Engineering Steroid 5β-Reductase Activity into Rat Liver 3α-Hydroxysteroid Dehydrogenase

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Abstract
Δ4-3-Ketosteroid-5β-reductase (5β-reductase) precedes 3α-hydroxysteroid dehydrogenase (3α-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 3α-HSD, the tetrad consists of Tyr55, Lys84, Asp50, and His117, but a glutamic acid replaces His117 in 5β-reductase. By introducing the H117E point mutation into 3α-HSD, we engineered 5β-reductase activity into the dehydrogenase. Homogeneous H117E 3α-HSD reduced the double bond in testosterone to form 5β-dihydrotestosterone with kcat = 0.25 min-1 and Km = 19.0 μM and reduced the double bond in progesterone to generate 5β-dihydroprogesterone with kcat = 0.97 min-1 and Km = 33.0 μM. These kinetic parameters were similar to those reported for homogeneous rat liver 5β-reductase [Okuda, A., and Okuda, R. (1984) J. Biol. Chem. 259, 7519−7524]. The H117E mutant also reduced 5β-dihydrosteroids to 5β,3α-tetrahydrosteroids with a 600−1000-fold decrease in kcat/Km versus wild-type 3α-HSD. The ratio of 5β-reductase:3α-HSD activity in the H117E mutant was approximately 1:1. Although the H117A mutant reduced Δ4-3-ketosteroids, the 3α-HSD activity predominated because the 5β-dihydrosteroids were rapidly converted to the 5β,3α-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 (pKb = 6.3) for both reactions. In wild-type 3α-HSD, the titratable group responsible for 3-ketosteroid reduction has a pKb = 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 3α-HSD and 5β-reductase activities. We suggest that a protonated Glu117 increases the acidity of Tyr55 to promote acid-catalyzed enolization of the Δ4-3-ketosteroid substrate. Further, the identity of amino acid 117 determines whether an AKR can function as a 5β-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.