CYP51 catalyzes the 3-step 14?-demethylation reaction that is essential in sterol biosynthesis, being required for conversion of lanosterol to cholesterol (animals), 24-methylenedihydrolanosterol to ergosterol (fungi/yeast) and obtusifoliol to phytosterols (plants). It is the most widely distributed member of the cytochrome P450 superfamily, also being found in lower eukaryotes and some bacteria. We have determined the first high resolution structure of a CYP51, the soluble form from Mycobacterium tuberculosis (MT). The goal of this grant application is to take advantage of this structural information and the relatively large number of known CYP51 sequences to provide a detailed understanding of CYP51 structure/function. This is a revision of a new application.
Aim 1 - Alignment of the more than 50 CYP51 sequences from different phyla shows about 40 amino acids completely or very highly conserved. We propose the conserved residues to be the minimal structural requirement for 14?-demethylases. Site-directed mutagenesis of MT and human CYP51 will permit analysis of the role of each of these residues. Levels of bacterial expression, spectral analysis of substrate binding and catalytic activity will be studied in each mutant. X-ray structure, fluorescence and circular dichroism analysis of substrate-induced conformational changes, and stopped flow analysis of substrate binding will also provide biophysical detail of selected mutations.
Aim 2 - CYP51 from plants metabolizes only a single substrate (obtusifoliol--the biological CYP51 substrate having just a single methyl group at C4) while forms from other phyla metabolize multiple sterols. Site-directed mutagenesis of a second set of residues conserved strictly in plants will be used to determine the structural basis of plant CYP51 substrate specificity, relying on methods cited above.
Aim 3 - Treatment of pathogenic Candida albicans infections with azole CYP51 inhibitors leads to mutant forms of CYP51 in some drug-resistant Candida strains. These mutant forms bind azole inhibitors less well while binding substrate normally, both in the single CYP51 active site. Investigation of biophysical properties of these mutations in MTCYP51 will provide an explanation for CYP51 drug resistance in pathogens. Together these aims will provide new and extensive insight into structure/function of CYP51, the first opportunity for such detailed analysis of a widely distributed and essential CYP family. In addition we expect that these studies will lead to general information about P450 structure/function and provide useful insight into drug design for treatment of pathogenic infections. ? ?
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