| With the exception of the estrogens, all steroid hormones contain a Delta4-3-ketosteroid in their A-ring, e.g., androgens, progestins, gluco- and mineralo-corticoids. An important step in their metabolism is the reduction of the Delta4-ene to produce either 5alpha-dihydro- or 5beta-dihydrosteroids in reactions catalyzed by steroid 5alpha-reductase or 5beta-reductase (AKR1D1), respectively. These human enzymes have been unavailable in purified recombinant form. Structural information on these enzymes is currently lacking and point mutations in both may be responsible for deficiency syndromes.;In order to understand the biological roles of AKR1D1 and perform structural studies, a His tag expression system was used to express and purify milligram quantities of active recombinant AKR1D1. A thorough substrate characterization showed that the enzyme was able to perform 5beta-reduction of bile acid precursors and Delta4-3-ketosteroids of the C18, C19, C21, and C27 series, suggesting a need for only one human enzyme with this activity.;Structural studies reported the first X-ray crystal structure of a mammalian steroid hormone carbon-carbon double bond reductase. The binary complex of AKR1D1·NADP+ was determined at 1.79 A resolution, and the ternary complexes of AKR1D1·NADP+·cortisone at 1.90 A resolution, AKR1D1·NADP+-progesterone at 2.03 A resolution, and AKR1D1·NADP+·testosterone at 1.62 A resolution. The catalytic tetrad mutants E120A, Y58F, and E120H gave a clearer understanding of their role in the catalytic mechanism. Additionally, the structure of the ternary AKR1D1·NADP+·finasteride complex at 1.70 A resolution was reported, providing an explanation for why this inhibitor does not act as a mechanism-based inactivator for AKR1D1.;Finally, the first disease-related mutant of a human steroid double bond reductase, AKR1D1 P133R, was purified in milligram quantities. The mutant was found to have a depressed Km and kcat for bile acid precursors. From these changes in kinetic parameters, it is predicted that bile acid precursors would accumulate and be directed to form hepatotoxic allo-bile acids, which is the clinical presentation observed. This change is unexpected in the P133R mutant since based on structures of AKRs in the PDB this residue is neither located in the catalytic tetrad nor directly associated with either cofactor or substrate binding. |
