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

结核分枝杆菌(Mtb)是一种引起结核病(TB)的病原菌，目前是世界卫生组织关注的一个主要原因，因为全世界有大量的人受到感染(约占世界人口的三分之一)，而且对现有抗生素具有广泛耐药性的细菌类型(菌株)不断繁殖。这往往是过度使用抗生素的必然后果，这意味着必须不断地生产新的治疗药物。就结核分枝杆菌而言，针对其复杂的脂肪酸代谢途径和细胞壁结构，传统上是非常有效的禁用和杀灭细菌的方法。结核分枝杆菌有一层致密的外层(称为包膜或壁)，其中包含一种复杂的碳水化合物和脂类交联物，为细菌提供了一种强大的屏障，有助于保护它免受免疫系统的攻击，并在感染状态下在人体内生存。了解复杂的包膜是如何构造的，可以掌握对抗结核分枝杆菌的关键，并提供有关新生物化学的有趣新数据。在这方面，最近有研究表明，两种不同类型的药物(苯并噻唑酮和二硝基苯甲酰胺)都通过抑制负责为细胞膜提供一些关键的‘构建块’的酶系统来作用于结核杆菌。这个系统由两种名为DprE1和DprE2的蛋白质组成，它们的作用是将糖链连接的脂类从一种构象转换为另一种构象，使其能够被用作“锚”，通过它可以将分枝杆菌(分枝杆菌)特有的长链脂肪酸连接到包膜的核心。这成为包膜的一个主要结构特征，对细菌保持存活和维持感染状态至关重要。然而，直到最近，DprE1和DprE2蛋白被证明几乎不可能以适合于研究其三维结构和询问其反应机制的可溶形式生产。在这一应用之前，我们已经克服了这些问题，方法是生产从相关细菌(耻垢分枝杆菌)克隆的可溶性DprE1/E2酶，以及通过在基因水平上将这些蛋白融合在一起来产生更稳定、更可溶的Mtb DprE1/E2形式。我们现在能够制造大量相关的蛋白质，在这个拟议的研究计划中，我们将利用这些突破来解决DprE1/DprE2蛋白质的结构(使用X射线结晶学技术，在目标蛋白质晶体的X射线照射下产生一种特定的衍射图案，该图案可以精确定位组成原子的位置并允许建立结构)，并将仔细描述它们的机制及其在产生细胞包膜组装的关键“构建块”中的单独作用。通过这种方式，我们将提供关于对导致结核病的细菌的生存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以新型抗生素针对DprE1/E2系统。

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.