Characterization of Saccharomyces cerevisiae CYP51 and a CYP51 fusion protein with NADPH cytochrome P-450 oxidoreductase expressed in Escherichia coli.
Saccharomyces cerevisiae CYP51, target of azole antifungal agents, and CYP51 fused with S. cerevisiae cytochrome P-450 oxidoreductase (FUS protein) were expressed in active forms in Escherichia coli by cloning into pET15b. The expression was monitored immunologically, catalytically, and by using reduced carbon monoxide difference and type II binding spectra. CYP51 and FUS enzymes were located in membranes and produced a Soret peak at 448 nm in the reduced CO difference spectrum. The cytochrome P-450 contents in the membrane fractions containing CYP51 and FUS proteins were 12.8 +/- 2.6 and 17.4 +/- 3.7 pmol/mg of protein, respectively. The NADPH cytochrome P-450 oxidoreductase (CPR) content was estimated to be 15.7 +/- 1.1 pmol/mg of protein in FUS membrane fractions. FUS protein catalyzed the demethylation of substrate at the 14alpha position, with a turnover number of 1.96 +/- 0.37 min(-1) in the presence of NADPH. No reductase activity was observed in membrane fractions containing CYP51, and therefore, CYP51 did not function catalytically in the presence of NADPH, but in the presence of an artificial electron donor, cumene hydroperoxide, activity was comparable to that of the FUS enzyme. Further support for a normal structure for the hemoproteins was obtained from type II binding spectra, in which the spectral response was saturated with an equimolar concentration of ketoconazole.
Venkateswarlu
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Department of Molecular Biology and Biotechnology, The University of Sheffield, United Kingdom.
Kelly
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Kelly
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D001393:Azoles / Q000378:metabolism; D015153:Blotting, Western; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology*; D005822:Genetic Vectors; D009251:NADPH-Ferrihemoprotein Reductase / Q000032:analysis / Q000096:biosynthesis*; D010088:Oxidoreductases / Q000032:analysis / Q000096:biosynthesis*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D058886:Sterol 14-Demethylase
D001393:Azoles; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase; D009251:NADPH-Ferrihemoprotein Reductase