Characterisation of the quinone-binding site of the plant alternative oxidase

Lead Research Organisation: University of Sussex
Department Name: Sch of Life Sciences

Abstract

Enzymes are proteins that facilitate the reactions that enable living organisms to acquire energy for growth, reproduction and maintenance. One significant class of enzymes that is present in all plants, some fungi, yeasts and trypansomes (responsible for African sleeping sickness) and more recently in animals are the alternative oxidases. Although the alternative oxidase plays a key role in respiration in all of these organisms its precise structure and function is still uncertain. Apart from it's role in the production of heat in thermogenic plant species, a general physiological role for the oxidase has, however, not been established conclusively. With respect to its structure, although no crystallographic data is currently available the generally adopted view is that the protein is inserted into one leaflet of the mitochondrial inner membrane and that the active-site of the alternative oxidase comprises a non-haem diiron centre. The overall objective of this research programme is to elucidate the molecular nature of the structure, specifically the substrate binding site, of the alternative oxidase. Clearly, such fundamental knowledge is of considerable industrial relevance, as it has the potential to greatly facilitate the rational design of phytopathogenic fungicides and anti-parasitic pharmaceuticals that are targeted at mitochondrial respiration. The structural and mechanistic insights that are gained from the proposed studies will furthermore improve our fundamental understanding of the (mitochondrial) energy metabolism of plants. Given the wasteful effect that alternative oxidase activity has on plant respiration, this enhanced understanding could also have potential future agro-biotechnological implications.

Technical Summary

We aim to investigate the quinone-binding site of the plant alternative oxidase (AOX) expressed in S. pombe and E.coli. We will carry out rational site-directed mutagenesis of six key residues in this potential site that we have identified through accumulated data and bioinformatic searches. Polarographic and voltametric studies will enable us to perform a detailed kinetic analysis of the mutants, which together with the use of quinol-analogue inhibitors will provide information about substrate-binding. Electron paramagnetic resonance (EPR) and Electron Nuclear Double Resonance (ENDOR) spectroscopies will be used to gain structural information on the orientation of substrates and inhibitors in the enzyme binding site. Perturbations introduced by mutagenesis will also allow us to investigate how these residues participate in quinone-binding. Crystallisation screening trials of the purified AOX protein, together with the initiation and development of a purification protocol for recombinant AOX, will be initiated the outcome of which will considerably enhance our understanding of the structure of this enigmatic, but poorly resolved, enzyme.

Publications

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Shiba T (2013) Structure of the trypanosome cyanide-insensitive alternative oxidase. in Proceedings of the National Academy of Sciences of the United States of America

 
Description As outlined in this proposal emerging fungal infectious diseases are now recognised as a worldwide threat to food security and new anti-fungal targets are being actively sought. The research summarised in this proposal aimed to investigate one such target, namely the alternative oxidase, with the research outcomes not only providing new insights into the mechanism and inhibition of this terminal oxidase but also ultimately the research may translate into practical applications through the generation of new drugs/fungicides.
Our research has led to the identification of catalytic intermediates within the AOX redox cycle, which have resulted in the design of less reactive, and potentially safer, compounds which could potentially, for instance, interact with such intermediates and thereby act as suitable lead candidates for further drug design. In particular the research led to the discovery that Colletochlorin B and its derivatives appear to be very promising AOX fungicides (GB patents WO2013160670 and WO2015110820). They can be synthesized by a simple two-step process and are very effective at inhibiting AOX activity. We have widened the patents to include a new family of fungicides which are not only effective on fungal pathogens which attack cereal crops but also those which cause human diseases such as candidiasis and cryptosporidiosis. We have also extended our coverage worldwide through lodging patents in 10 countries outside of the UK and Europe. This has led to a new partnership with Agform Ltd, further grants from BBSRC and the establishment of a new company to commercialise these findings.
Exploitation Route The development of suitable protocols for the expression and purification of recombinant AOX from a wide variety of organisms means we are now in position to design some very potent and novel inhibitors which may prove useful in the treatment of diseases such as trypanosomiasis, candidiasis and microsporidiasis.
Sectors Agriculture, Food and Drink,Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology