The functions of key residues in the inhibitor, substrate and cofactor sites of human 3beta-hydroxysteroid dehydrogenase type 1 are validated by mutagenesis

Abstract

In postmenopausal women, human 3beta-hydroxysteroid dehydrogenase type 1 (3beta-HSD1) is a critical enzyme in the conversion of DHEA to estradiol in breast tumors, while 3beta-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to determine if Arg195 in 3beta-HSD1 vs. Pro195 in 3beta-HSD2 in the substrate/inhibitor binding site is a critical structural difference responsible for the higher affinity of 3beta-HSD1 for inhibitor and substrate steroids compared to 3beta-HSD2 and whether Asp61, Glu192 and Thr8 are fingerprint residues for cofactor and substrate binding using site-directed mutagenesis. The R195P-1 mutant of 3beta-HSD1 and the P195R-2 mutant of 3beta-HSD2 have been created, expressed, purified and characterized kinetically. Dixon analyses of the inhibition of the R195P-1 mutant, P195R-2 mutant, wild-type 3beta-HSD1 and wild-type 3beta-HSD2 by trilostane has produced kinetic profiles that show inhibition of 3beta-HSD1 by trilostane (K(i)=0.10microM, competitive) with a 16-fold lower K(i) and different mode than measured for 3beta-HSD2 (K(i)=1.60microM, noncompetitive). The R195P-1 mutation shifts the high-affinity, competitive inhibition profile of 3beta-HSD1 to a low-affinity (trilostane K(i)=2.56microM), noncompetitive inhibition profile similar to that of 3beta-HSD2 containing Pro195. The P195R-2 mutation shifts the low-affinity, noncompetitive inhibition profile of 3beta-HSD2 to a high-affinity (trilostane K(i)=0.19microM), competitive inhibition profile similar to that of 3beta-HSD1 containing Arg195. Michaelis-Menten kinetics for DHEA, 16beta-hydroxy-DHEA and 16alpha-hydroxy-DHEA substrate utilization by the R195P-1 and P195R-2 enzymes provide further validation for higher affinity binding due to Arg195 in 3beta-HSD1. Comparisons of the Michaelis-Menten values of cofactor and substrate for the targeted mutants of 3beta-HSD1 (D61N, D61V, E192A, T8A) clarify the functions of these residues as well.

Fig. 1

Docking of trilostane with our structural model of human 3β-HSD1. (A) The proposed interaction between the 17β-hydroxyl group of trilostane and the guanidinium group of the Arg195 residue of the wild-type enzyme (4.0 Å) and the apparent proximity of the anchoring hydroxyl group of the Ser124 residue to the 2α-cyanogroup of docked trilostane (3.4 Å) are shown. (B) Docking of trilostane with the R195P mutant of 3β-HSD1 (containing Pro195) shows a binding shift of the inhibitor compared to the orientation of trilostane docked with wild-type 3β-HSD1 containing Arg195 in Panel A. The illustrations of catalytic residues, Tyr154 and Ser124, and cofactor, NAD⁺, indicate that this view represents the active site of the enzyme. The protein backbone (green), carbon (black), oxygen (red) and nitrogen (blue) atoms plus estimated bond distances (magenta) are shown.

Fig. 2

Docking of 16β-hydroxy-DHEA and DHEA with our structural model of human 3β-HSD1. (A) The proposed dual interaction of the guanidinium group of Arg195 with the 17-keto (3.7 Å) and 16β-hydroxyl (3.3 Å) groups of 16β-hydroxy-DHEA is shown. The apparent proximities of the 3β-hydroxyl group of the docked DHEA substrate to the catalytic hydroxyl group of Tyr154 (4.6 Å) and the hydroxyl group of Ser124 (4.5 Å) are shown. The apparent orientation of the catalytic nicotinamide carbon 4 of NAD⁺ (3.8 Å) to the catalytic hydroxyl group of Tyr154 is illustrated. (B) The R-carboxylate group of Glu192 is apparently positioned to interact with 17-keto-group of bound DHEA substrate (2.9 Å) as well as the R-guanidinium group of Arg195 (3.7 Å). The 6-amino group on the adenine of NAD⁺ is apparently oriented near the R-carboxylate group of Asp61 in human 3β-HSD1 (3.1 Å). The apparent bond distance between the nicotinamide C4 of NAD⁺ and the C3 of DHEA (4.2 Å) illustrates the orientation of the enzyme active site. The Thr8 residue in wild-type 3β-HSD1 is also shown. The protein backbone (green), carbon (black), oxygen (red) and nitrogen (blue) atoms plus estimated bond distances (magenta) are shown.

Fig. 3

Western immunoblots and SDS-Polyacrylamide gel electrophoresis of the expressed R195P-1, P195R-2 mutant and wild-type 3β-HSD1 and 3β-HSD2 enzymes. (A) The Sf9 cell homogenate (4.0 μg) containing the R195P-1 or P195R-2 mutant or the purified control wild-type 3β-HSD1 or 3β-HSD2 enzyme (0.05 μg) was separated by SDS-polyacrylamide (7.5%) gel electrophoresis in these western immunoblots. The 42.0 kDa band of the enzyme monomer was detected using anti-3β-HSD antibody as described in the text. (B) SDS-polyacrylamide (7.5%) gel electrophoresis of the purified R195P-1, P195R-2 mutant and wild-type enzymes. Each lane was overloaded with 4.0 μg of purified protein, and the bands were visualized by Coomassie Blue staining.

Fig. 4

Western immunoblots and SDS-Polyacrylamide gel electrophoresis of the expressed T8A, D61N, D61V, E192A mutant and wild-type 3β-HSD1 enzymes. (A) The Sf9 cell homogenate (4.0 μg) containing each mutant enzyme or the purified control wild-type 3β-HSD1 (0.05 μg) was separated by SDS-polyacrylamide (7.5%) gel electrophoresis in these western immunoblots. The 42.0 kDa band of the enzyme monomer was detected using anti-3β-HSD antibody as described in the text. (B) The purified mutant or control wild-type enzyme was separated by SDS-Polyacrylamide (7.5%) gel electrophoresis. Each lane was overloaded with 4.0μg of purified protein, and the bands were visualized by Coomassie Blue staining.