Document Detail

Phosphorylation of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein reductase MabA regulates mycolic acid biosynthesis.
MedLine Citation:
PMID:  20178986     Owner:  NLM     Status:  MEDLINE    
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 beta-ketoacyl-acyl 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.
Romain Veyron-Churlet; Isabelle Zanella-Cléon; Martin Cohen-Gonsaud; Virginie Molle; Laurent Kremer
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2010-02-23
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  285     ISSN:  1083-351X     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2010 Apr 
Date Detail:
Created Date:  2010-04-19     Completed Date:  2010-05-20     Revised Date:  2013-05-30    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  12714-25     Citation Subset:  IM    
Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université de Montpellier I, CNRS, UMR 5235, case 107, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France.
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MeSH Terms
Alcohol Oxidoreductases / chemistry,  genetics,  metabolism*
Gene Expression Regulation, Bacterial / physiology*
Gene Expression Regulation, Enzymologic / physiology*
Mutagenesis, Site-Directed
Mycobacterium bovis / enzymology,  genetics
Mycobacterium tuberculosis / enzymology*,  genetics
Mycolic Acids / metabolism*
Oxidoreductases / chemistry,  genetics,  metabolism*
Phosphorylation / physiology
Protein Processing, Post-Translational / physiology*
Protein-Serine-Threonine Kinases / genetics,  metabolism
Spectrometry, Fluorescence
Reg. No./Substance:
0/Mycolic Acids; EC 1.-/Oxidoreductases; EC 1.1.-/Alcohol Oxidoreductases; EC reductase; EC 2.7.1.-/PknB protein, Mycobacterium tuberculosis; EC Kinases

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