Characterization of Saccharomyces cerevisiae CYP51 and a CYP51 fusion protein with NADPH cytochrome P-450 oxidoreductase expressed in Escherichia coli

被引:13
作者
Venkateswarlu, K [1 ]
Kelly, DE [1 ]
Kelly, SL [1 ]
机构
[1] UNIV SHEFFIELD,DEPT MOL BIOL & BIOTECHNOL,KREBS INST BIOMOLEC RES,SHEFFIELD S10 2UH,S YORKSHIRE,ENGLAND
关键词
D O I
10.1128/AAC.41.4.776
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 14 alpha 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.
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页码:776 / 780
页数:5
相关论文
共 41 条
[1]  
[Anonymous], CYTOCHROME P 450
[2]   OCCURRENCE OF A P450 SHOWING HIGH HOMOLOGY TO YEAST LANOSTEROL 14-DEMETHYLASE (P450(14DM)) IN THE RAT-LIVER [J].
AOYAMA, Y ;
FUNAE, Y ;
NOSHIRO, M ;
HORIUCHI, T ;
YOSHIDA, Y .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1994, 201 (03) :1320-1326
[3]  
AOYAMA Y, 1984, J BIOL CHEM, V259, P1661
[4]   EXPRESSION AND ENZYMATIC-ACTIVITY OF RECOMBINANT CYTOCHROME-P450 17-ALPHA-HYDROXYLASE IN ESCHERICHIA-COLI [J].
BARNES, HJ ;
ARLOTTO, MP ;
WATERMAN, MR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (13) :5597-5601
[5]  
DURST F, COMMUNICATION
[6]  
FISCHER CW, 1992, P NATL ACAD SCI USA, V89, P817
[7]  
GONZALEZ FJ, 1995, ANN REV PHARM TOXICO, V35, P36
[8]   PURIFICATION AND PROPERTIES OF CYTOCHROME-P-450-DEPENDENT 14-ALPHA-STEROL DEMETHYLASE FROM CANDIDA-ALBICANS [J].
HITCHCOCK, CA ;
DICKINSON, K ;
BROWN, SB ;
EVANS, EGV ;
ADAMS, DJ .
BIOCHEMICAL JOURNAL, 1989, 263 (02) :573-579
[9]  
JEFCOATE JR, 1979, METHOD ENZYMOL, V52, P258
[10]  
JENKINS CM, 1994, J BIOL CHEM, V269, P27401