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

结核分枝杆菌(Mtb)是一种古老的人类病原体，在各种传染病中仍是人类死亡的主要原因。世界上大约三分之一的人口感染了结核分枝杆菌，其中许多人没有意识到这种“潜伏”的感染会在晚年重新激活，从而导致结核病(TB)。在第二次世界大战后的几年里，许多新的抗生素被开发出来，一系列药物被有效地用于清除患者的结核分枝杆菌感染-包括利福平、异烟肼和链霉素。然而，近些年来，结核分枝杆菌耐药性的发展带来了毁灭性的影响，这导致了严重的问题，如多药耐药，甚至对任何主要的结核分枝杆菌抗生素都不敏感的感染。虽然这激励了目前正在进行临床试验的新药的最近开发，但情况仍然非常严重，迫切需要新的结核分枝杆菌目标和战略来攻击结核分枝杆菌。最近的研究发现了Mtb中一种有吸引力的新酶，它最终可能被证明是一个重要的新抗生素靶点。MhuD(分枝杆菌血红素利用，降解剂)是一种对结核分枝杆菌在人类宿主内的生存至关重要的酶。在感染状态下，结核分枝杆菌会被人类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。结核分枝杆菌已经制定了在巨噬细胞的破坏中存活下来的策略，并可以在这种状态下保持较长时间的存活，直到最终被摧毁，或成功地脱离巨噬细胞而引起进一步感染。在巨噬细胞内，结核分枝杆菌能够从宿主细胞获得营养，而MhuD(一种所谓的血红素加氧酶)在从宿主结合血红素并分解它以释放结合在其中心的铁-使铁能够用于结核分枝杆菌细胞内的多种重要功能方面发挥着关键作用。本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的表征。已经进行了初步工作，以了解MhuD的结构特性和它在降解血红素时使用的部分复杂机制。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有很大的不同-因此MhuD成为一种有吸引力的抗生素靶标酶。在这个项目中，我们将使用快速反应方法在MhuD的两个主要反应周期的不同阶段“捕获”MhuD-并确定导致血红素降解的酶的反应形式。这些研究将在溶液中(利用血红素本身的着色性质以及它被酶攻击、经历切割和释放血红素铁时所发生的变化)和MhuD蛋白质晶体中进行，后者提供了一种减缓反应的途径，在其中我们将能够在血红素降解反应开始后的不同时间冻结反应MhuD晶体，然后使用X射线衍射的方法来探索在血红素分解过程中发生的结构和化学变化。这些研究将得到使用另一种结构方法--核磁共振的研究的补充。核磁共振将被用来研究MhuD在没有亚铁血红素和结合亚铁血红素时的动态性质。人们认为，血红素本身的扭曲对血红素的分解过程是至关重要的，这些也将被核磁共振研究。最后，我们将使用含有铁以外金属的血红素类似物，以及其他血红素模拟物，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。我们的研究将为一种关键病原体酶的结构和机制提供重要的新见解，并使其失活。

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.