Probing the molecular basis of oxygen reduction by the alternative oxidases.

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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.