Activities and kinetic mechanisms of native and soluble NADPH- cytochrome P450 reductase

K. Venkateswarlu, David Christopher Lamb, Andrew G. S. Warrilow, Diane Elizabeth Kelly, Steven Lewis Kelly

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40 Citations (SciVal)


Native yeast NADPH–cytochrome P450 oxidoreductase (CPR; EC and a soluble derivative lacking 33 amino acids of the NH2-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 Δ22-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 NH2-terminable membrane anchor is functionally dispensable.
Original languageEnglish
Pages (from-to)48-54
Number of pages7
JournalBiochemical and Biophysical Research Communications
Publication statusPublished - 10 Aug 2001


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