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)
| Autor: | Steven L. Kelly, D. L. Corina, J. Neville Wright, David C. Lamb, Diane E. Kelly, Akbar Z. Shyadehi, Wolf-Hagen Schunck, Muhammad Akhtar |
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| Rok vydání: | 1996 |
| Předmět: |
Formic acid
Stereochemistry Molecular Sequence Data Cleavage (embryo) Biochemistry Catalysis Mass Spectrometry Sterol 14-Demethylase chemistry.chemical_compound Cytochrome P-450 Enzyme System Microsomes Candida albicans Formate Molecular Biology Bond cleavage DNA Primers Base Sequence biology Lanosterol Cell Biology biology.organism_classification Recombinant Proteins Oxygen chemistry biology.protein Demethylase Oxidoreductases |
| Zdroj: | Journal of Biological Chemistry. 271:12445-12450 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.271.21.12445 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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