_site_cleft.png|300 px|left|thumb|Figure 2: This shows a single sub unit of MbtI, with the active site cleft located at the lower left hand side of the image.]]

The crystal asymmetric unit was found to contain <scene name='69/694235/3log/1'> four MbtI molecules</scene>, however crystal packing and size exclusion chromatography data suggest a monomeric enzyme. There are no significant structural changes between the four monomers excepts from the localized differences in the active site (3). 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 (3). The core of the protein is formed by 21 <scene name='69/694235/Beta_sheets/3'>beta-strands</scene> folded into a twisted beta-sandwich. The protein's core is then surrounded by 10 alpha helices(3).

== Disease ==

''Mycobacterium tuberculosis'' is the causative agent of Tuberculosis (TB), an infectious disease that affects one-third of the worlds population. Two TB-related conditions exist: latent TB infection and active TB disease. 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.TB disease can also be treated through various antibiotic regimens. There are 10 drugs currently approved by the FDA for treating TB disease. The first-line anti-TB agents are the antibiotics isoniazid, rifampin, ethambutol, and pyrazinamide <ref>Tuberculosis (TB). Ed. Sam Posner. Centers for Disease Control and Prevention, n.d. Web. 9 Apr. 2015.</ref>. Although various treatments for TB infection and TB disease exist, the emergence of multi-drug and extensively-drug resistant strains of ''M. tuberculosis'' has increased the need for anti-tubercular agents with novel modes of action.

Iron is essential for mycobacterial growth and pathogenesis, therefore the pathways for iron acquisition are potential targets for antibacterial therapies.''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'''.'''Mycobactin is a siderophore synthesized by the proteins encoded by the ''mbt'' and ''mbt2'' gene cluters <ref name="gamma chi"/>. MbtI is the first enzyme in the mycobactin biosynthesis pathway and is a potential target for inhibition. The salicylate synthase activity of MbtI produces salicylate and pyruvate from chorismate through an isochorismate intermediate. 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>De Voss, James J., Kerry Rutter, Benjamin G. Schroeder, Hua Su, and YaQi Zhu. The salicylate-derived mycobacterium siderophore of Mycobacterium tuberculosis are essential for growth in macrophages. "Proceedings of the National Science Academy" 97.3 (2000): 1252-57. Web. 5 Apr. 2015.</ref> .

[[Image:Screen Shot 2015-04-10 at 1.27.15 PM.png‎|500 px|center|thumb|Figure 3: Reaction catalyzed by MbtI in the mycobactin biosynthesis pathway<ref name= "manos-turvey"/>.]]

== Structural highlights ==

MtI structure has a mobile element (residues 323 to 227) that can adopt a <scene name='69/694235/Irp9_closed_state/2'>closed</scene> or <scene name='69/694235/2g5f_with_open_loop/1'>open conformation</scene> depending on whether or not ligands are bound to the active site. The closed conformation partially obstructs the active site. <ref name= "gamma chi"/>.

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. 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. Positioning of the active site residues of MbtI with the inhibitor AMT is highly similar to the closed form of MbtI <ref name= "gamma chi"/>.

==Molecular Mechanism==

'''Magnesium cation effect'''

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. Histidine residue (His334) was proposed to act as a base, abstracting the C2 proton of isochorismate through a second order elimination mechanism. 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. 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.

'''Isochorismate synthast (IS)'''

'''chorismate mutase (CM)'''

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.

==Inhibition Studies==

MbtI Inhibition studies aid in the future design of anti-tubercular agents and broad-spectrum antibiotics. Mimics of the enzyme-bound intermediate of MbtI, isochorismate, prove to be significantly more potent inhibitors than the substrate, chorismate mimics <ref name= "manos-turvey"/>. Specifically, <scene name='69/694235/3rv6_with_vae1/1'>2-hydroxybenzoate-based inhibitors</scene> that contain extended hydrophobic enol ether side chains at C3 in place of the enol-pyruvate side chain found in chorismate and isochorismate.