title: Activities and kinetic mechanisms of native and soluble NADPH-cytochrome P450 reductase.
abstract: Native yeast NADPH-cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) and a soluble derivative lacking 33 amino acids of the NH(2)-terminus have been overexpressed as recombinant proteins in Escherichia coli. The presence of a hexahistidine sequence at the N-terminus allowed protein purification in a single step using nickel-chelating affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the predicted molecular weights of the proteins and indicated a purity of >95%. Protein functionality was demonstrated by cytochrome c reduction and reconstitution of CYP61-mediated sterol Delta(22)-desaturation. Steady-state kinetics of cytochrome c reductase activity revealed a random Bi-Bi mechanism with NADPH donating electrons directly to CPR to produce a reduced intermediary form of the enzyme. The kinetic mechanism studies showed no difference between the two yeast CPRs in mechanism or after reconstitution with CYP61-mediated 22-desaturation, confirming that the retention of the NH(2)-terminable membrane anchor is functionally dispensable.
authors.affiliation: Institute of Biological Sciences, University of Wales-Aberystwyth, Aberystwyth SY23 3DA, Wales, United Kingdom.
authors.forename: D C
authors.initials: DC
authors.lastname: Lamb
authors.forename: A G
authors.initials: AG
authors.lastname: Warrilow
authors.forename: K
authors.initials: K
authors.lastname: Venkateswarlu
authors.forename: D E
authors.initials: DE
authors.lastname: Kelly
authors.forename: S L
authors.initials: SL
authors.lastname: Kelly
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C066577:ERG5 protein, S cerevisiae; D009251:NADPH-Ferrihemoprotein Reductase