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J. Biol. Chem., Vol. 282, Issue 15, 11000-11008, April 13, 2007

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The Reductase That Catalyzes Mycolic Motif Synthesis Is Required for Efficient Attachment of Mycolic Acids to Arabinogalactan^*

David J. Lea-Smith¹, James S. Pyke, Dedreia Tull, Malcolm J. McConville², Ross L. Coppel², and Paul K. Crellin³

From the Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, and Victorian Bioinformatics Consortium, Department of Microbiology, Monash University, Clayton, Victoria 3800 and the Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Sciences and Biotechnology, University of Melbourne, Parkville, Victoria 3010, Australia

Mycolic acids are essential components of the cell walls of bacteria belonging to the suborder Corynebacterineae, including the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae. Mycolic acid biosynthesis is complex and the target of several frontline antimycobacterial drugs. The condensation of two fatty acids to form a 2-alkyl-3-keto mycolate precursor and the subsequent reduction of this precursor represent two key and highly conserved steps in this pathway. Although the enzyme catalyzing the condensation step has recently been identified, little is known about the putative reductase. Using an extensive bioinformatic comparison of the genomes of M. tuberculosis and Corynebacterium glutamicum, we identified NCgl2385, the orthologue of Rv2509 in M. tuberculosis, as a potential reductase candidate. Deletion of the gene in C. glutamicum resulted in a slow growing strain that was deficient in arabinogalactan-linked mycolates and synthesized abnormal forms of the mycolate-containing glycolipids trehalose dicorynomycolate and trehalose monocorynomycolate. Analysis of the native and acetylated trehalose glycolipids by MALDI-TOF mass spectrometry indicated that these novel glycolipids contained an unreduced -keto ester. This was confirmed by analysis of sodium borodeuteride-reduced mycolic acids by gas chromatography mass spectrometry. Reintroduction of the NCgl2385 gene into the mutant restored the transfer of mature mycolic acids to both the trehalose glycolipids and cell wall arabinogalactan. These data indicate that NCgl2385, which we have designated CmrA, is essential for the production of mature trehalose mycolates and subsequent covalent attachment of mycolic acids onto the cell wall, thus representing a focus for future structural and pathogenicity studies.

Received for publication, September 8, 2006 , and in revised form, February 16, 2007.

^* This work was supported in part by the Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics and the National Health and Medical Research Council of Australia. 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.

¹ Supported by an Australian Postgraduate Award.

² Supported by the Howard Hughes Medical Institute International Scholars Program.

³ To whom correspondence should be addressed. Tel.: 61-3-9905-1468; Fax: 61-3-9905-4811; E-mail: paul.crellin{at}med.monash.edu.au.

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