Gurvitz Aner, Hiltunen J Kalervo, Kastaniotis Alexander J
Section of Physiology of Lipid Metabolism, Institute of Physiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Vienna, Austria.
Appl Environ Microbiol. 2008 Aug;74(16):5078-85. doi: 10.1128/AEM.00655-08. Epub 2008 Jun 13.
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 C(20) fatty acids to form C(60)-to-C(90) 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 etr1Delta 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 etr1Delta 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 (C(4) to C(8)) than was previously thought (>C(12)). 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.
我们描述了在酵母（酿酒酵母）线粒体中从头合成硫辛酸过程中异源表达的结核分枝杆菌InhA的生理功能。InhA代表2-反式烯酰-酰基载体蛋白还原酶，是一线抗结核药物异烟肼的作用靶点，它参与解离型2脂肪酸合酶（FASII）的活性，该酶将缔合型1脂肪酸合酶（FASI）衍生的C(20)脂肪酸延长形成C(60)至C(90)的分枝菌酸。分枝菌酸是病原体周围保护层的主要成分，有助于其致病性和对某些抗菌药物的抗性。与FASI不同，FASII被认为无法从头生物合成脂肪酸。在此，InhA（Rv1484）和四种相似蛋白（Rv0927c、Rv3485c、Rv3530c和Rv3559c）的基因在缺乏线粒体2-反式烯酰硫酯还原酶活性的酿酒酵母etr1Δ细胞中表达。酵母突变体的表型包括无法产生足够水平的硫辛酸、形成线粒体细胞色素、进行呼吸或在非发酵碳源上生长。表达线粒体InhA的酵母etr1Δ细胞能够进行呼吸、在甘油上生长并产生硫辛酸。与在线粒体从头脂肪酸生物合成中的作用相一致，InhA在体内能够接受比之前认为的（>C(12)）短得多的酰基硫酯（C(4)至C(8)）。此外，InhA在没有AcpM或与其天然FASII伙伴KasA、KasB、FabD和FabH的蛋白质-蛋白质相互作用的情况下发挥作用。四种与InhA相似的蛋白均不能互补酵母突变体表型。我们结合结核分枝杆菌中硫辛酸合成以及酵母FASII突变体在研究参与脂肪酸生物合成的药物靶向病原体酶的生理功能方面的潜在用途，讨论了我们研究结果的意义。
