Important role of the C-terminal region of pig aldo-keto reductase family 1 member C1 in the NADPH-dependent reduction of steroid hormones
The NADPH-dependent reduction activities of two paralogous pig AKR1C1s with and without 19 additional amino acid residues in C-terminus were evaluated against steroid hormones including 5alpha-dihydrotestosterone, testosterone, progesterone, androstenedione and 5alpha-androstane-3.17-dione, which act as substrates of the AKR1C1S. Among the hormones, the AKR1C1s exhibited the highest activity against 5alpha-dihydrotestosterone and the lowest activity against testosterone and progesterone. Furthermore, the AKR1C1s showed the largest differential activities against; 5alpha-dihydrotestosterone, but no such change of activities was found against progestrone and testosterone. These results suggest that the C-terminal region of AKR1C1 plays an important effect in the reduction activities of pig AKR1C1. Thus, the differential activities of two AKR1C1 paralogs observed in the present study provide important insights in understanding the molecular evolution.
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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 5¿-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.ResultsThe results of the enzyme kinetics revealed that V max and k cat 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 5¿-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 5¿-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 5¿-DHT, and further molecular mechanism for the enzyme kinetics of AKR1C1 proteins.
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