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{{Sandbox_Reserved_Butler_CH462_Sp2015_#}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE -->
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== Background ==
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==''Mycobacterium tuberculosis'' salicylate synthase (Mbt1)==
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<StructureSection load='3LOG' size='450' side='right' caption='Structure of MbtI ([[3LOG]])'>
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==Introduction==
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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.
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The salicylate synthase activity of MbtI catalyzes the first committed step in the synthesis of the iron chelating siderophore, mycobactin, in ''Mycobacterium tuberculosis'' (Figure 3)<ref name= "5a">PMID:22607697</ref>. This complex secondary metabolite is essential for both virulence and survival of ''M. tuberculosis''. Therefore, inhibitors of salicylate synthase may serve as potential TB therapies with a novel mode of action <ref name= "1a"> DOI: 10.1002/cmdc/201000137</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> (1,2,7,5,4,9)
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[[Image:Pathways.png|500 px|center|thumb|Figure 1: This is the pathway of reactions catilized by wild-type MbtI <ref>PMID:22307014</ref>.]]
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The antigen 85 (ag85) complex in Mycobacterium tuberculosis is composed of three intracellular membrane proteins: ag85A, B, and C. The ag85 complex is a major component of the cell wall, with each protein catalyzing the transfer of important cell wall constituents into the membrane. <ref>PMID: 10655617</ref> Ag85C is of particular interest due to its transfer of mycolic acids, which are major components in determining cell wall integrity. By targeting this mycoloyltransferase activity, inhibition of ag85C offers potential for cell wall disruption and subsequent antibiotic targeting for normally drug-resistant mycotaberia tuberculosis. <ref>PMID: 10200974</ref>
==Structure==
==Structure==
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[[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.]]
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<StructureSection load='1dqz' size='400' side='right' caption='Antigen 85C in Mycobacterium Tuberculosis' scene=''>
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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 <ref name= "3a"> DOI:10.1128/JB.0038-06</ref>. 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 <scene name='69/694235/Beta_sheets/4'>10 alpha helices</scene>(3).
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== General Structure ==
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== Disease ==
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Antigen 85C was crystallized in its dimeric form.<ref>PMID: 25028518</ref> The <scene name='69/694220/Secondary_structures/2'>secondary structure</scene> shown in the monomeric form is composed of helices with one interwoven beta sheet. Due to the serine hydrolase activity of Ag85C, the enzyme contains a ‡/? hydrolase fold with a central ?-sheet bordered by ‡?helices, and this tertiary conformation is highly conserved across enzymes that function in this capacity. <ref>PMID:10655617</ref> The substrate binding pocket of Ag85C is composed of two separate but equally important components; there is carbohydrate binding pocket for the trehalose, and there is a fatty acid binding pocket for the mycolic acid. As a result, trahalose monomycolate can effectively bind to the Ag85C binding pocket.
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''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.
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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="5a"/>. 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> .
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== Enzymatic Activity ==
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Mutagenesis studies have confirmed the Ag85C functions through a Glu-His-Ser <scene name='69/694220/Catalytic_triad/2'>catalytic triad</scene>, similar to that of chymotrypsin. By modifying each of the catalytic residues separately testing the enzyme?s relative activity, it has been shown that mutation of any one of these residues dramatically reduces activity (Figure #). The S124 alcohol?s nucleophilicity is inductively strengthened through H260 and E224, which allows S124 to hydrolyze trehalose 6, 6?-dimycolate. The formation of the functional catalytic triad relies on upon Van der Waals interaction between C209 and the peptide bond between L232 and T231. This interaction results in a kinked conformation of the ‡9 helix, which promotes that activity of the catalytic triad. As a result, Ag85C, a mycolyl transferase, can facilitate the modification of trehalose monomycolates to trehalose dimycolates, which are then transported to the bacterial cell wall.
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[[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"/>.]]
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[[Image:General_mechanism.jpg|400 px|center|General reaction catalyzed by Antigen 85C]]
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== Methods of Inhibition ==
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== Structural highlights ==
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[[Image:C209.jpeg|200 px|left|thumb|Cys209 stabilizing kinked formation of alpha-9 helix]]
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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= "5a"/>.
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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= "5a"/>.
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Due to the importance of Ag85C enzymatic activity in maintaining the integrity of the mycobacteria tuberculosis cell wall though mycolic acid modifications, the Ag85C enzyme represents a potentially effective avenue for inhibiting cell growth. The conformational sensitivity of the active site residues, H260, E228, and S124, relies entirely upon Van der Waals interaction between C209 and L232-T 231 (Figure #). The C209 facilitated interaction causes the <scene name='69/694220/Alpha_9_helix/2'>‡9 helix</scene> to acquire a kinked conformation that promotes optimal interaction distances between catalytic residues. As a result, C209 has been a specific target residue for Ag85C inhibition.>
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==Molecular Mechanism==
 
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'''Magnesium cation effect'''
 
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The presence of the 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 (8). The coordination shell of the magnesium cation in the active site of MbtI is composed of two water molecules, Glu434, Glu294, and the two oxygen atoms of the C1 carboxylate group of chorismate (8). 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. 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.
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[[Image:Ebselen_inhibition.jpeg|200 px|right|thumb|Ebselen inhibition relaxing the alpha-9 helix]]
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'''Isochorismate pyruvae 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. 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.
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Ag85C can be inhibited by ebselen covalently bound to the sulfur of C209. Ebselen is a thiol-modifying agent that serves as an electrophile for the C209 that results in a sulfur-selenium bond. Co-crystallization of ebselen with Ag85C provides an explanation for the mechanism of ebselen-based inhibition. The addition of ebselen increases the distance between C209 and L232-T31, which effectively disrupts the interaction that holds the ‡9 helix in the active conformation. Furthermore, the bulk of ebselen creates steric hindrance with the ‡9 helix residues (Figure #). Relaxation of the ‡9 helix removes E228 and H260, which now interacts with S148, from the active site. The absence of these residues decreases the nucleophilicity of the S124 alcohol which decreases serine hydrolytic activity.
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'''Isochorismate synthast (IS)'''
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===Inhibitors===
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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 <ref>PMID:14982443</ref>. Lys205 has been proposed to act as the catalytic base, activating a water molecule in the active site by abstracting one of its protons. 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. 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. Instead of Lys205, Glu297 residue has been proposed to act as a base in the activation of the water molecule. 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. 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. 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. The pKa of Gly252 also accounts for the accumulation of isochorismate at pH values below 7.5.
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Additional thiol-modifying agents, p-chloromercuribenzoic acid and iodoacetamide, were crystalized with Ag85C. The structures show that each of these thiol-reactive inhibitors covalently bound to C209 and caused a relaxation of the ‡9 helix in a similar fashion to ebselen.
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[[Image:Inhibitors_Ag85c.jpeg|400 px|center]]
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'''chorismate mutase (CM)'''
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</StructureSection>
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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.
 
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==Inhibition Studies==
 
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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.
 
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</StructureSection>
 
== References ==
== References ==
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{{reflist}}
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<references/>

Revision as of 14:13, 30 April 2015

This Sandbox is Reserved from 02/09/2015, through 05/31/2016 for use in the course "CH462: Biochemistry 2" taught by Geoffrey C. Hoops at the Butler University. This reservation includes Sandbox Reserved 1051 through Sandbox Reserved 1080.
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Background

The antigen 85 (ag85) complex in Mycobacterium tuberculosis is composed of three intracellular membrane proteins: ag85A, B, and C. The ag85 complex is a major component of the cell wall, with each protein catalyzing the transfer of important cell wall constituents into the membrane. [1] Ag85C is of particular interest due to its transfer of mycolic acids, which are major components in determining cell wall integrity. By targeting this mycoloyltransferase activity, inhibition of ag85C offers potential for cell wall disruption and subsequent antibiotic targeting for normally drug-resistant mycotaberia tuberculosis. [2]

Structure

Antigen 85C in Mycobacterium Tuberculosis

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References

  1. ↑ Ronning DR, Klabunde T, Besra GS, Vissa VD, Belisle JT, Sacchettini JC. Crystal structure of the secreted form of antigen 85C reveals potential targets for mycobacterial drugs and vaccines. Nat Struct Biol. 2000 Feb;7(2):141-6. PMID:10655617 doi:10.1038/72413
  2. ↑ Jackson M, Raynaud C, Laneelle MA, Guilhot C, Laurent-Winter C, Ensergueix D, Gicquel B, Daffe M. Inactivation of the antigen 85C gene profoundly affects the mycolate content and alters the permeability of the Mycobacterium tuberculosis cell envelope. Mol Microbiol. 1999 Mar;31(5):1573-87. PMID:10200974
  3. ↑ Favrot L, Lajiness DH, Ronning DR. Inactivation of the Mycobacterium tuberculosis Antigen 85 complex by covalent, allosteric inhibitors. J Biol Chem. 2014 Jul 14. pii: jbc.M114.582445. PMID:25028518 doi:http://dx.doi.org/10.1074/jbc.M114.582445
  4. ↑ Ronning DR, Klabunde T, Besra GS, Vissa VD, Belisle JT, Sacchettini JC. Crystal structure of the secreted form of antigen 85C reveals potential targets for mycobacterial drugs and vaccines. Nat Struct Biol. 2000 Feb;7(2):141-6. PMID:10655617 doi:10.1038/72413
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