Engineering of a functional human NADH-dependent cytochrome P450 system

Olaf Dohr, Mark J. I. Paine, Thomas Friedberg, Gordon C. K. Roberts, C. Roland Wolf

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    75 Citations (Scopus)


    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.
    Original languageEnglish
    Pages (from-to)81-86
    Number of pages6
    JournalProceedings of the National Academy of Sciences USA
    Issue number1
    Publication statusPublished - 2001


    • Monooxygenases
    • NADPH-cytochrome P450 reductase
    • Flavoproteins
    • Nitric oxide synthase
    • Ferredoxin reductase


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