Young, Luke, May, Benjamin, Pendlebury-Watt, Alice, Shearman, Julia, Elliott, Kate, Albury, Mary S, Shiba, Tomoo, Inaoka, Daniel Ken, Harada, Shigeharu, Kita, Kiyoshi and Moore, Anthony L (2014) Probing the ubiquinol-binding site of recombinant Sauromatum guttatum alternative oxidase expressed in E. coli membranes through site-directed mutagenesis. BBA - Biochimica et Biophysica Acta, 1837 (7). pp. 1219-1225. ISSN 0006-3002

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Abstract

In the present paper we have investigated the effect of mutagenesis of a number of highly conserved residues
(R159, D163, L177 and L267) which we have recently shown to line the hydrophobic inhibitor/substrate cavity in the alternative oxidases (AOXs). Measurements of respiratory activity in rSgAOX expressed in Escherichia coli FN102 membranes indicate that all mutants result in a decrease in maximum activity of AOX and in some cases (D163 and L177) a decrease in the apparent Km (O2). Of particular importance was the finding that when the L177 and L267 residues, which appear to cause a bottleneck in the hydrophobic cavity, are mutated to alanine the sensitivity to AOX antagonists is reduced. When non-AOX anti-malarial inhibitors were also tested against these mutants widening the bottleneck through removal of isobutyl side chain allowed access of these bulkier inhibitors to the active-site and resulted in inhibition. Results are discussed in terms of how these mutations have altered the way in which the AOX's catalytic cycle is controlled and since maximum activity is decreased
we predict that such mutations result in an increase in the steady state level of at least one O2-derived AOX intermediate. Such mutations should therefore prove to be useful in future stopped-flow and electron paramagnetic
resonance experiments in attempts to understand the catalytic cycle of the alternative oxidase which
may prove to be important in future rational drug design to treat diseases such as trypanosomiasis. Furthermore
since single amino acid mutations in inhibitor/substrate pockets have been found to be the cause of multi-drug
resistant strains of malaria, the decrease in sensitivity to main AOX antagonists observed in the L-mutants studied
in this report suggests that an emergence of drug resistance to trypanosomiasis may also be possible. Therefore we suggest that the design of future AOX inhibitors should have structures that are less reliant on the orientation by the two-leucine residues.

Item Type:	Article
Schools and Departments:	School of Life Sciences > Biochemistry
Depositing User:	Anthony Moore
Date Deposited:	11 Dec 2017 15:17
Last Modified:	02 Jul 2019 17:05
URI:	http://sro.sussex.ac.uk/id/eprint/71980

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