Modification of the nucleotide cofactor-binding site of cytochrome P-450 reductase to enhance turnover with NADH in Vivo.
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 wild-type 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 approximately 2-fold, suggesting that the ability to use NADH afforded a significant advantage in this in vivo assay.
Elmore
C Lee
CL
Graduate Center for Toxicology, University of Kentucky, Lexington 40536-0305, USA.
Porter
Todd D
TD
D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D009243:NAD; D009251:NADPH-Ferrihemoprotein Reductase