The mechanism of the acyl-carbon bond cleavage reaction catalyzed by recombinant sterol 14α-demethylase of Candida albicans (other names are: Lanosterol 14α-demethylase, P-45014DM, and CYP51)

Abstract

The Candida albicans sterol 14α-demethylase gene (P-45014DM, 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 μumol 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 14α-demethylase, 3β-hydroxylanost-7-en-32-al and 3βhydroxylanost-7-en-32-ol, variously labeled with 2H and 18O at C-32 were synthesized. In this study the conversion of [32-2H,32-18O]- and [32-2H,32-18O]3β-hydroxylanost-7-en-32-al with the recombinant 14α-demethylase was performed under 16O2 or 18O2 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-450arom) and 17α-hydroxylase-17,20-lyase (P-45017α,CYP17). In the light of these results the mechanism of the acyl-carbon bond cleavage step catalyzed by the 14α-demethylase is considered.