Engineering steroid hormone specificity into aldo-keto reductases

Steroid hormone transforming aldo-keto reductases (AKRs) include virtually all mammalian 3 alpha -hydroxysteroid dehydrogenases (3 alpha -HSDs), 20 al pha -HSDs, as well as the 5 beta -reductases. To elucidate the molecular de terminants of steroid hormone recognition we used rat liver 3 alpha -KSD (A KR1C9) as a starting structure to engineer either 5 beta -reductase or 20 a lpha -HSD activity. 5 beta -Reductase activity was introduced by a single p oint mutation in which the conserved catalytic His (H117) was mutated to Gl u117. The H117E mutant had a k(cat) comparable to that for homogeneous rat and human liver 5 beta -reductases. pH Versus k(cat) profiles show that thi s mutation increases the acidity of the catalytic general acid Tyr55. It is proposed that the increased TyrOH(2)(+) character facilitates enolization of the Delta (4)-3-ketosteroid and subsequent hydride transfer to C5. Since 5 beta -reductase precedes 3 alpha -HSD in steroid hormone metabolism it i s likely that this metabolic pathway arose by gene duplication and point mu tation. 3 alpha -HSD is positional and stereospecific for 3-ketosteroids an d inactivates androgens. The enzyme was converted to a robust 20 alpha -HSD , which is positional and stereospecific for 20-ketosteroids and inactivate s progesterone, by the generation of loop-chimeras. The shift in log(10)(k( cat)/K-m) from androgens to progestins was of the order of 10(11). This rep resents a rare example of how steroid hormone specificity can be changed at the enzyme level. Protein engineering with predicted outcomes demonstrates that the molecular determinants of steroid hormone recognition in AKRs wil l be ultimately rationalized. (C) 2001 Elsevier Science Ireland Ltd. All ri ghts reserved.