Characterization of two acyl-CoA carboxylase complexes of Mycobacterium tuberculo

描述（由申请人提供）：结核分枝杆菌是人类结核病（TB）的病原，每年在全球造成约300万人死亡，估计感染世界三分之一的人口。由于缺乏药物依从性，多重耐药菌株的出现以及艾滋病的流行，这种疾病的重新出现促使开发对多重耐药菌株有活性的新药，从而可以缩短当前治疗的持续时间和复杂性。酰基辅酶a羧化酶（ACCase）在大多数生物体的脂肪酸代谢中起着至关重要的作用，已被提出作为开发一类新型抗菌剂的良好靶点。在结核分枝杆菌中，从基因组序列中预测了6个推定的accase，其中两个，ACCase4和ACCase6，被认为参与霉菌酸的生物合成。这种复合脂质是结核分枝杆菌细胞包膜的主要成分之一，与结核分枝杆菌的存活和致病性有关。本研究的目的是在遗传、生化和结构水平上对结核分枝杆菌中的ACCases4和accases6进行表征，并以耻垢分枝杆菌为工作模型，了解它们在霉菌酸生物合成和这些微生物存活中的生理作用和相关性。此外，我们将在体内和体外对通过化学文库的计算机对接鉴定的假定的ACCase抑制剂进行表征，并使用它们来验证这些酶复合物作为未来抗真菌药物开发的靶标。作为一个长期的目标，我们也将把重点放在了解分子基础，决定底物特异性的每个复合物。为此，我们将对必需复合物的羧基转移酶亚基(2)进行基于结构的诱变，然后对突变酶亚基进行生化表征。公共卫生相关性：结核分枝杆菌造成的人类死亡人数超过任何其他单一传染性有机体，估计每年有800万新发结核病病例和200万人死亡。鉴于当前新出现的耐多药结核病（MDR-TB）的背景，结核病治疗正在进入一个新的和具有挑战性的时代，有效控制需要确定新的药物和新的药物靶点。在这个项目中，我们提出了表征结核分枝杆菌的两个酰基辅酶a羧化酶（ACCase）复合物参与霉菌酸的生物合成，细菌细胞包膜中存在的复杂脂质是病原体生存和致病性所必需的。我们期望这些酶复合物的生化、生理和结构表征将帮助我们实现本项目的一个更长远的目标，即鉴定ACCase抑制剂，最终可能开发成新的抗细菌药物。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB) in humans, is responsible for approximately three million deaths worldwide every year and estimated to infect one-third of the world's population. The re-emergence of this disease, caused by the lack of drug compliance, the appearance of multiple-drug-resistant (MDR) strains, and the AIDS epidemic, urge the development of new drugs with activity against MDR strains that could reduce the duration and complexity of current therapies. Acyl-CoA carboxylases (ACCase) have crucial roles in fatty acid metabolism in most living organisms and have been proposed as a good target for the development of a new class of antibacterial agents. In M. tuberculosis, six putative ACCases are predicted from the genome sequence and two of them, ACCase4 and ACCase6, are proposed to be involved in mycolic acid biosynthesis. This complex lipid is one of the main components of the M. tuberculosis cell envelope and has been associated with the survival and pathogenicity of this bacterium. The objective of the proposed research is to characterize ACCases4 and 6 from M. tuberculosis at the genetic, biochemical, and structural levels and understand, by using M. smegmatis as a working model, their physiological role and relevance in mycolic acid biosynthesis and in the survival of these microorganisms. Additionally, we will characterize in vivo and in vitro, putative ACCase inhibitors identified by in silico docking of chemical libraries, and use them to validate these enzyme complexes as target for the future development of antimycobacterial drugs. As a long term goal we will also put emphasis in understanding the molecular bases that determine the substrate specificity of each complex. For this, we will carry on structure based mutagenesis of the carboxyltransferase subunits (2) of the essential complex followed by the biochemical characterization of the mutated enzyme subunits. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis causes more human deaths that any other single infectious organism with an estimated eight million new tuberculosis cases and two million fatalities each year. Given the current backdrop of emerging multi-drug resistant tuberculosis (MDR-TB), TB treatment is entering a new and challenging era where effective control requires the identification of new drugs and novel drug targets. In this project, we propose the characterization of two acyl-CoA carboxylase (ACCase) complexes of M. tuberculosis involved in the biosynthesis of mycolic acids, complex lipids present in the bacterial cell envelope which are essential for the pathogen survival and pathogenicity. We expect that the biochemical, physiological and structural characterization of these enzyme complexes will help us to achieve a more long-term goal of this project, the identification of ACCase inhibitors that could eventually be developed into new antimycobacterial drugs.