Engineering of a functional human NADH-dependent cytochrome P450 system

Abstract

A functional human NADH-dependent cytochrome P450 system has been developed by altering the cofactor preference of human NADPH cytochrome P450 reductase (CPR), the redox partner for P450s. This has been achieved by a single amino acid change of the conserved aromatic amino acid Trp-676, which covers the re-side of the FAD isoalloxazine ring in the nicotinamide-binding site. Of the mutations made, the substitution of Trp-676 with alanine (W676A) resulted in a functional NADH-dependent enzyme, which catalyzed the reduction of cytochrome c and ferricyanide as well as facilitated the metabolism of 7-ethoxyresorufin by CYP1A2. Kinetic analysis measuring cytochrome c activity revealed that the NADH-dependent k(cat) of W676A is equivalent (90%) to the NADPH-dependent k(cat) of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH. The apparent K(M)(NADPH) and K(M)(NADH) values of W676A are 80- and 150-fold decreased, respectively. In accordance with structural data, which show a bipartite binding mode of NADPH, substitution of Trp-676 does not affect 2'-AMP binding as seen by the inhibition of both wild-type CPR and the W676A mutant. Furthermore, NADPH was a potent inhibitor of the W676A NADH-dependent cytochrome c reduction and CYP1A2 activity. Overall, the results show that Trp-676 of human CPR plays a major role in cofactor discrimination, and substitution of this conserved aromatic residue with alanine results in an efficient NADH-dependent cytochrome P450 system.

Figure 1

Visible absorption spectra of cytochrome P450 reductase and mutants. Semiquinone spectra of sCPR (A, D), sW676H (B, E) and sW676A (C, F) were obtained after the addition of a 5-fold molar excess of NADPH (A–C) or NADH (D–F) under aerobic conditions, and recorded after equilibration has been reached. The reoxidation of the semiquinone (Insets) was followed at 585 nm for 16 h (solid lines: oxidized spectra; broken lines: reduced spectra).

Figure 2

Inhibition of NADPH- or NADH-dependent cytochrome P450 reductase activities by nucleotide analogues. Cytochrome c reduction was measured as described in the legend to Table 1 by using CPR with NADPH (15 μM, filled symbols) or W676A with NADH (300 μM, open symbols). The concentrations of NADPH and NADH corresponded to the respective apparent K_M values (Table 3). Inhibition studies were performed with 2′-AMP (1 μM–0 mM, squares), NADH (100 μM–10 mM, filled circle) and NADPH (10 nM–100 μM, open circle).

Figure 3

Effect of NADPH on NADH-dependent 7-ethoxyresorufin metabolism. NADPH- and NADH-dependent EROD activities were measured with CYP1A2 (5 pmol) in E. coli membranes coexpressing either CPR or the mutants W676H and W676A, as described in the legend to Table 1. Reactions were started with NADH (500 μM). After 5 min, NADPH (50 μM) was added, and the reaction recorded for additional 5 min (CPR, circle; W676A, squares; W676H, asterix).

Figure 4

Schematic model of the bipartite NADPH-binding mode. The model is based on our data and those of others (, –30). The 2′-phosphate of NADPH is circled.