Conversion of human steroid 5β-reductase (AKR1D1) into 3β-hydroxysteroid dehydrogenase by single point mutation E120H: example of perfect enzyme engineering.

摘要: Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis steroid hormone metabolism. AKR1D1 catalyzes the 5β-reduction of Δ4-3-ketosteroids, whereas hydroxysteroid dehydrogenases (HSDs). These share high sequence identity catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue conserved AKR catalytic tetrad, His120 (AKR1D1 numbering), is substituted by a glutamate AKR1D1. We find E120H mutant abolishes 5β-reductase activity introduces HSD activity. However, unexpectedly favors dihydrosteroids with 5α-configuration and, unlike most enzymes, shows dominant stereochemical preference act as 3β-HSD opposed 3α-HSD. The efficiency achieved higher than observed any date. High resolution crystal structures complex epiandrosterone, 5β-dihydrotestosterone, Δ4-androstene-3,17-dione elucidated structural basis this functional change. glutamate-histidine substitution prevents 3-ketosteroid penetrating active site so hydride directed toward C3 carbonyl group rather Δ4-double bond confers on 5β-reductase. Structures indicate stereospecificity because flips over present its α-face A-face NADPH. This contrast which can invert stereochemistry when swings across binding pocket. studies show how single point mutation introduce unexpected configurational preference.