Engineering steroid hormone specificity into aldo-keto reductases

Steroid hormone transforming aldo-keto reductases (AKRs) include virtually all mammalian 3α-hydroxysteroid dehydrogenases (3α-HSDs), 20α-HSDs, as well as the 5β-reductases. To elucidate the molecular determinants of steroid hormone recognition we used rat liver 3α-HSD (AKR1C9) as a starting structure to engineer either 5β-reductase or 20α-HSD activity. 5β-Reductase activity was introduced by a single point mutation in which the conserved catalytic His (H117) was mutated to Glu117. The H117E mutant had a k_cat comparable to that for homogeneous rat and human liver 5β-reductases. pH versus k_cat profiles show that this mutation increases the acidity of the catalytic general acid Tyr55. It is proposed that the increased TyrOH₂⁺ character facilitates enolization of the Δ⁴-3-ketosteroid and subsequent hydride transfer to C5. Since 5β-reductase precedes 3α-HSD in steroid hormone metabolism it is likely that this metabolic pathway arose by gene duplication and point mutation. 3α-HSD is positional and stereospecific for 3-ketosteroids and inactivates androgens. The enzyme was converted to a robust 20α-HSD, which is positional and stereospecific for 20-ketosteroids and inactivates progesterone, by the generation of loop-chimeras. The shift in log₁₀(k_cat/K_m) from androgens to progestins was of the order of 10¹¹. This represents 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 will be ultimately rationalized.