The G464S amino acid substitution in Candida albicans sterol 14alpha-demethylase causes fluconazole resistance in the clinic through reduced affinity.
Fluconazole selectively inhibits fungal sterol 14alpha-demethylase, a cytochrome P450 enzyme found in plants, animals, fungi, and Mycobacteria. The mutation G464S, observed in the heme-binding domain of sterol 14alpha-demethylase in clinical strains of fluconazole-resistant Candida albicans, is shown here to cause resistance through substantially reducing the inhibitory effect of fluconazole and is associated with perturbation of the heme environment as indicated by spectral data. The protein exhibits 42% of the maximal enzymatic rate of the wild-type protein allowing continued production of the end product of fungal sterol biosynthesis, ergosterol, in resistant strains. This mutation may cause these phenotypes through altering the heme location, thus changing the ability of residues above the heme to bind the drug effectively. This perturbation would also account for the observation of reduced sterol demethylase catalytic activity by changing the location of the 14alpha-methyl group in relation to oxygen-bound heme during the catalytic cycle.
Kelly
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SL
Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales, SY23 3DA, United Kingdom. Steven.Kelley@aber.ac.uk
Lamb
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Loeffler
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Einsele
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Kelly
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects / Q000201:enzymology*; D002248:Carbon Monoxide / Q000378:metabolism; D002384:Catalysis; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004352:Drug Resistance, Microbial; D004875:Ergosterol / Q000096:biosynthesis; D015725:Fluconazole / Q000378:metabolism / Q000494:pharmacology*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013057:Spectrum Analysis; D058886:Sterol 14-Demethylase
D000935:Antifungal Agents; D065607:Cytochrome P-450 Enzyme Inhibitors; D006418:Heme; D002248:Carbon Monoxide; D015725:Fluconazole; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase; D004875:Ergosterol