Engineering of a functional human NADH-dependent cytochrome P450 system.
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.
Döhr
O
O
Biomedical Research Center, University of Dundee, and Imperial Cancer Research Fund Molecular Pharmacology Unit, Ninewells Hospital and Medical School, Dundee, DD1 9SY, United Kingdom.
Paine
M J
MJ
Friedberg
T
T
Roberts
G C
GC
Wolf
C R
CR
D019943:Amino Acid Substitution; D001665:Binding Sites; D003574:Cytochrome c Group / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D005292:Ferricyanides / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010078:Oxazines / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D005292:Ferricyanides; D010078:Oxazines; D011994:Recombinant Proteins; D009243:NAD; C007931:hexacyanoferrate III; D009249:NADP; C007649:ethoxyresorufin; D009251:NADPH-Ferrihemoprotein Reductase