Institut de Biologie et Chimie des Protéines, Université Lyon1, IFR128 BioSciences, Lyon-Gerland, Lyon Cedex 07, France.
Mol Microbiol. 2010 Dec;78(6):1591-605. doi: 10.1111/j.1365-2958.2010.07446.x. Epub 2010 Nov 9.
The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA_T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development.
结核分枝杆菌在感染宿主中的惊人生存能力与其细胞壁相关的类脂酸的存在有关。尽管它们很重要，但调节这些脂质在环境变化下表达的机制尚不清楚。在这里，我们证明了烯酰基-ACP 还原酶活性的 InhA（分枝酸生物合成途径的必需酶，也是抗结核药物异烟肼的主要靶点）是通过磷酸化来控制的。Thr-266 是体外和体内唯一的激酶磷酸受体。通过使用 inhA 磷酸化缺失（T266A）或磷酸模拟（T266D/E）突变体，证明了 Thr-266 磷酸化的生理相关性。在体外，由于与 NADH 的结合亲和力降低，模拟突变体中的烯酰还原酶活性严重受损。重要的是，inhA_T266D/E 的引入未能像异烟肼处理后观察到的那样，弥补 inhA 热敏型耻垢分枝杆菌菌株的生长和分枝酸缺陷。这项研究表明，InhA 的磷酸化可能代表一种允许结核分枝杆菌调节其分枝酸含量的特殊机制，为未来抗结核药物的开发提供了一种新的方法。
