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

Hydroxysteroid dehydrogenases (HSDs) regulate the occupancy and activation of steroid hormone receptors by converting potent steroid hormones into the ir cognate inactive metabolites. 3 alpha-HSD catalyzes the inactivation of androgens in the prostate by converting 5 alpha-dihydrotestosterone to 3 al pha-androstanediol, where excess 5 alpha-dihydrotestosterone is implicated in prostate disease. By contrast, 20 alpha-HSD catalyzes the inactivation o f progestins in the ovary and placenta by converting progesterone to 20 alp ha-hydroxyprogesterone, where progesterone is essential for maintaining pre gnancy, Mammalian 3 alpha-HSDs and 20 alpha-HSDs belong to the aldo-keto re ductase superfamily and share 67% amino acid sequence identity yet show pos itional and stereospecificity for the formation of secondary alcohols on op posite ends of steroid hormone substrates. The crystal structure of 3 alpha -HSD indicates that the mature steroid binding pocket consists of 10 residu es located on five loops, including loop A and the mobile loops B and C. 3 alpha-HSD was converted to 20 alpha-HSD by replacing these loops with those found in 20 alpha-HSD. However, when pocket residues in 3 alpha-HSD were m utated to those found in 20 alpha-HSD altered specificity was not achieved. Replacement of loop A created a 17 beta-HSD activity that was absent in ei ther 3 alpha- or 20 alpha-HSD, Once loops A and C were replaced, the chimer a had both 3 alpha- and 20 alpha-HSD activity. When loops A, B, and C were substituted, 3 alpha-HSD was converted to a stereospecific 20 alpha-HSD wit h a resultant shift in k(cat)/K-m for the desired reaction of 2 x 10(11). T his study represents an example where sex hormone specificity can be change d at the enzyme level.