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

• 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.

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（Mycobacterium tuberculosis，Mtb）是引起结核病（TB）的病原性细菌，并且由于全世界感染的人数巨大（约占世界人口的三分之一）以及对现有抗生素具有广泛抗性的细菌类型（菌株）的增殖，结核分枝杆菌目前是世界卫生组织关注的主要原因。这通常是过度使用抗生素的不可避免的后果，并意味着必须不断生产新的治疗药物。在结核分枝杆菌的情况下，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。结核分枝杆菌有一个致密的外层（称为包膜或壁），其中含有复杂的碳水化合物和脂质的交联混合物，在细菌周围提供了一个强大的屏障，有助于保护它免受免疫系统的影响，并在感染状态下在人体内存活。了解复杂的包膜是如何构建的可能是对抗结核病的关键，并为所涉及的新生物化学提供有趣的新数据。在这方面，最近显示，两种不同类型的药物（苯并噻嗪酮和二硝基苯甲酰胺）都通过抑制负责为细胞包膜提供一些关键“构建块”的酶系统来作用于结核分枝杆菌。该系统包括两种名为DprE 1和DprE 2的蛋白质，其作用是将糖连接的脂质从一种构象转化为另一种构象，以使其能够用作“锚”，通过该“锚”，分枝杆菌特有的长链脂肪酸（肌脂）可以附着到包膜的核心。这成为包膜的主要结构特征，对于细菌保持活力和维持感染状态至关重要。然而，直到最近，DprE 1和DprE 2蛋白质已经被证明几乎不可能以可溶形式产生，这将适合于研究它们的三维结构和询问它们的反应机制。在该应用之前，我们已经通过生产从相关细菌（耻垢分枝杆菌）克隆的可溶性DprE 1/E2酶以及通过在遗传水平上将这些蛋白质融合在一起来生产更稳定的可溶形式的Mtb DprE 1/E2来克服这些问题。我们现在能够制造大量的相关蛋白质，在这项拟议的研究计划中，我们将利用这些突破来解决DprE 1/DprE 2蛋白质的结构（使用X射线晶体学技术，其中X射线照射靶蛋白的晶体产生特定的衍射图案，可以精确定位组成原子的位置并允许构建结构）并且还将对它们的机制以及它们在细胞包膜组装的关键“构建块”的产生中的各自作用进行仔细表征。通过这种方式，我们将提供有关对结核菌生存力至关重要的酶系统的重要新信息，从而使进一步的策略能够用新型抗生素靶向该DprE 1/E2系统。

项目成果

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

• DOI: 10.1007/978-3-319-12108-6_6
• 发表时间: 2015
• 作者: McLean K

Encyclopedia of Biophysics

生物物理学百科全书

• DOI: 10.1007/978-3-642-16712-6_41
• 发表时间: 2013
• 作者: Munro A