Structure of the G225P/G226P mutant of mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) ternary complex: implications for the binding of inhibitor and substrate.
3(17)alpha-Hydroxysteroid dehydrogenase (AKR1C21) is a unique member of the aldo-keto reductase (AKR) superfamily owing to its ability to reduce 17-ketosteroids to 17alpha-hydroxysteroids, as opposed to other members of the AKR family, which can only produce 17beta-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 A resolution is presented. Kinetic analysis and molecular-modelling studies of 17alpha- and 17beta-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 3alpha- and 17alpha-hydroxysteroid substrates by up to 160-fold, indicating that these residues are critical for the binding of substrates.
Dhagat
Urmi
U
Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.
Endo
Satoshi
S
Mamiya
Hiroaki
H
Hara
Akira
A
El-Kabbani
Ossama
O
D019943:Amino Acid Substitution; D000818:Animals; D002384:Catalysis; D018360:Crystallography, X-Ray; D006589:Hexestrol / Q000378:metabolism / Q000494:pharmacology; D006913:Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006914:Hydroxysteroids / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009249:NADP / Q000737:chemistry; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D006914:Hydroxysteroids; D011993:Recombinant Fusion Proteins; D006589:Hexestrol; D009249:NADP; D006913:Hydroxysteroid Dehydrogenases; C035507:3(17)-hydroxysteroid dehydrogenase