Engineering steroid 5β-reductase activity into rat liver 3α-hydroxysteroid dehydrogenase

Delta(4)-3-Ketosteroid-5 beta-reductase (5 beta-reductase) precedes 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) in steroid hormone metabolism. Both enzymes are members of the aldo-keto reductase (AKR) superfamily and possess catalytic tetrads differing by a single amino acid, In 3 alpha-HSD, the tetrad consists of Tyr55, Lys84, Asp50, and His117, but glutamic acid replaces His117 in SP-reductase, By introducing the H117E point mutation into 3 alpha-HSD, we engineered 5 beta-reductase activity into the dehydrogenase. Homogeneous H117E 3 alpha-HSD reduced the double bond in testosterone to form SP-dihydrotestosterone with k(cat) = 0.25 min(-1) and K-m = 19.0 mu M and reduced the double bond in progesterone to generate 5 beta-dihydroprogesterone with k(cat) = 0.97 min(-1) and K-m = 33.0 mu M. These kinetic parameters were similar to those reported for homogeneous rat liver 5 beta-reductase [Okuda, A., and Okuda, R. (1984) J, Biol. Chem. 259, 7519-7524]. The H117E mutant also reduced 5 beta-dihydrosteroids to 5 beta,3 alpha-tetrahydrosteroids with a 600-1000-fold decrease in k(cat)/K-m versus wild-type 3 alpha-HSD. The ratio of 5 beta-reductase:3 alpha-HSD activity in the H117E mutant was approximately 1:1, Although the H117A mutant reduced Delta(4)-3-ketosteroids, the 3 alpha-HSD activity predominated because the 5 beta-dihydrosteroids were rapidly converted to the 5 beta,3 alpha-tetrahydrosteroids. The pH-rate profiles for carbon-carbon double-bond and ketone reduction catalyzed by the H117E mutant were superimposable, suggesting a common titratable group (pK(b) = 6.3) for both reactions. In wild-type 3 alpha-HSD, the titratable group responsible for 3-ketosteroid reduction has a pK(b) = 6.9 and is assignable to Tyr55. The pH-rate profiles for 3-ketosteroid reduction by the H117A mutant were pH-independent, Our data indicate that Tyr55 functions as a general acid for both 3 alpha-HSD and 5 beta-reductase activities. We suggest that a protonated Glu117 increases the acidity of Tyr55 to promote acid-catalyzed enolization of the Delta(4)-3-ketosteroid substrate. Further, the identity of amino acid 117 determines whether an AKR can function as a 5 beta-reductase by reorienting the substrate relative to the nicotinamide cofactor, This study,provides functional evidence that utilization of modified catalytic residues on an identical protein scaffold is important for evolution of enzymatic activities within the same metabolic pathway.