Mycobacterium tuberculosis Proteins Involved in Mycolic Acid Synthesis and Transport Localize Dynamically to the Old Growing Pole and Septum

ABSTRACT Isoxyl (ISO), a thiourea derivative that was successfully used for the clinical treatment of tuberculosis during the 1960s, is an inhibitor of the synthesis of oleic and mycolic acids in Mycobacterium tuberculosis. Its effect on oleic acid synthesis has been shown to be attributable to its inhibitory activity on the stearoyl-coenzyme A desaturase DesA3, but its enzymatic target(s) in the mycolic acid pathway remains to be identified. With the goal of elucidating the mode of action of ISO, we have isolated a number of spontaneous ISO-resistant mutants of M. tuberculosis and undertaken their genotypic characterization. We report here the characterization of a subset of these strains carrying mutations in the monooxygenase gene ethA. Through complementation studies, we demonstrate for the first time that the EthA-mediated oxidation of ISO is absolutely required for this prodrug to inhibit its lethal enzymatic target(s) in M. tuberculosis. An analysis of the metabolites resulting from the in vitro transformation of ISO by purified EthA revealed the occurrence of a formimidamide allowing the formulation of an activation pathway in which the oxidation of ISO catalyzed by EthA is followed by chemical transformations involving extrusion or elimination and, finally, hydrolysis.

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Antigen 85C Inhibition Restricts Mycobacterium tuberculosis Growth through Disruption of Cord Factor Biosynthesis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05742-11 ◽

2012 ◽

Vol 56 (4) ◽

pp. 1735-1743 ◽

Cited By ~ 44

Author(s):

Thulasi Warrier ◽

Marielle Tropis ◽

Jim Werngren ◽

Anne Diehl ◽

Martin Gengenbacher ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Cell Envelope ◽

Acid Synthesis ◽

Multidrug Resistant ◽

Mycolic Acid ◽

Broth Culture ◽

Nuclear Magnetic Resonance Analysis ◽

Transferase Activity ◽

Content Type ◽

Substrate Analogs

ABSTRACTThe antigen 85 (Ag85) protein family, consisting of Ag85A, -B, and -C, is vital forMycobacterium tuberculosisdue to its role in cell envelope biogenesis. The mycoloyl transferase activity of these proteins generates trehalose dimycolate (TDM), an envelope lipid essential forM. tuberculosisvirulence, and cell wall arabinogalactan-linked mycolic acids. Inhibition of these enzymes through substrate analogs hinders growth of mycobacteria, but a link to mycolic acid synthesis has not been established. In this study, we characterized a novel inhibitor of Ag85C, 2-amino-6-propyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile (I3-AG85). I3-AG85 was isolated from a panel of four inhibitors that exhibited structure- and dose-dependent inhibition ofM. tuberculosisdivision in broth culture. I3-AG85 also inhibitedM. tuberculosissurvival in infected primary macrophages. Importantly, it displayed an identical MIC against the drug-susceptible H37Rv reference strain and a panel of extensively drug-resistant/multidrug-resistantM. tuberculosisstrains. Nuclear magnetic resonance analysis indicated binding of I3-AG85 to Ag85C, similar to its binding to the artificial substrate octylthioglucoside. Quantification of mycolic acid-linked lipids of theM. tuberculosisenvelope showed a specific blockade of TDM synthesis. This was accompanied by accumulation of trehalose monomycolate, while the overall mycolic acid abundance remained unchanged. Inhibition of Ag85C activity also disrupted the integrity of theM. tuberculosisenvelope. I3-AG85 inhibited the division of and reduced TDM synthesis in anM. tuberculosisstrain deficient in Ag85C. Our results indicate that Ag85 proteins are promising targets for novel antimycobacterial drug design.

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AccD6, a Key Carboxyltransferase Essential for Mycolic Acid Synthesis in Mycobacterium tuberculosis, Is Dispensable in a Nonpathogenic Strain

Journal of Bacteriology ◽

10.1128/jb.05638-11 ◽

2011 ◽

Vol 193 (24) ◽

pp. 6960-6972 ◽

Cited By ~ 21

Author(s):

J. Pawelczyk ◽

A. Brzostek ◽

L. Kremer ◽

B. Dziadek ◽

A. Rumijowska-Galewicz ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Acid Synthesis ◽

Mycolic Acid ◽

Nonpathogenic Strain

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Kinase Targets for Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

Current Molecular Pharmacology ◽

10.2174/1874467211666181025141114 ◽

2019 ◽

Vol 12 (1) ◽

pp. 27-49 ◽

Cited By ~ 6

Author(s):

Shahinda S.R. Alsayed ◽

Chau C. Beh ◽

Neil R. Foster ◽

Alan D. Payne ◽

Yu Yu ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Enzymatic Activity ◽

Bacterial Pathogen ◽

Building Blocks ◽

Mycolic Acid ◽

Adaptive Responses ◽

Mycolic Acids ◽

Hydroxylated Fatty Acids

Background:Mycolic acids (MAs) are the characteristic, integral building blocks for the mycomembrane belonging to the insidious bacterial pathogen Mycobacterium tuberculosis (M.tb). These C60-C90 long α-alkyl-β-hydroxylated fatty acids provide protection to the tubercle bacilli against the outside threats, thus allowing its survival, virulence and resistance to the current antibacterial agents. In the post-genomic era, progress has been made towards understanding the crucial enzymatic machineries involved in the biosynthesis of MAs in M.tb. However, gaps still remain in the exact role of the phosphorylation and dephosphorylation of regulatory mechanisms within these systems. To date, a total of 11 serine-threonine protein kinases (STPKs) are found in M.tb. Most enzymes implicated in the MAs synthesis were found to be phosphorylated in vitro and/or in vivo. For instance, phosphorylation of KasA, KasB, mtFabH, InhA, MabA, and FadD32 downregulated their enzymatic activity, while phosphorylation of VirS increased its enzymatic activity. These observations suggest that the kinases and phosphatases system could play a role in M.tb adaptive responses and survival mechanisms in the human host. As the mycobacterial STPKs do not share a high sequence homology to the human’s, there have been some early drug discovery efforts towards developing potent and selective inhibitors.Objective:Recent updates to the kinases and phosphatases involved in the regulation of MAs biosynthesis will be presented in this mini-review, including their known small molecule inhibitors.Conclusion:Mycobacterial kinases and phosphatases involved in the MAs regulation may serve as a useful avenue for antitubercular therapy.

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The effect of substrate on inhibition of Corynebacterium lepus by isonicotinic acid hydrazide (Isoniazid)

Canadian Journal of Microbiology ◽

10.1139/m80-247 ◽

1980 ◽

Vol 26 (12) ◽

pp. 1498-1500 ◽

Cited By ~ 3

Author(s):

D. F. Gerson ◽

D. G. Cooper ◽

B. Ramsay ◽

J. E. Zajic

Keyword(s):

Isonicotinic Acid ◽

Acid Hydrazide ◽

Acid Synthesis ◽

Mycolic Acid ◽

Primary Effect ◽

Isonicotinic Acid Hydrazide ◽

Insoluble Substrate

Isonicotinic acid hydrazide (INH) inhibits the growth of Corynebacterium lepus on hexadecane but has no effect on its growth on fructose. INH also inhibits the production of the mycolic acid containing lipopeptide bioemulsifier normally produced by C. lepus in response to an insoluble substrate. The primary effect of INH appears to be inhibition of mycolic acid synthesis, which limits the growth of C. lepus on hexadecane by reducing the concentration of bioemulsifier.

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Crystal structure of hexanoyl-CoA bound to β-ketoacyl reductase FabG4 of Mycobacterium tuberculosis

Biochemical Journal ◽

10.1042/bj20121107 ◽

2013 ◽

Vol 450 (1) ◽

pp. 127-139 ◽

Cited By ~ 27

Author(s):

Debajyoti Dutta ◽

Sudipta Bhattacharyya ◽

Amlan Roychowdhury ◽

Rupam Biswas ◽

Amit Kumar Das

Keyword(s):

Mycobacterium Tuberculosis ◽

Active Site ◽

Ternary Complex ◽

Catalytic Mechanism ◽

Acyl Carrier Protein ◽

Structural Data ◽

Acid Synthesis ◽

Binary Complex ◽

C Terminus ◽

Covalently Linked

FabGs, or β-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/NADH-mediated conversion of β-oxoacyl-ACP (acyl-carrier protein) into β-hydroxyacyl-ACP. HMwFabGs (high-molecular-weight FabG) form a phylogenetically separate group of FabG enzymes. FabG4, an HMwFabG from Mycobacterium tuberculosis, contains two distinct domains, an N-terminal ‘flavodoxintype’ domain and a C-terminal oxoreductase domain. The catalytically active C-terminal domain utilizes NADH to reduce β-oxoacyl-CoA to β-hydroxyacyl-CoA. In the present study the crystal structures of the FabG4–NADH binary complex and the FabG4–NAD+–hexanoyl-CoA ternary complex have been determined to understand the substrate specificity and catalytic mechanism of FabG4. This is the first report to demonstrate how FabG4 interacts with its coenzyme NADH and hexanoyl-CoA that mimics an elongating fattyacyl chain covalently linked with CoA. Structural analysis shows that the binding of hexanoyl-CoA within the active site cavity of FabG significantly differs from that of the C16 fattyacyl substrate bound to mycobacterial FabI [InhA (enoyl-ACP reductase)]. The ternary complex reveals that both loop I and loop II interact with the phosphopantetheine moiety of CoA or ACP to align the covalently linked fattyacyl substrate near the active site. Structural data ACP inhibition studies indicate that FabG4 can accept both CoA- and ACP-based fattyacyl substrates. We have also shown that in the FabG4 dimer Arg146 and Arg445 of one monomer interact with the C-terminus of the second monomer to play pivotal role in substrate association and catalysis.

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