Human 3-alpha hydroxysteroid dehydrogenase type 3 (3α-HSD3): the V54L mutation restricting the steroid alternative...

Human 3-alpha hydroxysteroid dehydrogenase type 3 (3α-HSD3) has an essential role in the inactivation of 5α-dihydrotestosterone (DHT). Notably, human 3α-HSD3 shares 97.8% sequence identity with human 20-alpha hydroxysteroid dehydrogenase (20α-HSD) and there is only one amino acid difference (residue 54) that is located in their steroid binding pockets. However, 20α-HSD displays a distinctive ability in transforming progesterone to 20α-hydroxy-progesterone (20α-OHProg). In this study, to understand the role of residue 54 in the steroid binding and discrimination, the V54L mutation in human 3α-HSD3 has been created. We have solved two crystal structures of the 3α-HSD3·NADP(+)·Progesterone complex and the 3α-HSD3 V54L·NADP(+)·progesterone complex. Interestingly, progesterone adopts two different binding modes to form complexes within the wild type enzyme, with one binding mode similar to the orientation of a bile acid (ursodeoxycholate) in the reported ternary complex of human 3α-HSD3·NADP(+)·ursodeoxycholate and the other binding mode resembling the orientation of 20α-OHProg in the ternary complex of human 20α-HSD·NADP(+)·20α-OHProg. However, the V54L mutation directly restricts the steroid binding modes to a unique one, which resembles the orientation of 20α-OHProg within human 20α-HSD. Furthermore, the kinetic study has been carried out. The results show that the V54L mutation significantly decreases the 3α-HSD activity for the reduction of DHT, while this mutation enhances the 20α-HSD activity to convert progesterone.