Purification, characterization and inhibition of sterol C24-methyltransferase from Candida albicans

Solubilized sterol C24-methyltransferase (24-SMT) was purified to homogeneity from a cell extract of the yeast Candida albicans (Ca) by anion exchange chromatography, gel permeation chromatography and fast performance liquid chromatography using a Mono Q column. The purified enzyme has an apparent molecular mass of 178 kDa on gel permeation chromatography and 43 kDa on SDS/PAGE, indicating that it is composed of four identical subunits. The substrate requirement of the native enzyme has an optimal specificity for zymosterol with associated kinetic constants of K-m 50 mu M and k(cat) of 0.01 s(-1). The product of the enzyme incubated with zymosterol was fecosterol. Inhibition of the catalyst was observed with substrate analogs designed as transition state analogs (25-azalanosterol, K-i = 54 nM and 24 (R,S),25-epiminolanosterol, K-i = 11 nM) or as mechanism-based inactivators (26,27-dehydrozymosterol, K-i 9 mu M) and k(inact) = 0.03 min(-1)) of the C24-methylation reaction. Product analogs ergosterol and fecosterol, but neither cholesterol nor sitosterol, inhibited activity affording K-i values of 20 and 72 mu M, respectively. Ammonium and thia analogs of the intermediates of the sterol C24-methyl reaction sequence were effective growth inhibitors exhibiting IC50 values that ranged from 3 to 20 mu M. (C) 2010 Elsevier Inc. All rights reserved.