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Applied and Environmental Microbiology, August 2008, p. 5078-5085, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00655-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Function of Heterologous Mycobacterium tuberculosis InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C₂₀ Fatty Acids to C₆₀-to-C₉₀ Mycolic Acids, during De Novo Lipoic Acid Synthesis in Saccharomyces cerevisiae

Section of Physiology of Lipid Metabolism, Institute of Physiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Vienna, Austria,¹ Department of Biochemistry and Biocenter Oulu, University of Oulu, Oulu, Finland²

Received 18 March 2008/ Accepted 9 June 2008

We describe the physiological function of heterologously expressed Mycobacterium tuberculosis InhA during de novo lipoic acid synthesis in yeast (Saccharomyces cerevisiae) mitochondria. InhA, representing 2-trans-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous drug isoniazid, is involved in the activity of dissociative type 2 fatty acid synthase (FASII) that extends associative type 1 fatty acid synthase (FASI)-derived C₂₀ fatty acids to form C₆₀-to-C₉₀ mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen that contribute to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is thought 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 S. cerevisiae etr1 cells lacking mitochondrial 2-trans-enoyl-thioester reductase activity. The phenotype of the yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on nonfermentable carbon sources. Yeast etr1 cells expressing mitochondrial InhA were able 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 (C₄ to C₈) than was previously thought (>C₁₂). Moreover, InhA functioned in the absence of AcpM or protein-protein interactions with 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 implications of our findings with reference to lipoic acid synthesis in M. tuberculosis and the potential use of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes involved in fatty acid biosynthesis.

Published ahead of print on 13 June 2008.