Structure and mechanism of a key enzyme in M. tuberculosis cell envelope biogenesis

结核分枝杆菌细胞包膜生物合成关键酶的结构和机制

基本信息

批准号：
BB/I020160/1
负责人：
Andrew Munro
金额：
$ 49.89万
依托单位：
University of Manchester
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2011
资助国家：
英国
起止时间：
2011 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FI020160%2F1
关键词：
Structure mechanism key enzyme M.

项目摘要

Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that causes tuberculosis (TB) and which is currently a major cause of concern for the World Health Organization, due to the huge numbers of humans infected worldwide (about one third of the world's population) and to the proliferation of types (strains) of the bacterium that are widely resistant to existing antibiotics. This is an often inevitable consequence of the overuse of antibiotics, and means that a continual new pipeline of therapeutic drugs must be produced. In the case of Mtb, targeting its complicated fatty acid metabolism pathways and cell wall structure have traditionally been very effective ways of disabling and killing the bacterium. Mtb has a dense outer layer (termed the envelope or wall) which contains a complex cross-linked mixture of carbohydrates and lipids that provide a formidable barrier around the bacterium and that help to protect it from the immune system, and to survive in the human body in the infective state. Understanding how the complicated envelope is constructed could hold the key to combating Mtb, as well as providing interesting new data on the novel biochemistry involved. In this respect, it was shown recently that two different types of drugs (benzothiazinones and dinitrobenzamides) both act on Mtb by inhibiting an enzyme system that is responsible for providing some of the key 'building blocks' for the cell envelope. This system comprises two proteins named DprE1 and DprE2, whose role is to convert a sugar-linked lipid from one conformation to another, to enable it to be used as an 'anchor' by which peculiar long chain fatty acids unique to the mycobacteria (mycolipids) can be attached to the core of the envelope. This becomes a major structural feature of the envelope that is critical for the bacteria to remain viable and to sustain an infective state. However, until recently the DprE1 and DprE2 proteins had proven almost impossible to produce in a soluble form that would be appropriate for studying their 3-dimensional structure and for interrogating their reaction mechanism. In advance of this application, we have overcome these problems by producing soluble DprE1/E2 enzymes cloned from a related bacterium (Mycobacterium smegmatis) and also by producing more stable, soluble forms of Mtb DprE1/E2 by fusing these proteins together at the genetic level. We are now able to make large amounts of the relevant proteins, and in this proposed programme of research we will exploit these breakthroughs to solve the structures of the DprE1/DprE2 proteins (using the technique of X-ray crystallography, where X-ray irradiation of crystals of the target protein produces a specific diffraction pattern that can pinpoint the locations of the component atoms and allow the structure to be built) and will also perform a careful characterization of their mechanisms and their individual roles in the generation of the key 'building blocks' for cell envelope assembly. In this way, we will provide important new information on an enzyme system crucial for the viability of the TB-causing bacterium, enabling further strategies to target this DprE1/E2 system with novel antibiotics.

结核分枝杆菌（MTB）是一种致病细菌，导致结核病（TB），目前是世界卫生组织引起关注的主要原因，这是由于全球感染了大量的人类（大约三分之一的世界人口），并且是对现有抗抗菌的抗抗菌（大约三分之一）的（大约是世界人口的三分之一）。这通常是过度使用抗生素的必然结果，这意味着必须产生不断的新型治疗药物。在MTB的情况下，靶向其复杂的脂肪酸代谢途径和细胞壁结构，传统上是禁用和杀死细菌的非常有效的方法。 MTB具有密集的外层（称为包膜或壁），其中包含复杂的碳水化合物和脂质的交联混合物，可在细菌周围提供强大的屏障，并有助于保护其免疫系统，并在感染状态的人体中生存。了解如何构建复杂的信封可以持有对抗MTB的关键，并提供有关涉及的新型生物化学的有趣新数据。在这方面，最近表明，两种不同类型的药物（苯并噻嗪酮和二硝基苯甲酰基）都通过抑制负责为细胞包膜提供一些关键的“建筑块”的酶系统来对MTB作用。该系统包含两种名为DPRE1和DPRE2的蛋白质，其作用是将糖连接的脂质从一种构象转换为另一种构象，以使其可以用作“锚”，通过该构型，可以将其特殊的长链脂肪酸与Mycobacteria（Mycolipids）所特有的，可以将其连接到易启发器的核心。这成为信封的主要结构特征，这对于细菌保持生存和维持感染状态至关重要。然而，直到最近，DPRE1和DPRE2蛋白几乎不可能以可溶性形式产生，该形式适合研究其3维结构并询问其反应机制。在此应用之前，我们通过从相关细菌（分枝杆菌）克隆的可溶性DPRE1/E2酶来克服了这些问题，并通过在遗传水平上将这些蛋白质融合在一起，从而产生更稳定的MTB DPRE1/E2的稳定形式。 We are now able to make large amounts of the relevant proteins, and in this proposed programme of research we will exploit these breakthroughs to solve the structures of the DprE1/DprE2 proteins (using the technique of X-ray crystallography, where X-ray irradiation of crystals of the target protein produces a specific diffraction pattern that can pinpoint the locations of the component atoms and allow the structure to be built) and will also perform a careful表征其机制及其在生成细胞包络组件的关键“构建块”中的单个角色。通过这种方式，我们将提供有关酶系统对引起TB的细菌生存能力至关重要的重要新信息，从而有助于进一步的策略，以使用新型的抗生素来靶向该DPRE1/E2系统。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Novel Aryl Substituted Pyrazoles as Small Molecule Inhibitors of Cytochrome P450 CYP121A1: Synthesis and Antimycobacterial Evaluation.

DOI：
10.1021/acs.jmedchem.7b01562
发表时间：
2017-12-28
期刊：
Journal of medicinal chemistry
影响因子：
7.3
作者：
Taban IM;Elshihawy HEAE;Torun B;Zucchini B;Williamson CJ;Altuwairigi D;Ngu AST;McLean KJ;Levy CW;Sood S;Marino LB;Munro AW;de Carvalho LPS;Simons C
通讯作者：
Simons C

Cytochrome P450 - Structure, Mechanism, and Biochemistry

细胞色素 P450 - 结构、机制和生物化学