Protein–protein interactions within the Fatty Acid Synthase‐II system of <i>Mycobacterium tuberculosis</i> are essential for mycobacterial viability

Veyron‐Churlet, Romain; Guerrini, O; Mourey, Lionel; Daffé, Mamadou; Zerbib, Didier

doi:10.1111/j.1365-2958.2004.04334.x

Cited by 85 publications

(94 citation statements)

References 43 publications

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“…MabA has been reported to be essential in mycobacteria (56), and this study demonstrates that overexpression of MabA_WT under a strong promoter was also associated to a reduced M. bovis BCG colony size compared with the control strain. It was previously reported that each monomer of MabA can interact with each condensing enzyme and that its high propensity to multimerization (MabA is a tetramer in solution) might maintain the macromolecular organization of FAS-II complexes (57). Thus, a large excess of MabA may alter FAS-II activity, resulting in a slower growth rate.…”

Section: Discussionmentioning

confidence: 99%

“…Why would M. tuberculosis phosphorylate multiple enzymes that are known to interact together (57) and to act repetitively to ensure mycolic acid elongation? One explanation of the biological significance of this specific regulation could be that altering the activity of most of these enzymes may lead to a tightly regulated system to allow survival and/or adaptation under variable growth conditions.…”

Section: Discussionmentioning

confidence: 99%

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Phosphorylation of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis

Veyron‐Churlet

Zanella-Cléon

Cohen‐Gonsaud

et al. 2010

Journal of Biological Chemistry

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Mycolic acids are key cell wall components for the survival, pathogenicity, and antibiotic resistance of the human tubercle bacillus. Although it was thought that Mycobacterium tuberculosis tightly regulates their production to adapt to prevailing environmental conditions, the molecular mechanisms governing mycolic acid biosynthesis remained extremely obscure. Meromycolic acids, the direct precursors of mycolic acids, are synthesized by a type II fatty acid synthase from acyl carrier protein-bound substrates that are extended iteratively, with a reductive cycle in each round of extension, the second step of which is catalyzed by the essential ␤-ketoacylacyl carrier protein reductase, MabA. In this study, we investigated whether post-translational modifications of MabA might represent a strategy employed by M. tuberculosis to regulate mycolic acid biosynthesis. Indeed, we show here that MabA was efficiently phosphorylated in vitro by several M. tuberculosis Ser/Thr protein kinases, including PknB, as well as in vivo in mycobacteria. Mass spectrometric analyses using LC-ESI/MS/MS and site-directed mutagenesis identified three phosphothreonines, with Thr 191 being the primary phosphor-acceptor. A MabA_T191D mutant, designed to mimic constitutive phosphorylation, exhibited markedly decreased ketoacyl reductase activity compared with the wild-type protein, as well as impaired binding of the NADPH cofactor, as demonstrated by fluorescence spectroscopy. The hypothesis that phosphorylation of Thr 191 alters the enzymatic activity of MabA, and subsequently mycolic acid biosynthesis, was further supported by the fact that constitutive overexpression of the mabA_T191D allele in Mycobacterium bovis BCG strongly impaired mycobacterial growth. Importantly, conditional expression of the phosphomimetic MabA_T191D led to a significant inhibition of de novo biosynthesis of mycolic acids. This study provides the first information on the molecular mechanism(s) involved in mycolic acid regulation through Ser/Thr protein kinase-dependent phosphorylation of a type II fatty acid synthase enzyme.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Phosphorylation of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis

Veyron‐Churlet

Zanella-Cléon

Cohen‐Gonsaud

et al. 2010

Journal of Biological Chemistry

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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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“…There is now a strong body of evidence that several FAS-II components are phosphorylated and that STPK-dependent phosphorylation can induce either positive or negative signaling to the different interconnected FAS-II complexes. Indeed, a model based on co-exist-ence of multiple interconnected FAS-II systems has been proposed (44). It predicts the occurrence of four FAS-II systems responsible for the initiation, elongation, and termination steps of the mycolic acid pathway, each system characterized by a common enzyme core along with a specific condensase (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, it is conceivable that phosphorylation of Thr 45 affects the interaction of mtFabH with the acyl carrier protein AcpM, which also must bind in the vicinity of the narrow substrate binding channel of mtFabH. Thus, the structural analysis suggests that phosphorylation of Thr 45 may affect substrate binding and/or protein-protein interactions with other FAS-II partners (44). Therefore, the condensing activities of wild-type mtFabH with that of point mutants, Thr 45 to alanine and aspartic acid, were assessed and compared in vitro.…”

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confidence: 99%

The Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Synthase III Activity Is Inhibited by Phosphorylation on a Single Threonine Residue

Veyron‐Churlet

Molle

Taylor

et al. 2009

Journal of Biological Chemistry

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Mycolic acids are hallmark features of the Mycobacterium tuberculosis cell wall. They are synthesized by the condensation of two fatty acids, a C 56 -64 -meromycolyl chain and a C 24 -26 -fatty acyl chain. Meromycolates are produced via the combination of type I and type II fatty acid synthases (FAS-I and FAS-II). The ␤-ketoacyl-acyl carrier protein (ACP) synthase III (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-ACP. Because mtFabH represents a potential regulatory key point of the mycolic acid pathway, we investigated the hypothesis that phosphorylation of mtFabH controls its activity. Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently emerged as a major physiological mechanism of regulation in prokaryotes. We demonstrate here that mtFabH was efficiently phosphorylated in vitro by several mycobacterial STPKs, particularly by PknF and PknA, as well as in vivo in mycobacteria. Analysis of the phosphoamino acid content indicated that mtFabH was phosphorylated exclusively on threonine residues. Mass spectrometry analyses using liquid chromatography-electrospray ionization/tandem mass spectrometry identified Thr 45 as the unique phosphoacceptor. This was further supported by complete loss of PknF-or PknA-dependent phosphorylation of a mtFabH mutant. Mapping Thr 45 on the crystal structure of mtFabH illustrates that this residue is located at the entrance of the substrate channel, suggesting that the phosphate group may alter accessibility of the substrate and thus affect mtFabH enzymatic activity. A T45D mutant of mtFabH, designed to mimic constitutive phosphorylation, exhibited markedly decreased transacylation, malonyl-AcpM decarboxylation, and condensing activities compared with the wild-type protein or the T45A mutant. Together, these findings not only represent the first demonstration of phosphorylation of a ␤-ketoacyl-ACP synthase III enzyme but also indicate that phosphorylation of mtFabH inhibits its enzymatic activity, which may have important consequences in regulating mycolic acid biosynthesis.Within the infected host, Mycobacterium tuberculosis encounters numerous environmental conditions and induces or represses a number of genes for a quick adjustment to new conditions. The infection process of M. tuberculosis involves cross-talk of signals between the host and the bacterium, resulting in reprogramming of the host signaling network. Protein phosphorylation/dephosphorylation represent a central mechanism for distribution of signals to various parts of the cell, regulating growth, differentiation, mobility, and survival (1). In mycobacteria, a common signal transduction pathway is transmitted through membrane-embedded sensor kinases, enabling the pathogen to modify itself for survival in this hostile environment.Protein kinases can be classified into two families based on their similarities and enzymatic specification: (i) the histidine kinase superfamily, which relies on autophosphorylation of a conserved histidine residue ...

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Protein–protein interactions within the Fatty Acid Synthase‐II system of Mycobacterium tuberculosis are essential for mycobacterial viability

Cited by 85 publications

References 43 publications

Phosphorylation of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis

Phosphorylation of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis

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

The Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Synthase III Activity Is Inhibited by Phosphorylation on a Single Threonine Residue

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