Human placental 3ß-hydroxysteroid dehydrogenase/steroid Δ5,4-isomerase 1: Identity, regulation and environmental inhibitors.

Human placental 3ß-hydroxysteroid dehydrogenase/steroid Δ5, 4-isomerase 1 (HSD3B1), a high-affinity type I enzyme, uses pregnenolone to make progesterone, which is critical for maintenance of pregnancy. HSD3B1 is located in the mitochondrion and the smooth endoplasmic reticulum of placental cells and is encoded by HSD3B1 gene. HSD3B1 contains GATA and TEF-5 regulatory elements. Many endocrine disruptors, including phthalates, methoxychlor and its metabolite, organotins, and gossypol directly inhibit placental HSD3B1 thus blocking progesterone production. In this review, we discuss the placental HSD3B1, its gene regulation, biochemistry, subcellular location, and inhibitors from the environment.

Dynamics of loops at the substrate entry channel determine the specificity of iridoid synthases.

Iridoid synthases belong to the family of short-chain dehydrogenase/reductase involved in the biosynthesis of iridoids. Despite having high sequence and structural homology with progesterone 5ß-reductase, these enzymes exhibit differential substrate specificities. Previously, two loops, L1 and L2 at substrate-binding pocket, were suggested to be involved in generating substrate specificity. However, the structural basis of specificity determinants was elusive. Here, combining sequence and structural analysis, site-directed mutagenesis, and molecular dynamics simulations, we have shown that iridoid synthase contains two channels for substrate entry whose geometries are altered by L1-L2 dynamics, primarily orchestrated by interactions of residues Glu161 and Gly162 of L1 and Asn358 of L2. A complex interplay of these interactions confer the substrate specificity to the enzyme.

Development of monoclonal antibodies against the human 3ß-hydroxysteroid dehydrogenase/isomerase isozymes.

The human 3ß-hydroxysteroid dehydrogenase/isomerase (HSD3B) enzymes catalyze the conversion of 3ß-hydroxy Δ5-6 steroids into 3-keto Δ4-5 steroids, which is required for the synthesis of the mature steroid hormones secreted by the adrenal and gonads. The human has 2 isozymes, the HSD3B1 that is traditionally located in placenta and extra-adrenal tissues and the HSD3B2 that is expressed in the adrenal and gonads. Mice with both cryptochrome 1 and 2 genes deletion were recently found to have salt-sensitive hypertension and hyperaldosteronism. These deletions were also associated with overexpression of the Hsd3b6 enzyme, the homolog of the human HSD3B1, in the zona glomerulosa which was believed to explain the hyperaldosteronism. A report using antibodies against human HSD3B1 suggested that it was expressed in the zona glomerulosa of normal human adrenals and in patients with idiopathic hyperaldosteronism and the HSD3B2 expressed in both the zona fasciculata and glomerulosa. We have developed specific monoclonal antibodies against the human HSD3B1 and HSD3B2 isozymes and found that the main enzyme expressed in the zona glomerulosa was the HSD3B2. Faint staining of the adrenal was also obtained using the anti-HSD3B1antibody only at high concentrations of antibody. This study fails to confirm that HSD3B1 expression in the human zona glomerulosa and double immunofluorescence clearly shows that the HSD3B2 is expressed in the zona glomerulosa and fasciculata and in the zona glomerulosa HSD3B2 is co-expressed with aldosterone synthase (CYP11B2).

Ziram inhibits aromatase activity in human placenta and JEG-3 cell line.

Placenta produces progesterone and estradiol for maintaining pregnancy. Two critical enzymes are responsible for their production: 3ß-hydroxysteroid dehydrogenase 1 (HSD3B1) that catalyzes the formation of progesterone from pregnenolone and aromatase that catalyzes the production of estradiol from testosterone. Fungicide ziram may disrupt the placental steroid production. In the present study, we investigated the effects of ziram on steroid formation in human placental cell line JEG-3 cells and on HSD3B1 and aromatase in the human placenta. Ziram did not inhibit progesterone production in JEG-3 cells and HSD3B1 activity at 100µM. Ziram was a potent aromatase inhibitor with the half maximal inhibitory concentration (IC50) value of 333.8nM. When testosterone was used to determine the mode of action, ziram was found to be a competitive inhibitor. Docking study showed that ziram binds to the testosterone binding pocket of the aromatase. In conclusion, ziram is a potent inhibitor of human aromatase.

Cloning and Characterization of Two Iridoid Synthase Homologs from Swertia Mussotii.

Swertia mussotii is an important medicinal plant found on the Qinghai Tibetan Plateau that has great economic and medicinal value. This plant has enjoyed a long history of use as a curative for hepatitis. The biological activity of secoiridoids, including gentiopicroside and swertiamarin, has been mainly tested for its anti-hepatitis effects. Here, we identify two candidate genes (SmIS1 and SmIS2) that are homologues of iridoid synthase and that are components of the secoiridoid pathway in S. mussotii. Using sequencing and phylogenetic analyses, we confirm that SmIS1 and SmIS2 contain six conserved short-chain dehydrogenases/reductase (SDR) motifs and thus belong to the P5ßRs group. The two purified Escherichia coli-expressed proteins reduced 8-oxogeranial to both nepetalactol and iridodials. A comparison of the kinetic parameters of SmIS1 and SmIS2 recombinant proteins revealed that SmIS2 has a lower affinity than SmIS1 for 8-oxogeranial. Transcript levels of the two genes were analysed in three different tissues of S. mussotii using semi-quantitative RT-PCR and RT-qPCR. SmIS1 and SmIS2 expression levels were more abundant in leaves and stems. This investigation adds to our knowledge of P5ßRs genes in the secoiridoid synthesis pathway and provides candidate genes for genetically improving S. mussotii by enhancing secondary metabolite production.

Non-Virilizing Congenital Adrenal Hyperplasia in a Female Patient with a Novel HSD3B2 Mutation.

Classic 3ß-hydroxysteroid dehydrogenase type 2 (3ß-HSD II) deficiency causes congenital adrenal hyperplasia with glucocorticoid, mineralocorticoid, and sex steroid deficiency. We present a female patient with congenital adrenal hyperplasia detected in newborn screening due to elevated 17OH-progesterone. Female external genitalia and non-measurable androgen levels elicited the suspicion of a defect early in the steroid cascade. Two loss-of-function HSD3B2 mutations (1 novel) were detected and confirmed in silico. We argue that in a girl with glucocorticoid and mineralocorticoid deficiency without virilization, 3ß-HSD II deficiency is an important differential diagnosis. 17OH-progesterone may initially be elevated due to placental and peripheral activity of 3ß-HSD I, whereas dehydroepiandrosterone may not be increased.

A New Homozygous Frameshift Mutation in the HSD3B2 Gene in an Apparently Nonconsanguineous Italian Family.

BACKGROUND: 3ß-Hydroxysteroid dehydrogenase (3ß-HSD) deficiency is a rare cause of congenital adrenal hyperplasia (CAH) caused by inactivating mutations in the HSD3B2 gene. PATIENT AND METHODS: We report the molecular and structural analysis of the HSD3B2 gene in a 46,XY child born to apparently nonconsanguineous parents and presenting ambiguous genitalia and salt wasting. The steroid profile showed elevated concentrations of 17-hydroxyprogesterone, androstenedione, ACTH and plasma renin, but normal values of cortisol and dehydroepiandrosterone sulfate. Unexpectedly, plasma aldosterone was high. For structural and functional analyses, the three-dimensional structure of 3ß-HSD2 was modeled using the crystal structure of the short-chain dehydrogenase Gox2253 from Gluconobacter oxydans as a template. RESULTS: The direct DNA sequence of the child revealed a new homozygous frameshift mutation in exon 4 of the HSD3B2 gene, a single nucleotide deletion at codon 319 [GTC(Val)x2192;GC], yielding premature stop codon in position 367. Molecular homology modeling and secondary structure predictions suggested that the variant sequence might both alter the substrate-binding cleft and compromise the overall stability of the enzyme. CONCLUSION: We have described the first HSD3B2 gene mutation in the Italian population and analyzed its effect in the context of the 3ß-HSD2 structure and function.

Regulation of human 3-beta-hydroxysteroid dehydrogenase type-2 (3ßHSD2) by molecular chaperones and the mitochondrial environment affects steroidogenesis.

Human 3-ß-hydroxysteroid dehydrogenase/isomerase types 1 and 2 (3ßHSD1 and 3ßHSD2, respectively) are expressed in a tissue-specific pattern by different genes. Site-directed mutagenesis studies have confirmed the function of the catalytic amino acids (Tyr154, Lys 158, Ser124 in both isoenzymes), substrate/inhibitor isoform-specific residues (His156 and Arg195 in 3ßHSD1) and cofactor binding residues (Asp36 provides NAD(+) specificity in both isoenzymes). However, detailed analysis of isoform-specific organelle localization and characterization is difficult due to the 93% amino acid identity between the two isoforms. With recent advances in the knowledge of mitochondrial architecture and localization of the various translocases, our laboratory has studied the mechanisms regulating mitochondrial 3ßHSD2 localization. The mitochondrial N-terminal leader sequence of 3ßHSD2 directs its entry into the mitochondria where it is localized to the intermembrane space (IMS). Unlike other mitochondrial proteins, the N-terminal signal sequence of 3ßHSD2 is not cleaved upon mitochondrial import. 3ßHSD2 interacts with the mitochondrial translocase, Tim50, to regulate progesterone and androstenedione formation. Our studies suggest that its activity at the IMS is facilitated in a partially unfolded "molten globule" conformation by the proton pump between the matrix and IMS. The unfolded protein is refolded by the mitochondrial chaperones. The protons at the IMS are absorbed by the lipid vesicles, to maintain the proton pump and recycle 3ßHSD2. As a result, one molecule of 3ßHSD2 may participate in multiple catalytic reactions. In summary, the steroidogenic cell recycles 3ßHSD2 to catalyze the reactions needed to produce androstenedione, progesterone and 17α-hydroxyprogesterone on demand in coordination with the mitochondrial translocase, Tim50. This article is part of a Special Issue entitled 'Steroid/Sterol signaling'.

In silico structural, functional and pathogenicity evaluation of a novel mutation: an overview of HSD3B2 gene mutations.

Mutations of 3 beta hydroxysteroid dehydrogenase type II (HSD3B2) gene result in different clinical consequences. We explain a patient who demonstrated a salt wasting form of 3ßHSD deficiency in infancy. Signs of hyponatremia and hyperkalemia were recognized in the infant with ambiguous genitalia and perineal hypospadias. The 46,XY male was genotyped by direct sequencing of HSD3B2 gene. Steroid profiles showed elevated concentration of 17 hydroxyprogesterone, and decrease in concentration of cortisol, and testosterone. Dehydroepiandrotone (DHEA) to androstenedione ratio had 6 fold increases. Direct sequencing of the patient revealed homozygous missense A82P mutation in exon 3. This mutation was confirmed by segregation analysis of the parents. Bioinformatic tools were used for in silico structural and functional analyses. Also, the pathological effect of the mutation was validated by different software. Alanine is a conserved amino acid in the membrane binding domain of the enzyme and proline substitution was predicted to destabilize the protein. This report may highlight the importance of the screening programs of the disorder in Iran.

Mitochondrial 3ß-hydroxysteroid dehydrogenase enzyme activity requires reversible pH-dependent conformational change at the intermembrane space.

The inner mitochondrial membrane protein 3ß-hydroxysteroid dehydrogenase 2 (3ßHSD2) synthesizes progesterone and androstenedione through its dehydrogenase and isomerase activities. This bifunctionality requires 3ßHSD2 to undergo a conformational change. Given its proximity to the proton pump, we hypothesized that pH influences 3ßHSD2 conformation and thus activity. Circular dichroism (CD) showed that between pH 7.4 and 4.5, 3ßHSD2 retained its primarily α-helical character with a decrease in α-helical content at lower pH values, whereas the ß-sheet content remained unchanged throughout. Titrating the pH back to 7.4 restored the original conformation within 25 min. Metabolic conversion assays indicated peak 3ßHSD2 activity at pH 4.5 with ~2-fold more progesterone synthesized at pH 4.5 than at pH 3.5 and 7.4. Increasing the 3ßHSD2 concentration from 1 to 40 µg resulted in a 7-fold increase in progesterone at pH 4.5, but no change at pH 7.4. Incubation with guanidinum hydrochloride (GdmHCl) showed a three-step cooperative unfolding of 3ßHSD2 from pH 7.4 to 4.5, possibly due to the native state unfolding to the intermediate ion core state. With further decreases in pH, increasing concentrations of GdmHCl led to rapid two-step unfolding that may represent complete loss of structure. Between pH 4 and 5, the two intermediate states appeared stable. Stopped-flow kinetics showed slower unfolding at around pH 4, where the protein is in a pseudostable state. Based on our data, we conclude that at pH 4-5, 3ßHSD2 takes on a molten globule conformation that promotes the dual functionality of the enzyme.

Lipid-mediated unfolding of 3ß-hydroxysteroid dehydrogenase 2 is essential for steroidogenic activity.

For inner mitochondrial membrane (IMM) proteins that do not undergo N-terminal cleavage, the activity may occur in the absence of a receptor present in the mitochondrial membrane. One such protein is human 3ß-hydroxysteroid dehydrogenase 2 (3ßHSD2), the IMM resident protein responsible for catalyzing two key steps in steroid metabolism: the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione. Conversion requires that 3ßHSD2 serve as both a dehydrogenase and an isomerase. The dual functionality of 3ßHSD2 results from a conformational change, but the trigger for this change remains unknown. Using fluorescence resonance energy transfer, we found that 3ßHSD2 interacted strongly with a mixture of dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylcholine (DPPC). 3ßHSD2 became less stable when incubated with the individual lipids, as indicated by the decrease in thermal denaturation (T(m)) from 42 to 37 °C. DPPG, alone or in combination with DPPC, led to a decrease in α-helical content without an effect on the ß-sheet conformation. With the exception of the 20 N-terminal amino acids, mixed vesicles protected 3ßHSD2 from trypsin digestion. However, protein incubated with DPPC was only partially protected. The lipid-mediated unfolding completely supports the model in which a cavity forms between the α-helix and ß-sheet. As 3ßHSD2 lacks a receptor, opening the conformation may activate the protein.

The crystal structure of progesterone 5beta-reductase from Digitalis lanata defines a novel class of short chain dehydrogenases/reductases.

Progesterone 5beta-reductase (5beta-POR) catalyzes the stereospecific reduction of progesterone to 5beta-pregnane-3,20-dione and is a key enzyme in the biosynthetic pathway of cardenolides in Digitalis (foxglove) plants. Sequence considerations suggested that 5beta-POR is a member of the short chain dehydrogenase/reductase (SDR) family of proteins but at the same time revealed that the sequence motifs that in standard SDRs contain the catalytically important residues are missing. Here we present crystal structures of 5beta-POR from Digitalis lanata in complex with NADP(+) at 2.3A and without cofactor bound at 2.4A resolution together with a model of a ternary complex consisting of 5beta-POR, NADP(+), and progesterone. Indeed, 5beta-POR displays the fold of an extended SDR. The architecture of the active site is, however, unprecedented because none of the standard catalytic residues are structurally conserved. A tyrosine (Tyr-179) and a lysine residue (Lys-147) are present in the active site, but they are displayed from novel positions and are part of novel sequence motifs. Mutating Tyr-179 to either alanine or phenylalanine completely abolishes the enzymatic activity. We propose that the distinct topology reflects the fact that 5beta-POR reduces a conjugated double bond in a steroid substrate via a 1-4 addition mechanism and that this requires a repositioning of the catalytically important residues. Our observation that the sequence motifs that line the active site are conserved in a number of bacterial and plant enzymes of yet unknown function leads us to the proposition that 5beta-POR defines a novel class of SDRs.

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.

Molecular biology of the 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene family.

The 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (3beta-HSD) isoenzymes are responsible for the oxidation and isomerization of Delta(5)-3beta-hydroxysteroid precursors into Delta(4)-ketosteroids, thus catalyzing an essential step in the formation of all classes of active steroid hormones. In humans, expression of the type I isoenzyme accounts for the 3beta-HSD activity found in placenta and peripheral tissues, whereas the type II 3beta-HSD isoenzyme is predominantly expressed in the adrenal gland, ovary, and testis, and its deficiency is responsible for a rare form of congenital adrenal hyperplasia. Phylogeny analyses of the 3beta-HSD gene family strongly suggest that the need for different 3beta-HSD genes occurred very late in mammals, with subsequent evolution in a similar manner in other lineages. Therefore, to a large extent, the 3beta-HSD gene family should have evolved to facilitate differential patterns of tissue- and cell-specific expression and regulation involving multiple signal transduction pathways, which are activated by several growth factors, steroids, and cytokines. Recent studies indicate that HSD3B2 gene regulation involves the orphan nuclear receptors steroidogenic factor-1 and dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1). Other findings suggest a potential regulatory role for STAT5 and STAT6 in transcriptional activation of HSD3B2 promoter. It was shown that epidermal growth factor (EGF) requires intact STAT5; on the other hand IL-4 induces HSD3B1 gene expression, along with IL-13, through STAT 6 activation. However, evidence suggests that multiple signal transduction pathways are involved in IL-4 mediated HSD3B1 gene expression. Indeed, a better understanding of the transcriptional factors responsible for the fine control of 3beta-HSD gene expression may provide insight into mechanisms involved in the functional cooperation between STATs and nuclear receptors as well as their potential interaction with other signaling transduction pathways such as GATA proteins. Finally, the elucidation of the molecular basis of 3beta-HSD deficiency has highlighted the fact that mutations in the HSD3B2 gene can result in a wide spectrum of molecular repercussions, which are associated with the different phenotypic manifestations of classical 3beta-HSD deficiency and also provide valuable information concerning the structure-function relationships of the 3beta-HSD superfamily. Furthermore, several recent studies using type I and type II purified enzymes have elegantly further characterized structure-function relationships responsible for kinetic differences and coenzyme specificity.

Structure/function relationships responsible for coenzyme specificity and the isomerase activity of human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase.

Human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD/isomerase) catalyzes the two sequential enzyme reactions on a single protein that converts dehydroepiandrosterone or pregnenolone to androstenedione or progesterone, respectively, in placenta, mammary gland, breast tumors, prostate, prostate tumors, and other peripheral tissues. Our earlier studies show that the two enzyme reactions are linked by the coenzyme product, NADH, of the 3 beta-HSD activity. NADH activates the isomerase activity by inducing a time-dependent conformational change in the enzyme protein. The current study tested the hypothesis that the 3 beta-HSD and isomerase activities shared a common coenzyme domain, and it characterized key amino acids that participated in coenzyme binding and the isomerase reaction. Homology modeling with UDP-galactose-4-epimerase predicts that Asp36 is responsible for the NAD(H) specificity of human 3 beta-HSD/isomerase and identifies the Rossmann-fold coenzyme domain at the amino terminus. The D36A/K37R mutant in the potential coenzyme domain and the D241N, D257L, D258L, and D265N mutants in the potential isomerase domain (previously identified by affinity labeling) were created, expressed, and purified. The D36A/K37R mutant shifts the cofactor preference of both 3 beta-HSD and isomerase from NAD(H) to NADP(H), which shows that the two activities utilize a common coenzyme domain. The D257L and D258L mutations eliminate isomerase activity, whereas the D241N and D265N mutants have nearly full isomerase activity. Kinetic analyses and pH dependence studies showed that either Asp257 or Asp258 plays a catalytic role in the isomerization reaction. These observations further characterize the structure/function relationships of human 3 beta-HSD/isomerase 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 relationships responsible for the kinetic differences between human type 1 and type 2 3beta-hydroxysteroid dehydrogenase and for the catalysis of the type 1 activity.

Two distinct genes encode the 93% homologous type 1 (placenta, peripheral tissues) and type 2 (adrenals, gonads) 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD/isomerase) in humans. Mutagenesis studies using the type 1 enzyme have produced the Y154F and K158Q mutant enzymes in the Y(154)-P-H(156)-S-K(158) motif as well as the Y269S and K273Q mutants from a second motif, Y(269)-T-L-S-K(273), both of which are present in the primary structure of the human type 1 3beta-HSD/isomerase. In addition, the H156Y mutant of the type 1 enzyme has created a chimera of the type 2 enzyme motif (Y(154)-P-Y(156)-S-K(158)) in the type 1 enzyme. The mutant and wild-type enzymes have been expressed and purified. The K(m) value of dehydroepiandrosterone is 13-fold greater, and the maximal turnover rate (K(cat)) is 2-fold greater for wild-type 2 3beta-HSD compared with the wild-type 1 3beta-HSD activity. The H156Y mutant of the type 1 enzyme has substrate kinetic constants for 3beta-HSD activity that are very similar to those of the wild-type 2 enzyme. Dixon analysis shows that epostane inhibits the 3beta-HSD activity of the wild-type 1 enzyme with 14-17-fold greater affinity compared with the wild-type 2 and H156Y enzymes. The Y154F and K158Q mutants exhibit no 3beta-HSD activity, have substantial isomerase activity, and utilize substrate with K(m) values similar to those of wild-type 1 isomerase. The Y269S and K273Q mutants have low, pH-dependent 3beta-HSD activity, exhibit only 5% of the maximal isomerase activity, and utilize the isomerase substrate very poorly. From these studies, a structural basis for the profound differences in the substrate and inhibition kinetics of the wild-type 1 and 2 3beta-HSD, plus a catalytic role for the Tyr(154) and Lys(158) residues in the 3beta-HSD reaction have been identified. These advances in our understanding of the structure/function of human type 1 and 2 3beta-HSD/isomerase may lead to the design of selective inhibitors of the type 1 enzyme not only in placenta to control the onset of labor but also in hormone-sensitive breast, prostate, and choriocarcinoma tumors to slow their growth.

The engineered, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase: purification, characterization and crystallization.

Human type I 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD/isomerase) is an integral membrane protein of human placental trophoblast and of insect Sf9 cells transfected with recombinant baculovirus containing the cDNA encoding the enzyme. Purified native or wild-type enzyme remains in solution only in the presence of detergent that may prevent crystallization. The membrane-spanning domain (residues 283-310) of the enzyme protein was deleted in the cDNA using PCR-based mutagenesis. The modified enzyme was expressed by baculovirus in the cytosol instead of in the microsomes and mitochondria of the Sf9 cells. The cytosolic form of 3beta-HSD/isomerase was purified using affinity chromatography with Cibacron Blue 1000. The NAD(+) and NaCl used to elute the enzyme were removed by size-exclusion centrifugation. Hydroxylapatite chromatography yielded a 26-fold purification of the enzyme. SDS-PAGE revealed a single protein band for the purified cytosolic enzyme (monomeric molecular mass 38.8 kDa) that migrated just below the wild-type enzyme (monomeric molecular mass 42.0 kDa). Michaelis-Menten constants measured for 3beta-HSD substrate (dehydroepiandrosterone) utilization by the purified cytosolic enzyme (K(m)=4.5 microM, V(max)=53 nmol/min per mg) and the pure wild-type enzyme (K(m)=3.7 microM, V(max)=43 nmol/min per mg), for isomerase substrate (5-androstene-3,17-dione) conversion by the purified cytosolic (K(m)=25 microM, V(max)=576 nmol/min per mg) and wild-type (K(m)=28 microM, V(max)=598 nmol/min per mg) enzymes, and for NAD(+) reduction by the 3beta-HSD activities of the cytosolic (K(m)=35 microM, V(max)=51 nmol/min per mg) and wild-type (K(m)=34 microM, V(max)=46 nmol/min per mg) enzymes are nearly identical. The isomerase activity of the cytosolic enzyme requires allosteric activation by NADH (K(m)=4.6 microM, V(max)=538 nmol/min per mg) just like the wild-type enzyme (K(m)=4.6 microM, V(max)=536 nmol/min per mg). Crystals of the purified, cytosolic enzyme protein have been obtained. The inability to crystallize the detergent-solubilized, wild-type microsomal enzyme has been overcome by engineering a cytosolic form of this protein. Determining the tertiary structure of 3beta-HSD/isomerase will clarify the mechanistic roles of potentially critical amino acids (His(261), Tyr(253)) that have been identified in the primary structure.

Creation of a fully active, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase by the deletion of a membrane-spanning domain.

Human 3beta-hydroxysteroid dehydrogenase/steroid Delta(5)-Delta(4)-isomerase (3beta-HSD/isomerase) is a bifunctional, single enzyme protein that is membrane-bound in the endoplasmic reticulum (microsomes) and mitochondria of cells in the placenta (type I) and in the adrenals and gonads (type II). Two membrane-binding domains (residues 72-89 and 283-310) have been predicted by analyses of hydrophobicity in the type I and II isoenzymes (90% regional homology). These putative membrane domains were deleted in the cDNA by PCR-based mutagenesis, and the two mutant enzymes were expressed by baculovirus in insect Sf9 cells. Differential centrifugation of the Sf9 cell homogenate containing the 283-310 deletion mutant revealed that 94% of the 3beta-HSD and isomerase activities were in the cell cytosol, 6% of the activities were in the microsomes, and no activity was in the mitochondria. This is the opposite of the subcellular distribution of the wild-type enzyme with 94% of the activities in the microsomes and mitochondria and only 6% activity in the cytosol. The organelle distribution of the 72-89 deletion mutant lies between these two extremes with 72% of the enzyme activity in the cytosol and 28% in the microsomes/mitochondria. The integrity of the subcellular organelle preparations was confirmed by electron microscopy. Western immunoblots confirmed the presence of the 283-310 deletion mutant enzyme and the absence of the wild-type enzyme in the insect cell cytosol. The unpurified, cytosolic 383-310 deletion mutant exhibited 3beta-HSD (22 nmol/min per mg) and isomerase (33 nmol/min per mg) specific activities that were comparable with those of the membrane-bound, wild-type enzyme. The isomerase reaction of the cytosolic 283-311 deletion mutant requires activation by NADH just like the isomerase of the microsomal or mitochondrial wild-type enzyme. In contrast, the 72-89 deletion mutant had low 3beta-HSD and isomerase specific activities that were only 12% of the wild-type levels. This innovative study identifies the 283-310 region as the critical membrane domain of 3beta-HSD/isomerase that can be deleted without compromising enzyme function. The shorter 72-89 region is also a membrane domain, but deletion of this NH(2)-terminal region markedly diminishes the enzyme activities. Purification of the active, cytosolic 283-310 deletion mutant will produce a valuable tool for crystallographic studies that may ultimately determine the tertiary/quaternary structure of this key steroidogenic enzyme.

Isolation of a new mouse 3beta-hydroxysteroid dehydrogenase isoform, 3beta-HSD VI, expressed during early pregnancy.

The enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD) is a key enzyme in the biosynthesis of steroid hormones. To date, this laboratory has isolated and characterized five distinct 3beta-HSD complementary DNAs (cDNAs) in the mouse (3beta-HSD I through V). These different forms are expressed in a tissue- and developmentally-specific manner and fall into two functionally distinct enzymes. 3beta-HSD I and III, and most likely II, function as dehydrogenase/isomerases, whereas 3beta-HSD IV and V function as 3-ketosteroid reductases. This study describes the isolation, characterization, and tissue-specific expression of a sixth member of this gene family, 3beta-HSD VI. This new isoform functions as an NAD+-dependent dehydrogenase/isomerase exhibiting very low Michaelis-Menten constant (Km) values for pregnenolone (approximately 0.035 microM) and dehydroepiandrosterone (approximately 0.12 microM). 3beta-HSD VI is the earliest isoform to be expressed during embryogenesis in cells of embryonic origin at 7 and 9.5 days postcoitum (pc), and is the major isoform expressed in uterine tissue at the time of implantation (4.5 days pc) and continues to be expressed in uterine tissue at 6.5, 7.5, and 9.5 days pc. 3beta-HSD VI is expressed in giant trophoblasts at 9.5 days pc and is expressed in the placenta through day 15.5 pc. In the adult mouse, 3beta-HSD VI appears to be the only isoform expressed in the skin and also is expressed in the testis, but to a lesser extent than 3beta-HSD I. Mouse 3beta-HSD VI cDNA is orthologous to human 3beta-HSD I cDNA. Human type I 3beta-HSD has been shown to be the only isoform expressed in the placenta and skin. The demonstration that mouse 3beta-HSD VI functions as a dehydrogenase/isomerase and is the predominant isoform expressed during the first half of pregnancy in uterine tissue and in embryonic cells suggests that this isoform may be involved in local production of progesterone, which is needed for successful implantation of the blastocyst and/or maintenance of early pregnancy.

An NADH-induced conformational change that mediates the sequential 3 beta-hydroxysteroid dehydrogenase/isomerase activities is supported by affinity labeling and the time-dependent activation of isomerase.

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and steroid delta-isomerase were copurified as a single protein from human placental microsomes. Because NADH is an essential activator of isomerase (Kact = 2.4 microM, Vmax = 0.6 mumol/min/mg), the affinity alkylating nucleotide, 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate (8-BDB-TADP), was synthesized. 8-BDB-TADP activates isomerase (Kact = 338 microM, Vmax = 2.1 mumol/min/mg) prior to inactivating the enzyme. The inactivation kinetics for isomerase fit the Kitz and Wilson model for time-dependent, irreversible inhibition by 8-BDB-TADP (KI = 314 microM, first order maximal rate constant kobs = 7.8 x 10(-3) s-1). NADH (50 microM) significantly protects isomerase from inactivation by 8-BDB-TADP (100 microM). The isomerase activity is inactivated more rapidly by 8-BDB-TADP as the concentration of the affinity alkylator increases from 67 microM (t1/2 = 8.4 min) to 500 microM (t1/2 = 2.4 min). In sharp contrast, the 3 beta-HSD activity is inactivated more slowly as the concentration of 8-BDB-TADP increases from 67 microM (t1/2 = 4.8 min) to 500 microM (t1/2 = 60.0 min). We hypothesized that the paradoxical kinetics of 3 beta-HSD inactivation is a consequence of the activation of isomerase by 8-BDB-TADP via a nucleotide-induced shift in enzyme conformation. Biophysical support for an NADH-induced conformational change was obtained using stopped-flow fluorescence spectroscopy. The binding of NADH (10 microM) quenches the intrinsic fluorescence of the enzyme protein in a time-dependent manner (rate constant kapp = 8.1 x 10(-3) s-1, t1/2 = 85 s). A time lag is also observed for the activation of isomerase by NADH. This combination of affinity labeling and biophysical data using nucleotide derivatives supports our model for the sequential reaction mechanism; the cofactor product of the 3 beta-HSD reaction, NADH, activates isomerase by inducing a conformational change in the single, bifunctional enzyme protein.