title: Engineering steroid 5 beta-reductase activity into rat liver 3 alpha-hydroxysteroid dehydrogenase.
abstract: 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 a glutamic acid replaces His117 in 5 beta-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 5 beta-dihydrotestosterone with kcat = 0.25 min-1 and Km = 19.0 microM and reduced the double bond in progesterone to generate 5 beta-dihydroprogesterone with kcat = 0.97 min-1 and Km = 33.0 microM. 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 5beta-dihydrosteroids to 5 beta, 3 alpha-tetrahydrosteroids with a 600-1000-fold decrease in kcat/Km 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 (pKb = 6.3) for both reactions. In wild-type 3 alpha-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 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.
authors.affiliation: Departments of Biochemistry and Biophysics and of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Jez
authors.forename: T M
authors.initials: TM
authors.lastname: Penning
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000818:Animals; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D015202:Protein Engineering / Q000379:methods; D051381:Rats; D013379:Substrate Specificity / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
chemical_list: D014443:Tyrosine; D010088:Oxidoreductases; D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); C021936:3-oxo-5 beta-steroid delta 4-dehydrogenase