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Mechanisms of Resistance

The R467K Amino Acid Substitution in Candida albicans Sterol 14α-Demethylase Causes Drug Resistance through Reduced Affinity

David C. Lamb, Diane E. Kelly, Theodore C. White, Steven L. Kelly
David C. Lamb
Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth SY23 3DA, United Kingdom, and
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Diane E. Kelly
Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth SY23 3DA, United Kingdom, and
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Theodore C. White
Department of Pathobiology, School of Public Health and Community Medicine, University of Washington and Seattle Biomedical Research Institute, Seattle, Washington 98109
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Steven L. Kelly
Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth SY23 3DA, United Kingdom, and
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DOI: 10.1128/AAC.44.1.63-67.2000
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    Fig. 1.

    Schematic representation of the strategy used for the generation of CYP51(R467K). Site-directed PCR mutagenesis was performed to change the triplet at position 467 from AGA to GGA as described in Materials and Methods. The NsiI-HindIII mutant fragment was ligated into the corresponding region of the wild-type gene in the yeast expression vector YEp51 allowing expression from the GAL10 promoter.

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    Fig. 2.

    Reduced-carbon monoxide difference spectrum of microsomal cytochrome CYP51(R467K). Shown are the reduced-carbon monoxide difference spectra of microsomal fractions, prepared as described in Materials and Methods, after induction of heterologous expression in yeast transformants containing YEp51 alone (— • —), CYP51 (——), and CYP51 (R467K) (–––).

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    Fig. 3.

    Comparative analysis of type II binding spectra. The magnitudes of spectra obtained, as described in Materials and Methods, with different concentrations of fluconazole bound to 0.2 nmol of CYP51 (□) or CYP51(R467K) (▵) are shown. Data were reproducible in triplicate experiments.

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    Fig. 4.

    Inhibition of sterol 14α-demethylase activity. Comparative plot of sterol demethylase activity in the presence of various amounts of fluconazole for CYP51 and CYP51(R467K). P450 was used to examine the inhibitory effects of fluconazole for CYP51 (□) and CYP51(R467K) (○).

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    Fig. 5.

    Amino acid sequences of P450s in the heme-binding region. Shown are an alignment of CYP51 amino acid sequences from humans, rats, S. cerevisiae (S.C.), Sorghum bicolor (S.B.), Mycobacterium tuberculosis (M.T.), andC. albicans (C.A.) and a comparison to the same region in CYP51(R467K) and various mammalian CYP isoforms involved in foreign compound metabolism. The conserved arginine and lysine residues are in boldface.

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The R467K Amino Acid Substitution in Candida albicans Sterol 14α-Demethylase Causes Drug Resistance through Reduced Affinity
David C. Lamb, Diane E. Kelly, Theodore C. White, Steven L. Kelly
Antimicrobial Agents and Chemotherapy Jan 2000, 44 (1) 63-67; DOI: 10.1128/AAC.44.1.63-67.2000

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The R467K Amino Acid Substitution in Candida albicans Sterol 14α-Demethylase Causes Drug Resistance through Reduced Affinity
David C. Lamb, Diane E. Kelly, Theodore C. White, Steven L. Kelly
Antimicrobial Agents and Chemotherapy Jan 2000, 44 (1) 63-67; DOI: 10.1128/AAC.44.1.63-67.2000
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KEYWORDS

antifungal agents
Candida albicans
Cytochrome P-450 Enzyme System
fluconazole
Oxidoreductases

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