The <i>Mycobacterium tuberculosis</i> FAS‐II condensing enzymes: their role in mycolic acid biosynthesis, acid‐fastness, pathogenesis and in future drug development

Bhatt, Apoorva; Molle, Virginie; Besra, Gurdyal S.; Jacobs, William R.; Kremer, Laurent

doi:10.1111/j.1365-2958.2007.05761.x

Cited by 205 publications

(189 citation statements)

References 67 publications

(99 reference statements)

Supporting

Mentioning

184

Contrasting

Unclassified

Order By: Relevance

“…It catalyzes the condensation of an acyl-CoA substrate (a FAS-I end product) with malonyl-AcpM to generate a ␤-ketoacyl-AcpM product (32), which following reduction generates an acyl-AcpM with two more carbons. KasA and KasB are ␤-ketoacyl-AcpM synthases that further elongate the growing acyl chain in the mycobacterial FAS-II system (33)(34)(35)(36) and have been shown to undergo post-translational modification by phosphorylation (24). Herein, we investigated whether mtFabH would also represent a substrate for mycobacterial kinases.…”

Section: Resultsmentioning

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

Self Cite

View full text Add to dashboard Cite

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 ...

show abstract

Section: Resultsmentioning

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

Self Cite

View full text Add to dashboard Cite

show abstract

“…In our previous studies we demonstrated that all of the condensing enzymes of the M. tuberculosis FAS-II system (mtFabH, KasA, KasB, and Pks13) are STPK substrates (8,26,32). In particular, we showed that mtFabH is phosphorylated, in vitro and in vivo, on a single Thr residue (Thr 45 ), which alters the accessibility of the substrate and thus negatively affects mtFabH enzymatic activity (32).…”

Section: Discussionmentioning

confidence: 99%

“…The resulting ␤-hydroxyacyl-ACP is then dehydrated by a set of dehydratases, HadABC (21,22), and finally reduced by the enoyl-ACP reductase, InhA, the primary target of isoniazid (23). The succeeding steps of condensation of the elongating chain with malonyl-ACP units are performed by the ␤-ketoacyl-ACP synthases KasA and KasB (8,24,25), leading to very long-chain meromycolyl-ACPs (up to C 56 ), which are the direct precursors of mycolic acids. How this complex metabolic pathway is regulated, allowing M. tuberculosis to tightly adjust mycolic acid biosynthesis to allow survival under variable environmental conditions, is currently unknown.…”

mentioning

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

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…There is some debate as to whether an increase in cyclopropane content of bacterial membranes leads to a decrease in membrane fluidity (Loffhagen et al, 2007;Munoz-Rojas et al, 2006). Furthermore, CFAs are involved in the pathogenesis of Mycobacterium tuberculosis (Glickman et al, 2000;Rao et al, 2005) and have antifungal activity in the antagonistic fluorescent pseudomonads (Ayyadurai et al, 2007), making CFAs targets for the development of new drugs for the treatment of antibiotic-resistant strains of M. tuberculosis (Bhatt et al, 2007;Guianvarc'h et al, 2006;Sekanka et al, 2007). Since CFAs are important in bacteria-host interactions for some pathogens, we have examined their possible role in S. meliloti during the alfalfa root-nodulation symbiosis.…”

Section: Introductionmentioning

confidence: 99%

Cyclopropane fatty acyl synthase in Sinorhizobium meliloti

et al. 2009

View full text Add to dashboard Cite

Cyclopropane fatty acyl synthases (CFA synthases) are enzymes that catalyse the addition of a methylene group across cis double bonds of monounsaturated fatty acyl chains in lipids. We have investigated the function of two putative genes, cfa1 and cfa2, proposed to code for CFA synthases in Sinorhizobium meliloti. Total fatty acid composition and fatty acid distributions within lipid classes for wild-type and cfa1 and cfa2 mutant strains grown under P i starvation and in acidic culture conditions were obtained by GC/MS and by infusion ESI/MS/MS, respectively. For wildtype cells and the cfa1 mutant, total cyclopropane fatty acids (CFAs) increased by 10 % and 15 % under P i starvation and acidic conditions, respectively; whereas in the cfa2 mutant, CFAs were less than 0.1 % of wild-type under both growth conditions. Reporter gene fusion experiments revealed that cfa1 and cfa2 were expressed at similar levels in free-living cells. Thus under the conditions we examined, cfa2 was required for the cyclopropanation of lipids in S. meliloti whereas the role of cfa1 remains to be determined. Analysis of intact lipids revealed that cyclopropanation occurred on cis-11-octadecenoic acid located in either the sn-1 or the sn-2 position in phospholipids and that cyclopropanation in the sn-2 position occurred to a greater extent in phosphatidylcholines and sulfoquinovosyldiacylglycerols under acidic conditions than under P i starvation. The cfa2 gene was also required for cyclopropanation of non-phosphoruscontaining lipids. Principal components analysis revealed no differences in the cyclopropanation of four lipid classes. We concluded that cyclopropanation occurred independently of the polar head group. Neither cfa1 nor cfa2 was required for symbiotic nitrogen fixation.

show abstract

The Mycobacterium tuberculosis FAS‐II condensing enzymes: their role in mycolic acid biosynthesis, acid‐fastness, pathogenesis and in future drug development

Cited by 205 publications

References 67 publications

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

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

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

Cyclopropane fatty acyl synthase in Sinorhizobium meliloti

Contact Info

Product

Resources

About