The Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis Relies on Multiple Specialized Elongation Complexes Interconnected by Specific Protein–Protein Interactions

Veyron‐Churlet, Romain; Bigot, Sarah; Guerrini, O; Verdoux, Sébastien; Malaga, Wladimir; Daffé, Mamadou; Zerbib, Didier

doi:10.1016/j.jmb.2005.09.016

Cited by 63 publications

(65 citation statements)

References 36 publications

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“…Moreover, InhA has been shown to interact physically with the latter four proteins in a yeast twohybrid system and coimmunoprecipitation experiments (51,52). Nevertheless, InhA was physiologically functional in yeast cells lacking AcpM, as well as all four interacting partners.…”

Section: Discussionmentioning

confidence: 99%

Function of Heterologous Mycobacterium tuberculosis InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C ₂₀ Fatty Acids to C ₆₀ -to-C ₉₀ Mycolic Acids, during De Novo Lipoic Acid Synthesis in Saccharomyces cerevisiae

Gurvitz

Hiltunen

Kastaniotis

2008

Appl Environ Microbiol

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We describe the physiological function of heterologously expressed Mycobacterium tuberculosis InhA during de novo lipoic acid synthesis in yeast (Saccharomyces cerevisiae) mitochondria. InhA, representing 2-trans-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous drug isoniazid, is involved in the activity of dissociative type 2 fatty acid synthase (FASII) that extends associative type 1 fatty acid synthase (FASI)-derived C 20 fatty acids to form C 60 -to-C 90 mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen that contribute to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is thought to be incapable of de novo biosynthesis of fatty acids. Here, the genes for InhA (Rv1484) and four similar proteins (Rv0927c, Rv3485c, Rv3530c, and Rv3559c) were expressed in S. cerevisiae etr1⌬ cells lacking mitochondrial 2-trans-enoyl-thioester reductase activity. The phenotype of the yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on nonfermentable carbon sources. Yeast etr1⌬ cells expressing mitochondrial InhA were able to respire, grow on glycerol, and produce lipoic acid. Commensurate with a role in mitochondrial de novo fatty acid biosynthesis, InhA could accept in vivo much shorter acyl-thioesters (C 4 to C 8 ) than was previously thought (>C 12 ). Moreover, InhA functioned in the absence of AcpM or protein-protein interactions with its native FASII partners KasA, KasB, FabD, and FabH. None of the four proteins similar to InhA complemented the yeast mutant phenotype. We discuss the implications of our findings with reference to lipoic acid synthesis in M. tuberculosis and the potential use of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes involved in fatty acid biosynthesis.

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Section: Discussionmentioning

confidence: 99%

Function of Heterologous Mycobacterium tuberculosis InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C ₂₀ Fatty Acids to C ₆₀ -to-C ₉₀ Mycolic Acids, during De Novo Lipoic Acid Synthesis in Saccharomyces cerevisiae

Gurvitz

Hiltunen

Kastaniotis

2008

Appl Environ Microbiol

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“…At the molecular level, given the complexity of the MA biosynthetic machinery, which relies on multiprotein complexes wherein protein interactions govern the elongation and modification of the meromycolate chain (22)(23)(24), it is at present not possible to definitively conclude whether ISO and TAC preferentially inhibit HadAB or HadBC and whether they inhibit this (these) enzyme(s) directly (e.g. by competing for their substrate-binding sites) or through a less direct mechanism (e.g.…”

Section: Discussionmentioning

confidence: 99%

“…by perturbing heterotypic interactions involving HadAB/BC and other MA biosynthetic enzymes). The importance of protein interactions within the FAS-II interactome was clearly illustrated by the work of Veyron-Churlet et al (22) (23,24). In this light, it is tempting to speculate that affecting the level of expression or primary sequence of these MA methyltransferases may impact the structure of the FAS-II interactome in a way that prevents ISO and TAC from effectively binding to the dehydratases.…”

Section: Discussionmentioning

confidence: 99%

A Common Mechanism of Inhibition of the Mycobacterium tuberculosis Mycolic Acid Biosynthetic Pathway by Isoxyl and Thiacetazone

Grzegorzewicz

Korduláková

Jones

et al. 2012

Journal of Biological Chemistry

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103

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“…The FAS II complex in M. tuberculosis is located in a genetic locus that also contains a lone, uncharacterized acyl-CoA carboxylase carboxyltransferase subunit, accD6 (␤ 6 ) (6), which could provide malonylCoA to the ␤-ketoacyl-ACP synthases and to FAS I. MalonylCoA is converted to malonyl-ACP by the malonyl-CoA-ACP transacylase (FabD) (19) and is utilized by the ␤-ketoacyl-ACP synthases (KasA/KasB) (5,18,27) in successive reactions with the other enzymes of the FAS II complex to produce the meromycolic acids. Protein-protein interaction analyses have shown that FabD is a structural component of the FAS II complex (30) and that KasA, KasB, InhA, MabA (FabG1), and FabH (␤-ketoacyl-ACP synthase III) interact with each other (31).…”

Section: Discussionmentioning

confidence: 99%

AccD6, a Member of the Fas II Locus, Is a Functional Carboxyltransferase Subunit of the Acyl-Coenzyme A Carboxylase in Mycobacterium tuberculosis

Daniel

Lee

et al. 2007

J Bacteriol

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The Mycobacterium tuberculosis acyl-coenzyme A (CoA) carboxylases provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase I (FAS I) and for the elongation of FAS I end products by the FAS II complex to produce meromycolic acids. The M. tuberculosis genome contains three biotin carboxylase subunits (AccA1 to -3) and six carboxyltransferase subunits (AccD1 to -6), with accD6 located in a genetic locus that contains members of the FAS II complex. We found by quantitative real-time PCR analysis that the transcripts of accA3, accD4, accD5, and accD6 are expressed at high levels during the exponential growth phases of M. tuberculosis in vitro. Microarray analysis of M. tuberculosis transcripts indicated that the transcripts for accA3, accD4, accD5, accD6, and accE were repressed during later growth stages. AccD4 and AccD5 have been previously studied, but there are no reports on the function of AccD6. We expressed AccA3 (␣ 3 ) and AccD6 (␤ 6 ) in E. coli and purified them by affinity chromatography. We report here that reconstitution of the ␣ 3 -␤ 6 complex yielded an active acyl-CoA carboxylase. Kinetic characterization of this carboxylase showed that it preferentially carboxylated acetyl-CoA (1.1 nmol/mg/min) over propionyl-CoA (0.36 nmol/mg/min). The activity of the ␣ 3 -␤ 6 complex was inhibited by the subunit. The ␣ 3 -␤ 6 carboxylase was inhibited significantly by dimethyl itaconate, C75, haloxyfop, cerulenin, and 1,2-cyclohexanedione. Our results suggest that the ␤ 6 subunit could play an important role in mycolic acid biosynthesis by providing malonyl-CoA to the FAS II complex.Tuberculosis causes 2 million deaths each year, according to the World Health Organization. Mycobacterium tuberculosis, the pathogen that causes the disease, infects 8 million people each year and is one of the world's deadliest pathogens (9). The ongoing AIDS pandemic has developed a deadly synergy with tuberculosis, which is the leading cause of death among AIDS patients (2). Multidrug-resistant M. tuberculosis strains have been emerging rapidly (9), and the need for identifying novel drug targets in this pathogen has become urgent. The cell wall of M. tuberculosis is lipid enriched and acts as an impermeable barrier to many common broad-spectrum antibiotics (14).The first committed step of fatty-acid biosynthesis, which is the biotin-dependent carboxylation of acyl-coenzyme A (CoA) to produce malonyl-CoA and methylmalonyl-CoA, is catalyzed by the acyl-CoA carboxylase. The reaction consists of two catalytic steps, which involve the biotin carboxylase and the carboxyltransferase (8). In M. tuberculosis, the biotin carboxylation step is catalyzed by the ␣ subunit; there are three open reading frames (ORFs) that can encode the ␣ subunit (accA1 to -A3) in the genome. Carboxyl transfer is catalyzed by the ␤ subunit, and there are six ␤ subunits (accD1 to -D6) in the genome of the pathogen (6).Previously, the catalytic activities of the ␣ 3 , ␤ 4 , and ␤ 5 subunits were studied (10,11,22,24). However, the level...

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The Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis Relies on Multiple Specialized Elongation Complexes Interconnected by Specific Protein–Protein Interactions

Cited by 63 publications

References 36 publications

Function of Heterologous Mycobacterium tuberculosis InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C ₂₀ Fatty Acids to C ₆₀ -to-C ₉₀ Mycolic Acids, during De Novo Lipoic Acid Synthesis in Saccharomyces cerevisiae

Function of Heterologous Mycobacterium tuberculosis InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C ₂₀ Fatty Acids to C ₆₀ -to-C ₉₀ Mycolic Acids, during De Novo Lipoic Acid Synthesis in Saccharomyces cerevisiae

A Common Mechanism of Inhibition of the Mycobacterium tuberculosis Mycolic Acid Biosynthetic Pathway by Isoxyl and Thiacetazone

AccD6, a Member of the Fas II Locus, Is a Functional Carboxyltransferase Subunit of the Acyl-Coenzyme A Carboxylase in Mycobacterium tuberculosis

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The Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis Relies on Multiple Specialized Elongation Complexes Interconnected by Specific Protein–Protein Interactions

Cited by 63 publications

References 36 publications

Function of Heterologous Mycobacterium tuberculosis InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C 20 Fatty Acids to C 60 -to-C 90 Mycolic Acids, during De Novo Lipoic Acid Synthesis in Saccharomyces cerevisiae

Function of Heterologous Mycobacterium tuberculosis InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C 20 Fatty Acids to C 60 -to-C 90 Mycolic Acids, during De Novo Lipoic Acid Synthesis in Saccharomyces cerevisiae

A Common Mechanism of Inhibition of the Mycobacterium tuberculosis Mycolic Acid Biosynthetic Pathway by Isoxyl and Thiacetazone

AccD6, a Member of the Fas II Locus, Is a Functional Carboxyltransferase Subunit of the Acyl-Coenzyme A Carboxylase in Mycobacterium tuberculosis

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Function of Heterologous Mycobacterium tuberculosis InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C ₂₀ Fatty Acids to C ₆₀ -to-C ₉₀ Mycolic Acids, during De Novo Lipoic Acid Synthesis in Saccharomyces cerevisiae

Function of Heterologous Mycobacterium tuberculosis InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C ₂₀ Fatty Acids to C ₆₀ -to-C ₉₀ Mycolic Acids, during De Novo Lipoic Acid Synthesis in Saccharomyces cerevisiae