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

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.

The Mycobacterium tuberculosis (Mtb) MhuD enzyme is a heme oxygenase (HOX) enzyme essential for degradation of host-derived heme and for the extraction of iron for utilization in the Mtb cell. Heme is derived from the host by a secreted Mtb protein that passes heme though Mtb membrane proteins and on to MhuD. MhuD is structurally distinct from human HOXs and its catalytic mechanism is different to those of the canonical HOXs. Recent studies have indicated that MhuD may catalyze consecutive monooxygenase and dioxygenase reactions in the same active site, and MhuD has also been structurally characterized in both monoheme- and diheme-bound forms, where the latter is considered an inactive form that acts to store heme until required by Mtb. The aim of this programme is to undertake a detailed characterization of the catalytic properties of MhuD, including the application of NMR to analyse the dynamic nature of MhuD and its interactions with the heme substrate. The ruffled nature of the bound monoheme is considered important for the activation and hydroxylation of the heme in the first catalytic step. Fast reaction kinetics approaches (using UV-vis and EPR methods) will also be used in work to trap and provide evidence for formation of reactive iron-oxo and other intermediate species in the mono- and di-oxygenase reaction cycles, as well as being applied to analysis of rate constants for heme binding/dissociation and heme oxidation. Kinetic crystallography techniques will also be used to initiate reactions in MhuD crystals and to capture intermediate catalytic species. X-ray crystallography will also be used to determine MhuD structures in the heme-free state, as well as in complex with metal-substituted hemes and related tetrapyrroles, in order to identify novel substrates and potential inhibitor leads. Collectively, these data will give new insights into mechanism of an enzyme crucial to Mtb viability in the host cell, and will provide strategies for its inhibition.

This proposed research is focused on the enzyme MhuD from Mycobacterium tuberculosis (Mtb). MhuD is among a growing number of heme oxygenase (HOX) enzymes found in bacterial pathogens, and catalyzes oxidative degradation of bound heme by cleavage of the tetrapyrrole ring with release of the iron atom. This allows Mtb to assimilate iron from the host cell while engulfed in the macrophage. The MhuD structure is very different to that of mammalian HOX enzymes, and the catalytic mechanism is also distinct. These factors are important in considering MhuD as a potential new target for Mtb antibiotic development. The fact that its structural and mechanistic features do not resemble those of the host enzyme; that active site structure in the active form of MhuD reveals a distinct pocket for positioning an inhibitor; and that MhuD is crucial for iron assimilation all point to an excellent target for drug development. Development of a novel class of inhibitor that can bind and inactivate the MhuD HOX enzyme would have significant impact and would be focused on a completely novel Mtb enzyme and process; whereas many traditionally used Mtb antibiotics (for which antibiotic resistance is widespread) and some of the newer drugs in development target aspects of Mtb cell wall biosynthesis. The major aims of this project are to use a combination of structural (NMR and X-ray crystallography) and fast reaction kinetic methods to understand the mechanism of MhuD, to trap catalytically relevant intermediates (in solution by stopped-flow methods; through rapid freeze-quenching of reactions for EPR analysis; and in crystals following initiation of reactions in crystallo), and to interrogate dynamics of MhuD in its apo (heme-free) state and when in complex with either one (the catalytically competent form) or two hemes (the latter being an inactive, heme storage state). Analyses of MhuD binding to various metal-substituted hemes, other tetrapyrroles and heme iron-coordinating molecules will also be done to find novel substrates and inhibitors of MhuD, and potential probes of the reaction mechanism. Thus, the project will define structure and mechanistic details of a pathogen enzyme central to Mtb iron assimilation, as well as exploring its active site structure and making inroads into the identification of compounds that could provide the basis for antibiotic leads.
Beneficiaries from the research include the numerous academic and industrial researchers involved in characterization of Mtb redox and other enzymes, and who are striving to identify new drug target enzymes. The HOX enzymes in Mtb and other bacterial pathogens are viable new targets, and active site space in MhuD provides an obvious target for inhibitor development. Development of an effective MhuD inhibitor is a long term aim of our studies, and such an antibiotic could have major implications for the treatment of Mtb. Similar applications are possible for drugs targeting HOX enzymes in other bacterial pathogens - e.g. the Staphylococcus aureus IsdG/H enzymes, which cleave heme at a different position. Our work to exploit kinetic crystallography as a route to identifying intermediates in the MhuD catalytic process should also lead to other enzymology and structural biology groups using similar strategies in efforts to rationalise mechanisms in both cofactor-binding and other enzymes. Research staff working on this project will receive training in areas including protein expression, structural biology (NMR and crystallography), fast reaction methods and biophysical/spectroscopic applications. They will also obtain diverse training in other areas (including computational skills) that will be of use for their future employment in areas including Pharma (e.g. antibiotic development) and academic research (e.g. in enzyme structure and mechanism), and will further benefit from the overlapping skills sets of the PI and CoI's and their groups in enzymology and structural biology.