The mutation T315A in Candida albicans sterol 14alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity.
Sterol 14alpha-demethylase (P45051) is the target for azole antifungal compounds, and resistance to these drugs and agrochemicals is of significant practical importance. We undertook site-directed mutagenesis of the Candida albicans P45051 heterologously expressed in Saccharomyces cerevisiae to probe a model structure for the enzyme. The change T315A reduced enzyme activity 2-fold as predicted for the removal of the residue that formed a hydrogen bond with the 3-OH of the sterol substrate and helped to locate it in the active site. This alteration perturbed the heme environment, causing an altered reduced carbon monoxide difference spectrum with a maximum at 445 nm. The changes also reduced the affinity of the enzyme for the azole antifungals ketoconazole and fluconazole and after expression induced by galactose caused 4-5-fold azole resistance in transformants of S. cerevisiae. This is the first example of a single base change in the target enzyme conferring resistance to azoles through reduced azole affinity.
Lamb
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DC
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, S10 2UH, United Kingdom.
Kelly
D E
DE
Schunck
W H
WH
Shyadehi
A Z
AZ
Akhtar
M
M
Lowe
D J
DJ
Baldwin
B C
BC
Kelly
S L
SL
D000595:Amino Acid Sequence; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D002176:Candida albicans / Q000201:enzymology*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004352:Drug Resistance, Microbial; D004578:Electron Spin Resonance Spectroscopy; D015725:Fluconazole / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D015183:Restriction Mapping; D016415:Sequence Alignment; D058886:Sterol 14-Demethylase
D000935:Antifungal Agents; C548418:CYP51A1 protein, human; D015725:Fluconazole; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase