Engineering of a functional human NADH-dependent cytochrome P450 system

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
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…

nadhwiki has written 69 articles

Leave a Reply