Direct comparison of the ammo acid sequences of mlcrosomal and soluble epoxlde hydrolase superficially mdlcates that these enzymes are unrelated Both protems, however, share slgmficant sequence slmllanty to a bacterial haloalkane dehalogenase that has earlier been shown to belong to the a@ hydrolase fold family of enzymes The catalytic mechanism for the dehalogenase has been elucidated m detail [Verschueren et al (1993) Nature 363, 693-6981 and proceeds via an ester mtermedlate where the substrate IS covalently bound to the enzyme From these observations we conclude (I) that mlcrosomal and soluble epoxlde hydrolase are distantly related enzymes that have evolved from a common ancestral protein together with the haloalkane dehalogenase and a variety of other proteins specified m the present paper, (u) that these enzymes most likely belong to the a//3 hydrolase fold family of enzymes and (III) that the enzymatic epoxlde hydrolysis proceeds via a hydroxy ester mtermedlate, m contrast to the presently favoured base-catalyzed direct attack of the epoxlde by an activated water

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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 kcat of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH.