title: Probing the ubiquinol-binding site of recombinant Sauromatum guttatum alternative oxidase expressed in E. coli membranes through site-directed mutagenesis.
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. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.
authors: Biochemistry and Molecular Sciences, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK.
authors: Luke
authors: L
authors: Young
authors: Biochemistry and Molecular Sciences, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK.
authors: Benjamin
authors: B
authors: May
authors: Biochemistry and Molecular Sciences, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK.
authors: Alice
authors: A
authors: Pendlebury-Watt
authors: Biochemistry and Molecular Sciences, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK.
authors: Julia
authors: J
authors: Shearman
authors: Biochemistry and Molecular Sciences, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK.
authors: Catherine
authors: C
authors: Elliott
authors: Biochemistry and Molecular Sciences, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK.
authors: Mary S
authors: MS
authors: Albury
authors: Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto 606-8585, Japan.
authors: Tomoo
authors: T
authors: Shiba
authors: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Daniel Ken
authors: DK
authors: Inaoka
authors: Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto 606-8585, Japan.
authors: Shigeharu
authors: S
authors: Harada
authors: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Kiyoshi
authors: K
authors: Kita
authors: Biochemistry and Molecular Sciences, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK. Electronic address: a.l.moore@sussex.ac.uk.
authors: Anthony L
authors: AL
authors: Moore
mesh_terms: D000595:Amino Acid Sequence; D029064:Araceae / Q000201:enzymology*; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013729:Terpenes / Q000494:pharmacology; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000378:metabolism; D000099188:Alternative Oxidase
chemical_list: D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D011994:Recombinant Proteins; D013729:Terpenes; D014451:Ubiquinone; D000099188:Alternative Oxidase; C056181:colletochlorin B; C003741:ubiquinol
keywords: AOX inhibitors; Alternative oxidase; Escherichia coli membrane; Oxygen affinity; Quinol oxidase; Site-directed mutagenesis; Structure–function relation