Structure, function and mechanism of the alternative oxidases

Young, Luke (2014) Structure, function and mechanism of the alternative oxidases. Doctoral thesis (PhD), University of Sussex.

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Abstract

The alternative oxidase (AOX) is the terminal protein in the alternative oxidation pathway found in plants, fungi and some protozoa. One of the more prominent protozoa that contain AOX within the bloodstream form is Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT), in which the parasite has demonstrated to be totally dependent upon the protein for continued respiration. Given the lack of AOX in mammalian cells, the protein represents an attractive chemotherapeutic target for trypanosidal activity.

Ascofuranone is a known inhibitor of AOX, but its complex synthesis precludes it from industrial production. To this end colletochlorin B, an analogue of ascofuranone, was synthesised and its inhibitory efficacy against AOX examined. IC50 values obtained demonstrate that removal of the problematic furanone ring does not reduce inhibitor efficacy to a large degree. Derivatives of colletochlorin B were synthesised to assess the importance of structural moieties present. The compounds were also tested against the cytochrome bc1 complex, an important respiratory chain complex, and compared with known fungicides. Using these compounds assays against fungal species has yielded promising results for the use of colletochlorin B as a lead fungicide.

Recombinant wild type trypanosomal alternative oxidase (TAO) and Sauromatum guttatum alternative oxidase (SgAOX) have been expressed in E.coli. in addition to a number of mutants. Respiratory activities of these mutants were measured in order to assess the importance of highly conserved amino acids, with all mutants showing a decline in specific activity. Three of the mutants generated were also shown to affect the apparent affinity for oxygen, the implications of which are discussed.

Recent crystallisation of TAO has enabled a more detailed examination of the structure of all AOXs. Work is presented relating structure to the overall function of the protein, taking into account conservation throughout the entire AOX family. Comparisons to other di-iron proteins revealed a conserved His-Asp-Tryp motif that could facilitate proton coupled electron transport. A full catalytic cycle based on these findings has been postulated.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Depositing User: Library Cataloguing
Date Deposited: 16 Jun 2014 05:32
Last Modified: 12 Oct 2015 12:02
URI: http://sro.sussex.ac.uk/id/eprint/48915

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