Interrogation of the catalytic properties of MhuD - a crucial heme oxygenase in Mycobacterium tuberculosis

结核分枝杆菌（Mycobacterium tuberculosis，Mtb）是一种古老的人类病原体，是导致人类死亡的主要传染病之一。世界上大约三分之一的人口感染了结核分枝杆菌，其中许多人不知道这种“休眠”感染将在以后的生活中重新激活，导致结核病（TB）。在第二次世界大战后的几年里，开发了许多新的抗生素，并有效地使用了一系列药物来清除患者的结核病感染-包括利福平，异烟肼和链霉素。然而，近年来已经看到了耐药性在Mtb中的发展的破坏性影响，这导致了严重的问题，例如多药耐药性甚至总耐药性，其中感染对任何主要的Mtb抗生素都没有反应。虽然这激发了最近正在进行临床试验的新药的开发，但情况仍然非常严重，迫切需要新的Mtb靶标和攻击Mtb的策略。最近的研究发现了结核分枝杆菌中一种有吸引力的新酶，最终可能被证明是一种重要的新抗生素靶点。MhuD（Mycobacterium Heme Utilization，Degrader）是一种酶，对Mtb在人类宿主中的生存能力至关重要。在感染状态下，Mtb被人体免疫系统吞噬在位于肺部和其他组织中的称为巨噬细胞的细胞内。结核分枝杆菌已经发展出在巨噬细胞中存活的策略，并且在最终被破坏或成功地脱离巨噬细胞引起进一步感染之前，可以在这种状态下保持长时间的存活。在巨噬细胞内，Mtb能够从宿主细胞中获得营养物质，而MhuD（一种所谓的血红素加氧酶）在结合宿主血红素并将其分解以释放结合在其中心的铁方面起着至关重要的作用-使铁能够用于Mtb细胞内的多种重要功能。该计划中提出的研究目的是对MhuD酶及其降解血红素以释放铁的机制进行详细表征。已经做了初步工作，以了解MhuD的结构特性和它在降解血红素中使用的复杂机制的一部分。重要的是，MhuD的结构及其明显的两步作用机制与人类形式的血红素加氧酶非常不同，因此MhuD成为一种有吸引力的抗生素靶酶。在这个项目中，我们将使用快速反应方法在两个主要反应周期的不同阶段“捕获”MhuD-并确定负责血红素降解的酶的反应形式。这些研究将在溶液中进行，（利用血红素本身的有色性质和当它被酶攻击时所经历的变化，经历裂解并释放血红素铁）和MhuD蛋白质晶体中，其提供了减缓反应的途径，并且其中我们将能够在血红素降解反应开始后的不同时间冷冻反应的MhuD晶体，然后用X射线衍射的方法来探讨血红素断裂过程中发生的结构和化学变化。这些研究将通过使用另一种结构方法- NMR的研究来补充。NMR将用于研究在不存在血红素的情况下以及当与血红素结合时MhuD的动态性质。它被认为是血红素本身的扭曲是至关重要的血红素分解过程中，这些也将通过核磁共振研究。最后，我们将使用含有铁以外金属的血红素类似物，以及血红素的其他模拟物，以抑制反应，并评估这是否可以提供一种最终可用于抗生素治疗的策略。我们的研究将为关键病原体酶的结构和机制及其失活途径提供重要的新见解。

Mycobacterium tuberculosis (Mtb) is an ancient human pathogen that remains as the major cause of human mortality among infectious diseases. Approximately one third of the world's population is infected by Mtb, many of whom are unaware of the "dormant" infection that will reactivate later in life to cause the disease tuberculosis (TB). In the years after the second world war, many new antibiotics were developed, and a series of drugs were used effectively to clear Mtb infections from patients - including rifampicin, isoniazid and streptomycin. However, recent years have seen the devastating effects of development of drug resistance in Mtb, which has led to serious issues such as multidrug resistance and even total drug resistance where the infection is not responsive to any leading Mtb antibiotics. While this has inspired the recent development of new drugs that are currently undergoing clinical trials, the situation remains very serious and new Mtb targets and strategies to attack Mtb are desperately needed. Recent studies have uncovered an attractive new enzyme in Mtb that could ultimately prove an important new antibiotic target. MhuD (Mycobacterial Heme Utilization, Degrader) is an enzyme that is crucial to the viability of Mtb within its human host. In the infective state, Mtb becomes engulfed by the human immune system inside cells called macrophages located in the lungs and often in other tissues. Mtb has developed strategies to survive destruction in the macrophage, and can remain viable for extended periods of time in this state before finally being destroyed, or successfully breaking free of the macrophage to cause further infection. While inside the macrophage, Mtb is able to obtain nutrients from the host cell, and MhuD (a so-called heme oxygenase) plays a crucial role in binding heme from the host and breaking it down to release the iron bound at its centre - enabling the iron to be used for multiple important functions within the Mtb cell. The purpose of the research proposed in this programme is to perform a detailed characterization of the MhuD enzyme and the mechanism by which it degrades heme to liberate the iron. Preliminary work has been done to understand aspects of MhuD's structural properties and parts of the complex mechanism it uses in degrading heme. Importantly, both the structure of MhuD and its apparent two-step mechanism of action are very different from those of the human forms of heme oxygenase - and as such MhuD becomes an attractive antibiotic target enzyme. In this programme we will use fast reaction methods to "trap" the MhuD in different stages of its two major reaction cycles - and identify reactive forms of the enzyme responsible for heme degradation. These studies will be done both in solution (exploiting the coloured nature of the heme itself and the changes that are undergone as it is attacked by the enzyme, undergoes cleavage and releases the heme iron) and in MhuD protein crystals, which provide a route to slowing down the reactions and in which we will be able to freeze reacting MhuD crystals at different times following initiation of the heme degradation reaction, and thereafter use the method of X-ray diffraction to explore the structural and chemical changes occurring during heme breakdown. These studies will be complemented by studies using another structural method - NMR. NMR will be used to study the dynamic nature of MhuD in absence of heme and when bound to heme. It is considered that distortions of the heme itself are crucial to the heme breakdown process, and these will also be investigated by NMR. Finally we will use analogues of heme that contain metals other than iron, as well as other mimics of the heme in order to inhibit the reaction and to evaluate if this can provide a strategy that could ultimately be used in antibiotic therapy. Our studies will provide crucial new insights into structure and mechanism of a crucial pathogen enzyme, and routes to its inactivation.