TY - JOUR
T1 - Activities and kinetic mechanisms of native and soluble NADPH- cytochrome P450 reductase
AU - Venkateswarlu, K.
AU - Lamb, David Christopher
AU - Warrilow, Andrew G. S.
AU - Kelly, Diane Elizabeth
AU - Kelly, Steven Lewis
N1 - Lamb, D. C., Warrilow, A. G. S., Venkateswarlu, K., Kelly, D. E., Kelly, S. L. (2001). Activities and kinetic mechanisms of native and soluble NADPH- cytochrome P450 reductase. Biochemical and Biophysical Research Communications, 286, (1), 48-54.
Sponsorship: BBSRC
PY - 2001/8/10
Y1 - 2001/8/10
N2 - Native yeast NADPH–cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) 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.
AB - Native yeast NADPH–cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) 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.
U2 - 10.1006/bbrc.2001.5338
DO - 10.1006/bbrc.2001.5338
M3 - Article
SN - 1090-2104
SP - 48
EP - 54
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
ER -