Background: Pig aldo-keto reductase family 1 member C1 (AKR1C1) belongs to AKR superfamily which catalyzes the NAD(P)H-dependent reduction of various substrates including steroid hormones. Previously we have reported two paralogous pig AKR1C1s, wild-type AKR1C1 (C-type) and C-terminal-truncated AKR1C1 (T-type). Also, the C-terminal region significantly contributes to the NADPH-dependent reductase activity for 5α-DHT reduction. Molecular modeling studies combined with kinetic experiments were performed to investigate structural and enzymatic differences between wild-type AKR1C1 C-type and T-type.
Results: The results of the enzyme kinetics revealed that Vmax and kcat values of the T-type were 2.9 and 1.6 folds higher than those of the C-type. Moreover, catalytic efficiency was also 1.9 fold higher in T-type compared to C-type. Since x-ray crystal structures of pig AKR1C1 were not available, three dimensional structures of the both types of the protein were predicted using homology modeling methodology and they were used for molecular dynamics simulations. The structural comparisons between C-type and T-type showed that 5α-DHT formed strong hydrogen bonds with catalytic residues such as Tyr55 and His117 in T-type. In particular, C3 ketone group of the substrate was close to Tyr55 and NADPH in T-type.
Conclusions: Our results showed that 5α-DHT binding in T-type was more favorable for catalytic reaction to facilitate hydride transfer from the cofactor, and were consistent with experimental results. We believe that our study provides valuable information to understand important role of C-terminal region that affects enzymatic properties for 5α-DHT, and further molecular mechanism for the enzyme kinetics of AKR1C1 proteins.