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PMID:11485306
Citation |
Lamb, DC, Warrilow, AG, Venkateswarlu, K, Kelly, DE and Kelly, SL (2001) Activities and kinetic mechanisms of native and soluble NADPH-cytochrome P450 reductase. Biochem. Biophys. Res. Commun. 286:48-54 |
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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. |
Links |
PubMed Online version:10.1006/bbrc.2001.5338 |
Keywords |
Cytochrome P-450 Enzyme System/metabolism; Electrophoresis, Polyacrylamide Gel; Kinetics; NADP/metabolism; NADPH-Ferrihemoprotein Reductase/isolation & purification; NADPH-Ferrihemoprotein Reductase/metabolism; Oxidoreductases/metabolism; Saccharomyces cerevisiae/enzymology; Saccharomyces cerevisiae Proteins |
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Significance
Annotations
Gene product | Qualifier | GO ID | GO term name | Evidence Code | with/from | Aspect | Notes | Status |
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