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Arginine 276 Controls the Directional Preference of AKR1C9 (Rat Liver 3-Hydroxysteroid Dehydrogenase) in Human Embryonic Kidney 293 Cells

Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8857

Address all correspondence and requests for reprints to Richard J. Auchus, Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8857. E-mail: Richard.Auchus{at}UTSouthwestern.edu.

Rat liver AKR1C9 is the best-studied 3-hydroxysteroid dehydrogenase (3HSD) of the aldo-keto reductase superfamily. The physiologic function of AKR1C9 is to catalyze the reduction of 5-androstane-17ß-ol-3-one (dihydrotestosterone) to 5-androstane-3,17ß-diol (androstanediol) rather than the reverse reaction, and all of the known AKR1C enzymes with 3HSD activity also preferentially catalyze dihydrotestosterone reduction in intact cells. Because the utilization of pyridine-nucleotide cofactors NAD(P)(H) primarily governs the directional preference of HSD enzymes in intact cells, and because R276 participates in NADP(H) binding, we hypothesized that mutation of R276 would alter directional preference in intact cells. To test this model, we constructed stable lines of human embryonic kidney 293 cells expressing wild-type AKR1C9 and mutations R276M, R276G, and R276E. Mutations R276M and R276G retained reductive preference with slightly reduced magnitude compared with wild-type AKR1C9. NADPH depletion by glucose deprivation minimally altered the equilibrium steroid distribution for wild-type AKR1C9 but further reduced the reductive preference of mutations R276M and R276G. Mutation R276E, in contrast, showed an oxidative preference under all conditions. The intrinsic rates of the reductive and oxidative reactions for all four enzymes were similar at the functional equilibrium states. We conclude the R276 maximizes the reductive preference of AKR1C9 in intact cells and maintains this strong preference despite NADPH depletion; mutation R276E reverses the directional preference.

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