Serine 124 completes the Tyr, Lys and Ser triad responsible for the catalysis of human type 1 3beta-hydroxysteroid dehydrogenase

Abstract

Human 3 -hydroxysteroid dehydrogenase/isomerase (3 -HSD) is a key steroidogenic enzyme that catalyzes the first step in the conversion of circulating dehydroepiandrosterone (DHEA), pregnenolone or 17 -hydroxypregenolone to produce the appropriate, active steroid hormone(s): estradiol, testosterone, progesterone, aldosterone or cortisol respectively. Our mutagenesis studies have identified Tyr154 and Lys158 as catalytic residues for the 3 -HSD reaction. Our three-dimensional homology model of 3 -HSD shows that Tyr154 and Lys158 are oriented near the 3 -hydroxyl group of the bound substrate steroid, and predicts that Ser123 or Ser124 completes a Tyr-Lys-Ser catalytic triad that operates in many other dehydrogenases. The S123A and S124A mutants of human type 1 3 -hydroxysteroid dehydrogenase/ isomerase (3 -HSD1) were created by PCR-based mutagenesis, expressed in insect cells using baculovirus and purified to homogeneity. The S124A mutant exhibits no 3 -HSD activity and has a K m value (83·6 µM) for the isomerase substrate that is threefold greater than that of wild-type 1 isomerase. In contrast, S123A has substantial 3 -HSD activity (DHEA K m =11·2 µM; k cat =0·8 min -1 ) and utilizes isomerase substrate, 5-androstene-3,17-dione, with a K m value (27·6 µM) that is almost identical to wild-type. The K m value (4·3 µM) of S124A for NADH as an allosteric activator of isomerase is similar to that of the wild-type 1 enzyme, indicating that Ser124 is not involved in cofactor binding. S123A utilizes NAD as a cofactor for 3 -HSD and NADH as the activator for isomerase with K m values that are similar to wild-type. The 3 -HSD activities of S123A and wild-type 3 -HSD increase by 2·7-fold when the pH is raised from 7·4 to the optimal pH 9·7, but S124A exhibits very low residual 3 -HSD activity that is pH-independent.These kinetic analyses strongly suggest that the Ser124 residue completes the catalytic triad for the 3 -HSD activity. Since there are 29 Ser residues in the primary structure of human 3 -HSD1, our homology model of the catalytic domain has been validated by this accurate prediction. A role for Ser124 in the binding of the isomerase substrate, which is the 3 -HSD product-steroid of the bifunctional enzyme protein, is also suggested. These observations further characterize the structure/function relationships of human 3 -HSD and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta to control the timing of labor or in hormone-sensitive breast tumors to slow their growth.

Structure/function of the inhibition of human 3β-hydroxysteroid dehydrogenase type 1 and type 2 by trilostane

“…However, the K m values for the coenzymes of 3β-HSD1 and isomerase measured for the S124T mutant are very similar to those for wild-type 3β-HSD1 (Table 3). These data with S124T support a critical role for Ser124 as a key residue that interacts with the 3-oxo group of the substrate steroids and are consistent with our previous report in which the S124A mutation abolished 3β-HSD activity [7]. Although Thr124 of the S124T mutant can function as a substrate recognition residue for human 3β-HSD1, the presence of the methyl group on Thr124 may sterically hinder the interaction of the amino acid hydroxyl group with the 3-oxo group of the substrate to increase the substrate K m value as well as hinder interaction with the 2α-cyano group of trilostane, as discussed above.…”

“…Site-directed mutagenesis has confirmed the function of the catalytic residues and key substrate and cofactor binding residues as predicted by the structural model [5,7,8,18,19]. In the current study, trilostane was docked in the active site of our structural model of human 3β-HSD1 using Autodock 3.0.…”

“…1A, the 2α-cyano group of trilostane is positioned to interact with the hydroxyl group of Ser124 (3.4 Å) wild-type 3β-HSD1. Ser124 was previously identified as a key recognition residue of the 3-oxo group of steroid substrate by human 3β-HSD [7]. To test the prediction that Ser124 also functions as a key recognition residue for inhibition by trilostane, the S124T mutant of 3β-HSD1 was created, expressed and purified.…”

“…Docking studies of trilostane with our structural model of human 3β-HSD1 predicts that the 2α-cyano side chain of trilostane (2α-cyano-4α,5α-epoxy-17β-ol-androstane-3-one) may interact with the Ser124 residue of 3β-HSD1. Because Ser124 was reported to be a critical residue for substrate binding in human 3β-HSD1 [7], the S124T mutation of 3β-HSD has been produced, expressed and purified to determine how steric hindrance by the methyl group of Thr124 affects the kinetics of 3β-HSD inhibition by trilostane. In addition, an analog of trilostane that lacks the 2α-cyano group, 4α,5α-epoxy-testosterone, has been synthesized, docking of this analog with 3β-HSD1 has been performed and the inhibition kinetics of 4α,5α-epoxy-testosterone have been compared to those of trilostane.…”

Homology Modeling and Site-Directed Mutagenesis Reveal Catalytic Key Amino Acids of 3β-Hydroxysteroid-Dehydrogenase/C4-Decarboxylase from Arabidopsis

“…Ser-131 is a homolog to the essential Ser-124 of the mammalian 3bHSD (Thomas et al, 2004) and Ser-138 is a homolog of 3b,17bHSD (Filling et al, 2002) 2003; Wu et al, 2007). The residual activity of the S131A mutation, which led to a complete loss of activity in several other SDRs, suggests that formation of the Arg-326-substrate carboxylate salt bridge can fulfill the stabilization of the substrate docking in the case of 3bHSD/D, in accord with the strict substrate specificity of 3bHSD/D that required a free C-4 carboxyl group (Rahier et al, 2006).…”