Structure of the G225P/G226P mutant of mouse 3(17)α-hydroxysteroid dehydrogenase (AKR1C21) ternary complex: implications for the binding of inhibitor and substrate

3(17)α-Hydroxysteroid dehydrogenase (AKR1C21) is a unique member of the aldo-keto reductase (AKR) superfamily owing to its ability to reduce 17-ketosteroids to 17α-hydroxysteroids, as opposed to other members of the AKR family, which can only produce 17β-hydroxysteroids. In this paper, the crystal structure of a double mutant (G225P/G226P) of AKR1C21 in complex with the coenzyme NADP⁺ and the inhibitor hexoestrol refined at 2.1 Å resolution is presented. Kinetic analysis and molecular-modelling studies of 17α- and 17β-hydroxysteroid substrates in the active site of AKR1C21 suggested that Gly225 and Gly226 play an important role in determining the substrate stereospecificity of the enzyme. Additionally, the G225P/G226P mutation of the enzyme reduced the affinity (K_m) for both 3α- and 17α-hydroxysteroid substrates by up to 160-fold, indicating that these residues are critical for the binding of substrates.