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), CYP51) was transferred to the yeast plasmid YEp51 placing it under the control of the GAL10 promoter. The resulting construct (YEp51:CYP51) when 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 contained up to 2.5 nmol of P-450/mg of protein. Two oxygenated precursors 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 recombinant 14 alpha-demethylase was performed under O-16(2) or O-18(2) and the released formic acid analyzed by mass spectrometry. The results showed that in the acyl-carbon bond cleavage step (i.e. the deformylation process) the original carbonyl oxygen at C-32 of the precursor is retained in formic acid and the second oxygen of formate is derived from molecular oxygen; precisely the same scenario that has previously been observed for the acyl-carbon cleavage steps catalyzed by aromatase (P-450(arom)) and 17 alpha-hydroxylase-17,20-lyase (P-450(17 alpha),CYP17). In the light of these results the mechanism of the acyl-carbon bond cleavage step catalyzed by the 14 alpha-demethylase is considered.