TY - JOUR
T1 - Engineering of a functional human NADH-dependent cytochrome P450 system
AU - Dohr, Olaf
AU - Paine, Mark J. I.
AU - Friedberg, Thomas
AU - Roberts, Gordon C. K.
AU - Wolf, C. Roland
N1 - dc.publisher: National Academy of Sciences
Senior author of paper demonstrating that the C-terminal Trp-676 of human NADPH cytochrome P450 reductase plays critical role in nicotinamide binding and that cofactor specificity can be profoundly altered by alteration of this single residue while retaining a significant level of catalytic function.
PY - 2001
Y1 - 2001
N2 - 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 kcat of W676A is equivalent (90%) to the NADPH-dependent kcat of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH. The apparent KM NADPH and KM 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.
AB - 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 kcat of W676A is equivalent (90%) to the NADPH-dependent kcat of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH. The apparent KM NADPH and KM 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.
KW - Monooxygenases
KW - NADPH-cytochrome P450 reductase
KW - Flavoproteins
KW - Nitric oxide synthase
KW - Ferredoxin reductase
U2 - 10.1073/pnas.98.1.81
DO - 10.1073/pnas.98.1.81
M3 - Article
C2 - 11136248
SN - 1091-6490
VL - 98
SP - 81
EP - 86
JO - Proceedings of the National Academy of Sciences USA
JF - Proceedings of the National Academy of Sciences USA
IS - 1
ER -