Functional and structural characterization of the fatty acid synthase type I from Mycobacterium tuberculosis

脂肪酸合成酶I(Fas I)系统是一种在一条多肽链上具有多种酶活性的多功能蛋白质。Fas I蛋白以其特有的形式存在于真核细胞的细胞质中。最近，真菌和哺乳动物中Fas I复合体的X射线结构被解决，并使人们能够深入了解脂肪酸的合成和这种多功能蛋白质的一般工作模式。细菌脂肪酸合成通常发生在分离的蛋白质中(类型II系统)；然而，类型I系统已在分枝杆菌属(放线菌目)的细菌中得到特征。结核分枝杆菌Fas I具有产生C16/C18和C24/C26脂肪酸的双峰产物谱。分枝杆菌Fas I是合理药物设计的高要求靶点。抑制该蛋白是结核病(TB)6个月标准治疗方案的一部分，结核病是一种人类传染病，通常由结核分枝杆菌引起。这个项目的目标是建立一个用于研究抑制结核分枝杆菌Fas I的功能和结构的工具包。我们追求一种创新的方法，在大肠杆菌中重组表达结核分枝杆菌Fas I。由于天然的II型脂肪酸代谢，大肠杆菌的生长实际上独立于重组Fas I的生产，这使得不受限制的蛋白质工程和体外突变分析可以指导用结构和功能方法设计结核分枝杆菌Fas I抑制剂。

Fatty acid synthase type I (FAS I) systems are large multifunctional proteins which harbor several enzymatic activities on one polypeptide chain. FAS I proteins occur characteristically in the cytoplasm of eukaryotic cells. Recently, the X-ray structures of the FAS I complexes in fungi and in mammals were solved, and enabled deep insight into fatty acid synthesis and the general working mode of such multifunctional proteins. Bacterial fatty acid synthesis generally occurs in separated proteins (type II system); however, type I systems have been characterized in bacteria of e.g. genus Mycobacterium (order Actinomycetales). Mycobacterium tuberculosis FAS I has a bimodal product spectrum producing C16/C18 and C24/C26 fatty acids. The mycobacterial FAS I is a highly demanded target for rational drug design. The inhibition of this protein is part of the 6 months standard treatment regime of tuberculosis (TB), a human infectious disease which is usually caused by M. tuberculosis. The goal of this project is to establish a toolkit for functional and structural studies to the inhibition of the M. tuberculosis FAS I. We pursue an innovative approach, where we recombinantly express M. tuberculosis FAS I in Escherichia coli. Due to a native type II fatty acid metabolism, E. coli growth is virtually independent from recombinant FAS I production which allows unrestricted protein engineering and in vitro mutational analysis for guiding M. tuberculosis FAS I inhibitor design by structural and functional methods.