Structure/function of human type 1 3beta-hydroxysteroid dehydrogenase: An intrasubunit disulfide bond in the Rossmann-fold domain and a Cys residue in the active site are critical for substrate and coenzyme utilization.

The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) are key enzymes in steroidogenic pathways leading to the production of all active steroid hormones. Kinetic analyses of purified 3beta-HSD1 show that the Michaelis-Menten constants (Km) for substrates and cofactor are decreased dramatically (three- to eight-fold) by the addition of beta-mercaptoethanol (BME), which suggest that a disulfide bond may be critical to ligand utilization. Western immunoblots and SDS-PAGE of purified 3beta-HSD1 in the presence or absence of BME showed a lack of intersubunit disulfide bonds in the dimeric enzyme. The Rossmann-fold domain of 3beta-HSD1 contains two Cys residues, Cys72 and Cys111, which are capable of forming an intrasubunit disulfide bond based on their proximity in our structural model. Our structural model also predicts that Cys83 may affect the orientation of substrate and cofactor. To test these predictions, the C72S, C72F, C111S, C111A, C83S and C83A mutants of 3beta-HSD1 were produced, expressed, and purified. BME failed to diminish the Km values of substrate and cofactor for C72S, C72F, C111S and C111A but produced a 2.5 decrease in Km values for C83A ligands similar to wild-type 3beta-HSD. Thus, our results support the presence of an intrasubunit disulfide bond between Cys72 and Cys111 that participates in the tertiary structure of the Rossmann-fold domain. Although C83S had no enzyme activity, the C83A mutant enzyme exhibited two- to five-fold higher Km values for substrate and cofactor but had similar K(cat) values compared to wild-type 3beta-HSD. These data characterize the roles of Cys residues in 3beta-HSD and validate the predictions of our structural model.

Rational proteomics V: structure-based mutagenesis has revealed key residues responsible for substrate recognition and catalysis by the dehydrogenase and isomerase activities in human 3beta-hydroxysteroid dehydrogenase/isomerase type 1.

Mammalian 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) is a member of the short chain dehydrogenase/reductase. It is a key steroidogenic enzyme that catalyzes the first step of the multienzyme pathway conversion of circulating dehydroepiandrosterone and pregnenolone to active steroid hormones. A three dimensional model of a ternary complex of human 3beta-HSD type 1 (3beta-HSD_1) with an NAD cofactor and androstenedione product has been developed based upon X-ray structures of the ternary complex of E. coli UDP-galactose 4-epimerase (UDPGE) with an NAD cofactor and substrate (PDB_AC: 1NAH) and the ternary complex of human type 1 17beta-hydroxysteroid dehydrogenase (17beta-HSD_1) with an NADP cofactor and androstenedione (PDB_AC: 1QYX). The dimeric structure of the enzyme was built from two monomer models of 3beta-HSD_1 by respective 3D superposition with A and B subunits of the dimeric structure of Streptococcus suis DTDP-D-glucose 4,6-dehydratase (PDB_AC: 1KEP). The 3D model structure of 3beta-HSD_1 has been successfully used for the rational design of mutagenic experiments to further elucidate the key substrate binding residues in the active site as well as the basis for dual function of the 3beta-HSD_1 enzyme. The structure based mutant enzymes, Asn100Ser, Asn100Ala, Glu126Leu, His232Ala, Ser322Ala and Asn323Leu, have been constructed and functionally characterized. The mutagenic experiments have confirmed the predicted roles of the His232 and Asn323 residues in recognition of the 17-keto group of the substrate and identified Asn100 and Glu126 residues as key residues that participate for the dehydrogenase and isomerization reactions, respectively.

Identification of key amino acids responsible for the substantially higher affinities of human type 1 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1) for substrates, coenzymes, and inhibitors relative to human 3beta-HSD2.

The human type 1 (placenta, breast tumors, and prostate tumors) and type 2 (adrenals and gonads) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1 and 3beta-HSD2) are encoded by two distinct genes that are expressed in a tissue-specific pattern. Our recent studies have shown that His156 contributes to the 14-fold higher affinity that 3beta-HSD1 exhibits for substrate and inhibitor steroids compared with human 3beta-HSD2 containing Tyr156 in the otherwise identical catalytic domain. Our structural model of human 3beta-HSD localizes His156 or Tyr156 in the subunit interface of the enzyme homodimer. The model predicts that Gln105 on one enzyme subunit has a higher probability of interacting with His156 on the other subunit in 3beta-HSD1 than with Tyr156 in 3beta-HSD2. The Q105M mutant of 3beta-HSD1 (Q105M1) shifts the Michaelis-Menten constant (Km) for 3beta-HSD substrate and inhibition constants (Ki) for epostane and trilostane to the much lower affinity profiles measured for wild-type 3beta-HSD2 and H156Y1. However, the Q105M2 mutant retains substrate and inhibitor kinetic profiles similar to those of 3beta-HSD2. Our model also predicts that Gln240 in 3beta-HSD1 and Arg240 in 3beta-HSD2 may be responsible for the 3-fold higher affinity of the type 1 isomerase activity for substrate steroid and cofactors. The Q240R1 mutation increases the isomerase substrate Km by 2.2-fold to a value similar to that of 3beta-HSD2 isomerase and abolishes the allosteric activation of isomerase by NADH. The R240Q2 mutation converts the isomerase substrate, cofactor, and inhibitor kinetic profiles to the 4-14-fold higher affinity profiles of 3beta-HSD1. Thus, key structural reasons for the substantially higher affinities of 3beta-HSD1 for substrates, coenzymes, and inhibitors have been identified. These structure and function relationships can be used in future docking studies to design better inhibitors of the 3beta-HSD1 that may be useful in the treatment of hormone-sensitive cancers and preterm labor.

The higher affinity of human type 1 3beta-hydroxysteroid dehydrogenase (3beta-HSD1) for substrate and inhibitor steroids relative to human 3beta-HSD2 is validated in MCF-7 tumor cells and related to subunit interactions.

Two distinct genes encode the tissue-specific expression of the two isoforms of human 3beta-hydroxysteroid dehydrogenase: 3beta-HSD1 (placenta, mammary gland, breast tumors) and 3beta-HSD2 (gonads, adrenals). Purified 3beta-HSD1 utilizes DHEA as a substrate with 13-fold lower Km than 3beta-HSD2. Using homogenates of human MCF-7 Tet-off breast tumor cells stably transfected with human 3beta-HSD1 or 3beta-HSD2, DHEA is utilized as substrate by 3beta-HSD1 (Km = 4.8 microM) much more avidly than by 3beta-HSD2 (Km = 43 microM). In addition, the 3beta-HSD inhibitor, epostane, binds to purified 3beta-HSD1 with a 17-fold higher affinity compared to 3beta-HSD2. In the MCF-7 cells, two 3beta-HSD inhibitors block 3beta-HSD1 activity (Ki = 0.06 microM, epostane; 0.08 microM, trilostane) with 12- to 14-fold higher affinities compared to the inhibition of 3beta-HSD2 (Ki = 0.85 microM, epostane; 1.01 microM, trilostane). Thus, the substantially higher affinities of human 3beta-HSD1 for substrate and inhibitor steroids measured using the pure isoenzymes have been validated using microsome-bound 3beta-HSD1 and 3beta-HSD2 in the MCF-7 cells. Similar to our previously reported H156Y mutant of 3beta-HSD1, the Q105M mutant of 3beta-HSD1 shifts the substrate and inhibitor kinetic profiles to those of wild-type 3beta-HSD2. However, the Q105M mutant of 3beta-HSD2 retains the substrate and inhibitor kinetic profiles of wild-type 3beta-HSD2. Our structural homology model of human 3beta-HSD predicts that Gln105 on one enzyme subunit hydrogen-binds to His156 on the other subunit of the enzyme homodimer. The higher affinity of 3beta-HSD1 for the steroids may be related to different subunit interactions in the quaternary structures of the two isoenzymes. It may be possible to exploit these kinetic differences to selectively inhibit the conversion of DHEA ultimately to estradiol by 3beta-HSD1 and slow the growth of breast tumor cells.

Agonist-induced endocytosis of lysophosphatidic acid-coupled LPA1/EDG-2 receptors via a dynamin2- and Rab5-dependent pathway.

Lysophosphatidic acid (LPA) is a serum-borne phospholipid that exerts a pleiotropic range of effects on cells through activation of three closely related G-protein-coupled receptors termed LPA1/EDG-2, LPA2/EDG-4 and LPA3/EDG-7. Of these receptors, the LPA1 receptor is the most widely expressed. In this study, we investigated the agonist-induced endocytosis of the human LPA1 receptor, bearing an N-terminal FLAG epitope tag, in stably transfected HeLa cells. Treatment with LPA induced the rapid endocytosis of approximately 40% of surface LPA1 within 15 minutes. Internalization was both dose dependent and LPA specific since neither lysophophatidylcholine nor sphingosine-1-phosphate induced LPA1 endocytosis. Removal of agonist following 30 minutes incubation resulted in recycling of LPA1 back to the cell surface. LPA1 internalization was strongly inhibited by dominant-inhibitory mutants of both dynamin2 (K44A) and Rab5a (S34N). In addition, both dynamin2 K44A and Rab5 S34N mildly inhibited LPA1-dependent activation of serum response factor. Finally, our results also indicate that LPA1 exhibits basal, LPA-dependent internalization in the presence of serum-containing medium.