Conversion of mammalian 3α-hydroxysteroid dehydrogenase to 20α-hydroxysteroid dehydrogenase using loop chimeras: Changing specificity from androgens to progestins

Abstract

Hydroxysteroid dehydrogenases (HSDs) regulate the occupancy and activation of steroid hormone receptors by converting potent steroid hormones into their cognate inactive metabolites. 3α-HSD catalyzes the inactivation of androgens in the prostate by converting 5α-dihydrotestosterone to 3α-androstanediol, where excess 5α-dihydrotestosterone is implicated in prostate disease. By contrast, 20α-HSD catalyzes the inactivation of progestins in the ovary and placenta by converting progesterone to 20α-hydroxyprogesterone, where progesterone is essential for maintaining pregnancy. Mammalian 3α-HSDs and 20α-HSDs belong to the aldo-keto reductase superfamily and share 67% amino acid sequence identity yet show positional and stereospecificity for the formation of secondary alcohols on opposite ends of steroid hormone substrates. The crystal structure of 3α-HSD indicates that the mature steroid binding pocket consists of 10 residues located on five loops, including loop A and the mobile loops B and C. 3α-HSD was converted to 20α-HSD by replacing these loops with those found in 20α-HSD. However, when pocket residues in 3α-HSD were mutated to those found in 20α-HSD altered specificity was not achieved. Replacement of loop A created a 17β-HSD activity that was absent in either 3α-or 20α-HSD. Once loops A and C were replaced, the chimera had both 3α- and 20α-HSD activity. When loops A, B, and C were substituted, 3α-HSD was converted to a stereospecific 20α-HSD with a resultant shift in kcat/Km for the desired reaction of 2 x 1011. This study represents an example where sex hormone specificity can be changed at the enzyme level.