The Mycobacterium Tuberculosis FAS-II Dehydratases and Methyltransferases Define the Specificity of the Mycolic Acid Elongation Complexes

BackgroundThe human pathogen Mycobacterium tuberculosis (Mtb) has the originality of possessing a multifunctional mega-enzyme FAS-I (Fatty Acid Synthase-I), together with a multi-protein FAS-II system, to carry out the biosynthesis of common and of specific long chain fatty acids: the mycolic acids (MA). MA are the main constituents of the external mycomembrane that represents a tight permeability barrier involved in the pathogenicity of Mtb. The MA biosynthesis pathway is essential and contains targets for efficient antibiotics. We have demonstrated previously that proteins of FAS-II interact specifically to form specialized and interconnected complexes. This finding suggested that the organization of FAS-II resemble to the architecture of multifunctional mega-enzyme like the mammalian mFAS-I, which is devoted to the fatty acid biosynthesis.
Principal FindingsBased on conventional and reliable studies using yeast-two hybrid, yeast-three-hybrid and in vitro Co-immunoprecipitation, we completed here the analysis of the composition and architecture of the interactome between the known components of the Mtb FAS-II complexes. We showed that the recently identified dehydratases HadAB and HadBC are part of the FAS-II elongation complexes and may represent a specific link between the core of FAS-II and the condensing enzymes of the system. By testing four additional methyltransferases involved in the biosynthesis of mycolic acids, we demonstrated that they display specific interactions with each type of complexes suggesting their coordinated action during MA elongation.
SignificanceThese results provide a global update of the architecture and organization of a FAS-II system. The FAS-II system of Mtb is organized in specialized interconnected complexes and the specificity of each elongation complex is given by preferential interactions between condensing enzymes and dehydratase heterodimers. This study will probably allow defining essential and specific interactions that correspond to promising targets for Mtb FAS-II inhibitors.

研究背景：结核分枝杆菌（Mycobacterium tuberculosis, Mtb）作为人类致病菌，其独特之处在于同时拥有多功能巨型酶I型脂肪酸合酶（Fatty Acid Synthase-I, FAS-I）与多蛋白组成的II型脂肪酸合酶（Fatty Acid Synthase-II, FAS-II）系统，共同完成通用长链脂肪酸以及特异性长链脂肪酸——分枝菌酸（mycolic acids, MA）的生物合成。分枝菌酸是外层分枝菌膜的主要组成成分，而该膜是构成结核分枝杆菌致病性的关键致密渗透屏障。分枝菌酸生物合成通路是必需通路，且包含高效抗生素的作用靶点。我们此前的研究已证实，FAS-II的蛋白质可发生特异性相互作用，形成特化且相互连接的复合物。这一发现提示，FAS-II的组织结构类似于专司脂肪酸生物合成的哺乳动物mFAS-I的架构。
主要研究结果：本研究基于酵母双杂交（yeast-two hybrid）、酵母三杂交（yeast-three-hybrid）以及体外免疫共沉淀（in vitro Co-immunoprecipitation）等常规可靠实验方法，完成了对结核分枝杆菌FAS-II复合物已知组分之间相互作用组的组成与架构的分析。我们证实，新近鉴定的脱水酶HadAB与HadBC属于FAS-II延伸复合物的组分，可作为连接FAS-II核心与系统内缩合酶的特异性纽带。通过对另外四种参与分枝菌酸生物合成的甲基转移酶进行实验验证，我们发现这些酶可与各类复合物发生特异性相互作用，提示其在分枝菌酸延伸过程中存在协同作用。
研究意义：本研究结果为II型脂肪酸合酶系统的架构与组织模式提供了全局性的更新梳理。结核分枝杆菌的FAS-II系统以特化的相互连接复合物形式存在，各延伸复合物的特异性由缩合酶与脱水酶异二聚体之间的偏好性相互作用所决定。本研究有望明确那些可作为结核分枝杆菌FAS-II抑制剂潜在候选靶点的必需且特异性相互作用。