Re-examination of inhibitor resistance conferred by Qo-site mutations in cytochrome b using yeast as a model system.
Cytochrome b from yeast (Saccharomyces cerevisiae Meyer ex Hansen) provides a convenient model system for the study of Qo-site inhibitor (QoI) resistance mutations from a variety of organisms. QoI resistance mutations from fungal plant pathogens (G143A and F129L), malaria agent Plasmodium sp (Y279C/S), and Pneumocystis carinii (L275F), an opportunistic pathogenic fungus of man, were introduced into yeast cytochrome b and their effect on the binding of a variety of natural (myxothiazol and stigmatellin) and synthetic (atovaquone, azoxystrobin and pyraclostrobin) inhibitors to the bc1 complex monitored. L275S (from a myxothiazol-resistant yeast) was also re-examined. Stigmatellin binding was relatively unaffected by the introduction of these mutations. Significant increases in resistance were observed for the strobilurin-class inhibitors myxothiazol, azoxystrobin and pyraclostrobin, with the largest increase in resistance conferred by G143A. In contrast, atovaquone binding was most effected by Y279C/S and L275S. Notably, F129L, G143A and L275S had a minor effect on bc1 activity, and so are unlikely to confer significant fitness penalties in vivo. These data are discussed in the light of the atomic structures for myxothiazol- and azoxystrobin-inhibited bovine bc1 which have recently become available. We propose that QoI resistance due to G143A arises from steric hindrance between the inhibitor and cytochrome b, whereas the mechanism of resistance for the other mutations is due to an increase in binding energy between the protein and inhibitor molecule. Site-directed mutagenesis was also used to model selected regions of the mammalian Qo site in yeast cytochrome b in order to further understand the differential efficacy of these QoI in the mammalian and pathogen bc1 complexes.
Fisher
Nicholas
N
The Wolfson Institute for Biomedical Research, UCL, Gower Street, London, WC1E 6BT, UK.
Meunier
Brigitte
B
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D045303:Cytochromes b / Q000235:genetics* / Q000378:metabolism*; D004351:Drug Resistance / Q000235:genetics*; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D045303:Cytochromes b