AEM Accepts, published online ahead of print on 13 June 2008

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Appl. Environ. Microbiol. doi:10.1128/AEM.00655-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Function of heterologous M. tuberculosis InhA, a FASII enzyme involved in extending C₂₀ fatty acids to C_60-90 mycolic acids, during de novo lipoic acid synthesis in yeast

Aner Gurvitz^*, J. Kalervo Hiltunen, and Alexander J. Kastaniotis

Section of Physiology of Lipid Metabolism, Institute of Physiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Austria; and Department of Biochemistry and Biocenter Oulu, University of Oulu, Finland

^* To whom correspondence should be addressed. Email: aner.gurvitz{at}meduniwien.ac.at.

We report on the physiological function of heterologously expressed Mycobacterium tuberculosis InhA during de novo lipoic acid synthesis in yeast mitochondria. InhA, representing 2-trans-enoyl-ACP reductase and target for the front-line antituberculous drug isoniazid, is involved in FASII that extends FASI-derived C₂₀ fatty acids to form C_60-90 mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen, lending to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is claimed to be incapable of de novo biosynthesis of fatty acids. Here, the genes for InhA (Rv1484) and four similar proteins (Rv0927c, Rv3485c, Rv3530c, and Rv3559c) were expressed in Saccharomyces cerevisiae etr1 cells lacking mitochondrial 2-trans-enoyl-thioester reductase activity. The phenotype of these yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on non-fermentable carbon sources. Yeast etr1 cells expressing mitochondrial InhA recovered their ability to respire, grow on glycerol, and produce lipoic acid. Commensurate with a role in mitochondrial de novo fatty acid biosynthesis, InhA could accept in vivo much shorter acyl-thioesters (C_4-8) than was hitherto thought (>C₁₂). Moreover, InhA functioned in the absence of AcpM or protein-protein interactions to its native FASII partners KasA, KasB, FabD, and FabH. None of the four proteins similar to InhA complemented the yeast mutant phenotype. We discuss the implication of our finding with reference to lipoic acid synthesis in M. tuberculosis, and the potential application of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes of fatty acid biosynthesis.

This article has been cited by other articles:

• Gurvitz, A., Hiltunen, J. K., Kastaniotis, A. J. (2009). Heterologous Expression of Mycobacterial Proteins in Saccharomyces cerevisiae Reveals Two Physiologically Functional 3-Hydroxyacyl-Thioester Dehydratases, HtdX and HtdY, in Addition to HadABC and HtdZ. J. Bacteriol. 191: 2683-2690 [Abstract] [Full Text]