Probing the molecular basis of oxygen reduction by the alternative oxidases

Enzymes are proteins that facilitate the reactions that enable living organisms to acquire energy for growth, reproduction and maintenance. A key challenge in understanding the structure-function relationship of one such group of enzymes, the alternative oxidases (AOX), rests upon the identification of its substrate and inhibitor-binding site and its mechanism of action. A detailed knowledge of the nature of this binding site is important since it will reveal whether or not there is a common architecture that can be applied to substrate and inhibitor-binding sites in general and hence provide an insight into the mechanism of binding. More importantly, this knowledge will assist in the suitable rational design of phytopathogenic and anti-parasitic drugs that are specifically targeted to the alternative oxidase. It is now recognized that the distribution of the alternative oxidase is substantially wider than previously thought. No longer restricted to plants, some fungi and protists, the alternative oxidase is also widespread amongst human parasites such as Trypanosoma brucei (the causative agent of African sleeping sickness), intestinal parasites such as Cryptosporidium parvum (responsible for an airborne intestinal infection cryptosporidiosis) and opportunistic human pathogens such as Candida albicans (causes candidiasis or 'thrush'). With respect to the role of AOX in fungi, the development of resistance to agrochemicals by plant fungal pathogens is an international problem that affects all major crops. Indeed fungicide resistance is an important factor in the successful cultivation of cereals in the UK. It is estimated that the UK market for fungicides in cereals is approximately £200m (worldwide $3bn) with winter wheat being the main crop. Fungicides are used against a number of diseases, the major one of winter wheat being caused by Septoria tritici. The main chemical classes of fungicides used to treat UK cereals include the sterol biosynthesis inhibitors. The most important and successful group of these fungicides that have proved effective in the control of plant pathogens are the strobilurin fungicides which are specifically targeted to the mitochondrial respiratory chain (Qo site) thereby inhibiting fungal respiration. Unfortunately resistance to this fungicide often develops resulting in an inability to control fungal pathogens through continued application. Although the mechanism for conferring resistance to Qo fungicides is still controversial there is growing evidence to suggest that the addition of inhibitors, such as azoxystrobin, to fungal pathogens results in a strong induction of the alternative oxidase (AOX). AOX is a mitochondrial terminal oxidase which by-passes the Qo site and is induced in all plants, fungal pathogens and protists following stress induction. We have previously demonstrated that fungal plant pathogens such as Septoria tritici, a fungus that causes major leaf spot diseases in wheat & the wheat "Take-all" fungus, Gaeumannomyces graminis var. tritici have the capacity to express an alternative oxidase when treated with respiratory inhibitors thereby allowing a strobilurin-resistant respiratory pathway to develop which may account for the varying efficacy of strobilurin fungicides. The objectives of the present study are to gain further detailed structural knowledge of the mechanism of oxygen reduction, the nature of the protein-ligand interaction and kinetics through the use of mutants and in the presence and absence of inhibitors. Such information will also be important for further catalytic tuning of the alternative oxidases for future gene therapy strategies and will place us in a very powerful position to undertake future rational inhibitor design which will act as specific and potent phytopathogenic and anti-parasitic drugs specifically targeted at the AOX.

The program of work described in this proposal is an attempt to understand the structural nature and mechanism of oxygen reduction of the alternative oxidase (AOX) the outcome of which will deliver new insights into the mechanism and inhibition of this enzyme, an important consequence for future rational drug design. It builds upon very recent structural data and will address important questions of how the AOX operates and is regulated, how do inhibitors interact with the enzyme, and the nature of the catalytic site. Importantly, it continues our quest to find suitable compounds that not only allow a characterisation of this important but enigmatic protein but will also potentially result in an effective translation of academic research into practical applications. Specifically, the work will probe the mechanism of oxygen reduction by the AOXs through characterisation of recombinant and mutant forms of AOX from Trypansomal brucei (we have recently crystallised this protein). It is centred around five closely integrated and synergistic strands which utilize cutting-edge techniques. The work outlined in this proposal will investigate the mechanism of oxygen reduction to water by the alternative oxidases through spectral characterisation of rAOX mutants which possess mutations that affect either the catalytic cycle or the substrate and inhibitor binding sites through:
Strand 1. Characterisation of a number of site-specific mutant AOXs possessing mutations in the quinol and inhibitor-binding domain(s)
Strand 2. Kinetic characterisation of wt and mutant forms of AOX using polarographic and voltametric techniques.
Strand 3. Spectroscopic investigations using FTIR and UV/Vis techniques to characterise the redox cofactors and the quinol/inhibitor binding site
Strand 4. EPR, ENDOR and ESEEM to characterise the intermediates of the catalytic cycle and the quinol binding sites.
Strand 5 Crystallographic studies in collaboration with Prof. Kita (Tokyo University)

1 Who will benefit from this research?
The alternative oxidase is a respiratory enzyme that decreases the efficiency of mitochondrial energy conservation but which is poorly understood. Our research, which is aimed at improving the structural and mechanistic understanding of the alternative oxidase, will contribute to the elucidation of the molecular basis of the energy metabolism of plants and parasites, clearly a key biological process in both organisms. The research would utilize the expertise in Fourier Transfer Infrared spectroscopy at UCl (P. Rich) and EPR spectroscopy at QMUL (P. Heathcote) and complement pre-existing research programmes on the structure and function of alternative oxidases in Sussex. In addition to researchers in the immediate professional circle carrying out similar research, other wider beneficiaries include:
Within the commercial private sector those in the agrochemical (for treatment of plant fungal pathogens such as DowAgroSciences, Syngenta, BASF etc) and pharmaceutical industries in the development of drugs to treat trypanosomiasis and cryptosporidiosis;
Those working in infectious and tropical disease institutes- in particular those working with intestinal parasites such as Blastocystis and opportunistic human pathogens such as Candida;
Potential policy-makers, within international, national, local or devolved government and government agencies who are actively involved in delivering financial aid in developing countries, such as DfID, for the treatment of the above diseases;
Other beneficiaries within the public sector who might use the results to their advantage include organisations such as the Eden Project (and Kew Gardens) who are keen to get publicly-funded scientists to disseminate the impact of their results to a wider audience. Results from this type of project are of particular interest since information such as this informs the public how research from plants can be used to generate drugs to treat human diseases.
2 How will they benefit from this research?
The fundamental knowledge that is gained by the project could very well find industrial application, since the alternative oxidase has been implicated as a potential target for both phytopathogenic fungicides and certain anti-parasitic pharmaceuticals. The rational design and development of such compounds, particularly dual-mode anti-fungals, will benefit through an enhanced molecular insight of the alternative oxidase structure and catalysis. Furthermore information on the substrate and inhibitor binding sites will prove invaluable in the design of rational inhibitors.
Should the project prove successful and result in the identification of drugs which specifically target the alternative oxidase the research has the real potential to impact on the nation's health, wealth and culture. It has the potential to impact upon the economic competitiveness of the United Kingdom through the development of drugs to treat trypanosomiasis and cryptosporidiosis by pharmaceutical companies and fungicides, by agrochemical companies, to treat plant pathogens which attack economically important cereals thereby increasing the UK's global economic performance. It is estimated that the UK market for fungicides in cereals is approximately £200m (worldwide $10bn) with winter wheat being the main crop.
3 Timescales
The design and development of suitable phytopathogenic fungicides and anti-parasitic pharmaceuticals will take a considerable time (probably >10 yrs) but nevertheless should this prove successful then it will have considerable impact upon the UK's global economic performance since, for instance, the current global annual expenditure on the above fungicides is in excess of $10bn.
4 Staff Development
The type of research and professional skills which staff working on this project will develop includes:the ability to multi-task, work in a team, communication skills both to non-scientists and to non-English speaking scientists