BIOSYNTHETIC MODIFICATIONS OF MYCOLIC ACIDS IN MYCOBACTERIUM TUBERCULOSIS

这项研究的目标是开发新的，更 治疗结核病的有效疗法，包括 耐多药结核病及相关非典型分枝杆菌 感染.这些革兰氏阳性菌的复杂细胞壁 放线菌是它们最具特色的特征， 生物合成的目标是一些最有效的 抗分枝杆菌剂。更好地理解生物化学 细胞壁加工中使用的转换将使我们能够设计 通过合理用药的工具， 设计和组合化学。该项目的重点是 了解脂肪酸合成后的修饰， 发生在包含分枝杆菌最外层的脂质 细胞壁分枝菌酸这些脂质是α-分支的， β-羟基脂肪酸是分枝杆菌所特有的， 被各种官能团大量修饰。修改 分枝菌酸的反应对微生物的正常功能至关重要。 细胞壁作为渗透性屏障和作为介质 分枝杆菌-宿主细胞相互作用。其中的一部分 修饰是致病性分枝杆菌所独有的， 已经表明这些与各种致病性 这些细菌的特性（例如，耐受 氧化爆发）。我们发现一家六口 负责引入这些修饰的酶 并描述了它们的功能令人惊讶的是所有这些酶 似乎利用了一种共同的化学机制， 在甲基加成到烯烃上之后的阳离子形成 霉菌酸盐前体。这种常见的中间体提供了独特的 脆弱的干预目标，因为抑制它会影响六个 酶的同时，使耐药性的出现， 不太可能我们已经克隆、过表达和纯化了 催化这些反应的酶，表征了它们的活性 并开始表征底物、产物和酰基 参与这些反应的载体。这些研究导致了 详细了解分枝菌酸是如何生物合成的， 在修饰反应发生的点， 以前未知的。我们未来的研究将使用这些酶， 相应的体外测定，和精确的结构， 化工产品和基板，以指导设计新颖的 化疗剂。

The goal of this research is to develop novel, more effective therapies for the treatment of tuberculosis, including multidrug-resistant tuberculosis and related atypical mycobacterial infections. The complex cell wall of these gram-positive actinomycetes is their most characteristic feature and its biosynthesis is the target of some of the most effective antimycobacterial agents. A better understanding of the biochemical transformations used in cell wall elaboration will allow us to design novel interventions through the use of the tools of rational drug design and combinatorial chemistry. This project has focused on understanding the post-fatty acid synthetic modifications which occur to lipids comprising the outermost layer of the mycobacterial cell wall, the mycolic acids. These lipids are alpha-branched, beta-hydroxy fatty acids that are unique to mycobacteria which are heavily modified by a variety of functional groups. Modification reactions of mycolic acids are essential to proper functioning of the cell wall as a permeability barrier and as a mediator of mycobacteria-host cell interactions. A certain subset of these modifications are unique to the pathogenic mycobacteria and we have shown that these are associated with various pathogenic properties of these bacteria (for example, the ability to withstand an oxidative burst). We have identified a family of six highly-related enzymes which are responsible for introducing these modifications and characterized their function. Surprisingly all of these enzymes appear to utilize a common chemical mechanism involving initial cation formation following methyl group addition to an olefinic mycolate precursor. This common intermediate offers a uniquely vulnerable target for intervention since inhibiting it would affect six enzymes simultaneously, making the emergence of drug resistance very unlikely. We have cloned, overexpressed, and purified the enzymes which catalyze these reactions, characterized their activity in vitro and begun characterizing the substrate, product, and acyl carrier involved in these reactions. These studies have led to a detailed picture of how mycolic acids are biosynthesized and the point at which the modification reactions take place, both previously unknown. Our future studies will use these enzymes, the corresponding in vitro assays, and the precise structures of the chemical products and substrates to guide the design of novel chemotherapeutic agents.