Graduation Year

2011

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Medicine

Major Professor

Andreas Seyfang, Ph.D.

Co-Major Professor

Robert Deschenes, Ph.D.

Committee Member

Michael J. Barber, D.Phil

Committee Member

Gloria Ferreira, Ph.D.

Committee Member

Alberto van Olphen, Ph.D.

Keywords

Fungal drug resistance, protein biochemistry and structure, ergosterol biosynthesis, yeast knockouts, oxidoreductases

Abstract

The opportunistic fungus Candida albicans is a commensal member of the human microflora and is the most common causative agent of fungal-related disease with particular significance in immunocompromised individuals. Emerging drug resistance is a major problem in Candida, contributed by enzymes involved in the detoxification of xenobiotics and pharmacological agents. One such enzyme, cytochrome b5 reductase (cb5r), has a high pharmacological significance owing to its role in fatty acid elongation, ergosterol (or cholesterol in mammals) biosynthesis, and cytochrome P450-mediated detoxification of xenobiotics.

We have compared the kinetic, biochemical, and pharmacological characteristics of C. albicans cb5r isoforms, Cbr1 and Mcr1, as compared to the mammalian control, rat cb5r. We have observed two key structural differences between the fungal and mammalian proteins that may account for decreased thermal stability and inhibitor specificity of C. albicans Cbr1. Substrate binding affinity and catalytic efficiencies, as well as investigation in the flavin-binding environment, were comparable between the fungal and rat enzymes. In S. cerevisiae, CBR1 and MCR1 knockout strains have been challenged with environmental stressors and subsequently shown to have a role in azole and amphotericin B resistance. Our results of potential protein interactions of C. albicans Cbr1 describe proteins involved in the weak acid stress response, implying a novel role of the protein in pathogenicity. Conclusively, this report describes potential inhibitors of the fungal protein, as well as elaborating upon its important role in ergosterol biosynthesis and possible mechanisms of CYP450-mediated drug detoxification.

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