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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-45014DM, and CYP51)

Item Type:Article
Title: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-45014DM, and CYP51)
Creators Name:Shyadehi, A.Z. and Lamb, D.C. and Kelly, S.L. and Kelly, D.E. and Schunck, W.H. and Wright, J.N. and Corina, D. and Akhtar, M.
Abstract:The Candida albicans sterol 14 alpha-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 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 2H and 18O at C-32 were synthesized. In this study the conversion of [32-2H,32-16O]- and [32-2H,32-18O]3 beta-hydroxylanost-7-en-32-al with the recombinant 14 alpha-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 17 alpha-hydroxylase-17,20-lyase (P-45017 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.
Keywords:Base Sequence, Candida Albicans, Catalysis, Cytochrome P-450 Enzyme System, DNA Primers, Mass Spectrometry, Microsomes, Molecular Sequence Data, Oxidoreductases, Oxygen, Recombinant Proteins, Sterol 14-Demethylase
Source:Journal of Biological Chemistry
ISSN:0021-9258
Publisher:American Society for Biochemistry and Molecular Biology (U.S.A.)
Volume:271
Number:21
Page Range:12445-12450
Date:24 May 1996
Official Publication:https://doi.org/10.1074/jbc.271.21.12445
PubMed:View item in PubMed

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