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

点击翻译按钮获取中文摘要

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.