Activities and kinetic mechanisms of native and soluble NADPH-cytochrome P450 reductase.
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.
Lamb
D C
DC
Institute of Biological Sciences, University of Wales-Aberystwyth, Aberystwyth SY23 3DA, Wales, United Kingdom.
Warrilow
A G
AG
Venkateswarlu
K
K
Kelly
D E
DE
Kelly
S L
SL
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
D029701:Saccharomyces cerevisiae Proteins; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C066577:ERG5 protein, S cerevisiae; D009251:NADPH-Ferrihemoprotein Reductase