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The mutation t315a in candida albicans sterol 14 alpha demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity

Item Type:Article
Title:The mutation t315a in candida albicans sterol 14 alpha demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity
Creators Name:Lamb, D.C. and Kelly, D.E. and Schunck, W.H. and Shyadehi, A.Z. and Akhtar, M. and Lowe, D.J. and Baldwin, B.C. and Kelly, S.L.
Abstract: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.
Keywords:Amino Acid Sequence, Antifungal Agents, Candida Albicans, Cytochrome P-450 Enzyme System, Electron Spin Resonance Spectroscopy, Fluconazole, Kinetics, Microbial Drug Resistance, Molecular Sequence Data, Mutagenesis, Oxidoreductases, Restriction Mapping, Sequence Alignment, Sterol 14-Demethylase, Animals, Rats
Source:Journal of Biological Chemistry
ISSN:0021-9258
Publisher:American Society for Biochemistry and Molecular Biology (U.S.A.)
Volume:272
Number:9
Page Range:5682-5688
Date:28 February 1997
Official Publication:http://www.jbc.org/content/272/9/5682.abstract
PubMed:View item in PubMed

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