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).
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.
Shyadehi
A Z
AZ
Department of Biochemistry, University of Southampton, United Kingdom.
Lamb
D C
DC
Kelly
S L
SL
Kelly
D E
DE
Schunck
W H
WH
Wright
J N
JN
Corina
D
D
Akhtar
M
M
D001483:Base Sequence; D002176:Candida albicans / Q000201:enzymology*; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers; D013058:Mass Spectrometry; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D010100:Oxygen / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D058886:Sterol 14-Demethylase
D017931:DNA Primers; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase; D010100:Oxygen