title: Conversion of human steroid 5β-reductase (AKR1D1) into 3β-hydroxysteroid dehydrogenase by single point mutation E120H: example of perfect enzyme engineering.
abstract: Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5β-reduction of Δ(4)-3-ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His(120) (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5β-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5α-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3β-HSD as opposed to a 3α-HSD. The catalytic efficiency achieved for 3β-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5β-dihydrotestosterone, and Δ(4)-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the Δ(4)-double bond and confers 3β-HSD activity on the 5β-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its α-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference.
authors.affiliation: Department of Pharmacology and Center of Excellence in Environmental Toxicology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Mo
authors.initials: M
authors.lastname: Chen
authors.forename: Jason E
authors.initials: JE
authors.lastname: Drury
authors.forename: David W
authors.initials: DW
authors.lastname: Christianson
authors.forename: Trevor M
authors.initials: TM
authors.lastname: Penning
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D006801:Humans; D020125:Mutation, Missense; D009249:NADP / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D009249:NADP; D010088:Oxidoreductases; D015096:3-Hydroxysteroid Dehydrogenases; C021936:3-oxo-5 beta-steroid delta 4-dehydrogenase