THE MECHANISM OF THE ACYL-CARBON BOND-CLEAVAGE REACTION CATALYZED BY RECOMBINANT STEROL 14-ALPHA-DEMETHYLASE OF CANDIDA-ALBICANS (OTHER NAMES ARE LANOSTEROL-14-ALPHA-DEMETHYLASE, P-450(14DM), AND CYP51)

The Candida albicans sterol 14 alpha-demethylase gene (P-450(14DM), CY P51) was transferred to the yeast plasmid YEp51 placing it under the c ontrol of the GAL10 promoter. The resulting construct (YEp51:CYP51) wh en transformed into the yeast strain GRF18 gave a clone producing 1.5 mu mol of P-450/liter of culture, the microsomal fraction of which con tained up to 2.5 nmol of P-450/mg of protein. Two oxygenated precursor s for the 14 alpha-demethylase, 3 beta-hydroxylanost-7-en-32-al and 3 beta-hydroxylanost-7-en-32-ol, variously labeled with H-2 and O-18 at C-32 were synthesized. In this study the conversion of [32-H-2,32-O-16 ]- and [32-H-2,32-O-18]3 beta-hydroxylanost-7-en-32-al with the recomb inant 14 alpha-demethylase was performed under O-16(2) or O-18(2) and the released formic acid analyzed by mass spectrometry. The results sh owed that in the acyl-carbon bond cleavage step (i.e. the deformylatio n process) the original carbonyl oxygen at C-32 of the precursor is re tained in formic acid and the second oxygen of formate is derived from molecular oxygen; precisely the same scenario that has previously bee n observed for the acyl-carbon cleavage steps catalyzed by aromatase ( P-450(arom)) and 17 alpha-hydroxylase-17,20-lyase (P-450(17 alpha),CYP 17). In the light of these results the mechanism of the acyl-carbon bo nd cleavage step catalyzed by the 14 alpha-demethylase is considered.