Modification of the Nucleotide Cofactor-binding Site of Cytochrome P-450 Reductase to Enhance Turnover with NADH in Vivo

Abstract

NADPH-cytochrome P-450 reductase is the electron transfer partner for the cytochromes P-450, heme oxygenase, and squalene monooxygenase and is a component of the nitric-oxide synthases and methionine-synthase reductase. P-450 reductase shows very high selectivity for NADPH and uses NADH only poorly. Substitution of tryptophan 677 with alanine has been shown to yield a 3-fold increase in turnover with NADH, but profound inhibition by NADP ؉ makes the enzyme unsuitable for in vivo applications. In the present study site-directed mutagenesis of amino acids in the 2-phosphate-binding site of the NADPH domain, coupled with the W677A substitution, was used to generate a reductase that was able to use NADH efficiently without inhibition by NADP ؉ . Of 11 single, double, and triple mutant proteins, two (R597M/W677A and R597M/K602W/W677A) showed up to a 500-fold increase in catalytic efficiency (k cat /K m ) with NADH. Inhibition by NADP ؉ was reduced by up to 4 orders of magnitude relative to the W677A protein and was equal to or less than that of the wildtype reductase. Both proteins were 2-3-fold more active than wild-type reductase with NADH in reconstitution assays with cytochrome P-450 1A2 and with squalene monooxygenase. In a recombinant cytochrome P-450 2E1 Ames bacterial mutagenicity assay, the R597M/ W677A protein increased the sensitivity to dimethylnitrosamine by ϳ2-fold, suggesting that the ability to use NADH afforded a significant advantage in this in vivo assay.NADPH-cytochrome P-450 reductase (EC 1.6.2.4) is the electron-donating partner for the cytochromes P-450, squalene monooxygenase, and heme oxygenase and is a component of the nitric-oxide synthases and methionine-synthase reductase. P-450 reductase is essential to cholesterol and steroid synthesis, and its importance is underscored by the developmental abnormalities and embryonic lethality observed in P-450 reductase-null mice (1). P-450 reductase contains 1 mol of FAD/ mol of enzyme and 1 mol of FMN/mol of enzyme. Reduced NADPH transfers two electrons as a hydride ion to the FAD of the enzyme, and FMN then accepts single electrons from the FAD and acts as the exit point to the protein acceptor such as the cytochromes P-450. P-450 reductase is a multi-domain protein, with the FAD and NADP(H) domains homologous with ferredoxin-NADP ϩ reductase and the FMN domain homologous with flavodoxin, suggesting that P-450 reductase was formed from an ancestral gene fusion event (2, 3). A fourth domain acts as a hinge to orient the two flavin-containing domains for electron transfer, and an NH 2 -terminal membrane-binding domain anchors the protein to the cytosolic side of the endoplasmic reticulum. The NADP(H) domain adopts the typical dinucleotide fold structure consisting of alternating ␣-helices and ␤-strands, with the nicotinamide cofactor binding in a cleft between the FAD and NADP(H) domains (4).Although expression of mammalian P-450/reductase systems in bacteria has been shown to be a valuable research tool and has promise as a tool for biodegradation (...

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Involvement of NADP(H) in the Interaction between Heme Oxygenase-1 and Cytochrome P450 Reductase

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A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases

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