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Probing the ubiquinol-binding site of recombinant Sauromatum guttatum alternative oxidase expressed in E. coli membranes through site-directed mutagenesis

journal contribution
posted on 2023-06-09, 09:18 authored by Luke Young, Benjamin May, Alice Pendlebury-Watt, Julia Shearman, Kate Elliott, Mary Albury, Tomoo Shiba, Daniel Ken Inaoka, Shigeharu Harada, Kiyoshi Kita, Anthony Moore
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.

History

Publication status

  • Published

File Version

  • Published version

Journal

BBA - Biochimica et Biophysica Acta

ISSN

0006-3002

Publisher

Elsevier

Issue

7

Volume

1837

Page range

1219-1225

Department affiliated with

  • Biochemistry Publications

Full text available

  • No

Peer reviewed?

  • Yes

Legacy Posted Date

2017-12-11

First Compliant Deposit (FCD) Date

2017-12-11

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