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J. Biol. Chem., Vol. 280, Issue 10, 8862-8874, March 11, 2005

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The Acyl-AMP Ligase FadD32 and AccD4-containing Acyl-CoA Carboxylase Are Required for the Synthesis of Mycolic Acids and Essential for Mycobacterial Growth

IDENTIFICATION OF THE CARBOXYLATION PRODUCT AND DETERMINATION OF THE ACYL-CoA CARBOXYLASE COMPONENTS^*

Damien Portevin, Célia de Sousa-D'Auria¶, Henri Montrozier, Christine Houssin¶, Alexandre Stella||, Marie-Antoinette Lanéelle, Fabienne Bardou, Christophe Guilhot**, and Mamadou Daffé

From the Département "Mécanismes Moléculaires des Infections Mycobactériennes" and ^||Plateforme "Protéomique IPBS-CNRS," Institut de Pharmacologie et Biologie Structurale, CNRS and Université Paul Sabatier (Unité Mixte de Recherche 5089), 205 route de Narbonne, 31077 Toulouse Cedex, France and ^¶Laboratoire de Biotechnologie des Microorganismes d'Intérêt Industriel, Institut de Génétique et Microbiologie, Centre National de la Recherche Scientifique and Université Paris Sud (UMR 8621), Bat 360, 91405 Orsay Cedex, France

Mycolic acids are major and specific long-chain fatty acids of the cell envelope of several important human pathogens such as Mycobacterium tuberculosis, M. leprae, and Corynebacterium diphtheriae. Their biosynthesis is essential for mycobacterial growth and represents an attractive target for developing new antituberculous drugs. We have previously shown that the pks13 gene encodes condensase, the enzyme that performs the final condensation step of mycolic acid biosynthesis and is flanked by two genes, fadD32 and accD4. To determine the functions of the gene products we generated two mutants of C. glutamicum with an insertion/deletion within either fadD32 or accD4. The two mutant strains were deficient in mycolic acid production and exhibited the colony morphology that typifies the mycolate-less mutants of corynebacteria. Application of multiple analytical approaches to the analysis of the mutants demonstrated the accumulation of a tetradecylmalonic acid in the fadD32::km mutant and its absence from the accD4::km strain. The parental corynebacterial phenotype was restored upon the transfer of the wild-type fadD32 and accD4 genes in the mutants. These data demonstrated that both FadD32 and AccD4-containing acyl-CoA carboxylase are required for the production of mycolic acids. They also prove that the proteins catalyze, respectively, the activation of one fatty acid substrate and the carboxylation of the other substrate, solving the long-debated question of the mechanism involved in the condensation reaction. We used comparative genomics and applied a combination of molecular biology and proteomic technologies to the analysis of proteins that co-immunoprecipitated with AccD4. This resulted in the identification of AccA3 and AccD5 as subunits of the acyl-CoA carboxylase. Finally, we used conditionally replicative plasmids to show that both the fadD32 and accD4 genes are essential for the survival of M. smegmatis. Thus, in addition to Pks13, FadD32 and AccD4 are promising targets for the development of new antimicrobial drugs against pathogenic species of mycobacteria and related microorganisms.

Received for publication, July 28, 2004 , and in revised form, December 23, 2004.

^* This work was supported by the CNRS (France) and the Ministère Délégué à la Recherche et aux Nouvelles Technologies (ACI "Molécules et Cibles Thérapeutiques"). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Holds a Fellowship from the Ministère Délégué à la Recherche et aux Nouvelles Technologies.

^** To whom correspondence may be addressed. Tel.: 33-561-175-845; Fax: 33-561-175-994; E-mail: guilhot{at}ipbs.fr. To whom correspondence may be addressed. Tel.: 33-561-175-569; Fax: 33-561-175-580; E-mail: daffe{at}ipbs.fr.

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