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.     

Mapping of steroids binding to 17 beta-hydroxysteroid dehydrogenase type 1 using Monte Carlo energy minimization reveals alternative binding modes

Crystallographic studies of ligand-protein complexes reveal most preferable ligand binding modes, but do not show less populated modes that may contribute to measurable biochemical and biophysical characteristics of the complexes. In some cases, a ligand may bind a protein in essentially different modes. An example is 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), a steroidogenic enzyme that catalyzes reduction of estrone to estradiol in gonadal and peripheral tissues. The enzyme exhibits a high specificity for estrogens which bind with their C17 atom in the proximity of the NADP(H) cofactor. 17Beta-HSD1 can also bind androgens, but in a reverse binding mode, in which the steroid C3 atom is the closest carbon atom to the cofactor. Here we map the interaction energy of estradiol and dihydrotestosterone binding to 17beta-HSD1. Positions and orientations of the steroids in the ligand-binding tunnel were sampled systematically, and at each combination of these generalized coordinates, the energy was Monte Carlo minimized. The computed maps show energy minima corresponding to the X-ray structures and predict alternative binding modes, in particular, an upside-down orientation in which steroidal face alpha is exposed to protein residues that normally interact with face beta. The methodology can be used for mapping ligand-receptor interactions in various systems, for example, in ion channels and G-protein-coupled receptors that bind elongated ligands in confined space between transmembrane helices.     

Chemical synthesis of 16beta-propylaminoacyl derivatives of estradiol and their inhibitory potency on type 1 17beta-hydroxysteroid dehydrogenase and binding affinity on steroid receptors

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are members of a family of enzymes that catalyze the interconversion of weakly active sexual hormones (ketosteroids) and potent hormones (17beta-hydroxysteroids). Among the known isoforms of 17beta-HSD, the type 1 catalyzes the NAD(P)H-mediated reduction of estrone (E(1)) to estradiol (E(2)), a predominant mitogen for the breast cancer cells. Therefore, the inhibition of this particular enzyme is a logical approach to reduce the concentration of estradiol in breast tumors. To develop inhibitors of type 1 17beta-HSD activity, we hypothesized that molecules containing both hydrophobic and hydrophilic components should be interesting candidates for interacting with both the steroid binding domain and some amino acid residues of the cofactor binding domain of the enzyme. Firstly, a conveniently protected 16beta-(3-aminopropyl)-E(2) derivative was synthesized from commercially available E(1). Then, a representative of all class of NHBoc-protected amino acids (basic, acid, aromatic, aliphatic, hydroxylated) were coupled using standard procedures to the amino group of the precursor. Finally, cleavage of all protecting groups was performed in a single step to generate a series of 16beta-propylaminoacyl derivatives of E(2). The enzymatic screening revealed that none of the novel compounds can inhibit the reductive activity of type 1 17beta-HSD. On the other hand, all of these E(2) derivatives did not show any significant binding affinity on four steroid receptors including the estrogen receptor. Additional efforts aimed at improving the inhibitory potency of these steroidal derivatives on type 1 17beta-HSD without providing estrogenic activities is under investigation using a combinatorial chemistry approach.     

Rational design of novel mutants of fungal 17beta-hydroxysteroid dehydrogenase

Reduction of 17-ketosteroids is a biocatalytic process of economic significance for the production of steroid drugs. This reaction can be catalyzed by different microbial 17beta-hydroxysteroid dehydrogenases (17beta-HSD), like the 17beta-HSD activity of Saccharomyces cerevisiae, Pichia faranosa and Mycobacterium sp., and by purified 3beta,17beta-HSD from Pseudomonas testosteroni. In addition to the bacterial 3beta,17beta-HSD the 17beta-HSD of the filamentous fungus Cochliobolus lunatus is the only microbial 17beta-HSD that has been expressed as a recombinant protein and fully characterized. On the basis of its modeled 3D structure, we selected several positions for the replacement of amino acids by site-directed mutagenesis to change substrate specificity, alter coenzyme requirements, and improve overall catalytic activity. Replacement of Val161 and Tyr212 in the substrate-binding region by Gly and Ala, respectively, increased the initial rates for the conversion of androstenedione to testosterone. Replacement of Tyr49 within the coenzyme binding site by Asp changed the coenzyme specificity of the enzyme. This latter mutant can convert the steroids not only in the presence of NADP(+) and NADPH, but also in the presence of NADH and NAD(+). The replacement of His164, located in the non-flexible part of the 'lid' covering the active center resulted in a conformation of the enzyme that possessed a higher catalytic activity.     

Structure of bacterial 3beta/17beta-hydroxysteroid dehydrogenase at 1.2 A resolution: a model for multiple steroid recognition

The enzyme 3beta/17beta-hydroxysteroid dehydrogenase (3beta/17beta-HSD) is a steroid-inducible component of the Gram-negative bacterium Comamonas testosteroni. It catalyzes the reversible reduction/dehydrogenation of the oxo/beta-hydroxy groups at positions 3 and 17 of steroid compounds, including hormones and isobile acids. Crystallographic analysis at 1.2 A resolution reveals the enzyme to have nearly identical subunits that form a tetramer with 222 symmetry. This is one of the largest oligomeric structures refined at this resolution. The subunit consists of a monomer with a single-domain structure built around a seven-stranded beta-sheet flanked by six alpha-helices. The active site contains a Ser-Tyr-Lys triad, typical for short-chain dehydrogenases/reductases (SDR). Despite their highly diverse substrate specificities, SDR members show a close to identical folding pattern architectures and a common catalytic mechanism. In contrast to other SDR apostructures determined, the substrate binding loop is well-defined. Analysis of structure-activity relationships of catalytic cleft residues, docking analysis of substrates and inhibitors, and accessible surface analysis explains how 3beta/17beta-HSD accommodates steroid substrates of different conformations.     

Structural basis for a change in substrate specificity: crystal structure of S113E isocitrate dehydrogenase in a complex with isopropylmalate, Mg2+, and NADP

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate and has negligible activity toward other (R)-malate-type substrates. The S113E mutant of IDH significantly improves its ability to utilize isopropylmalate as a substrate and switches the substrate specificity (k(cat)/K(M)) from isocitrate to isopropylmalate. To understand the structural basis for this switch in substrate specificity, we have determined the crystal structure of IDH S113E in a complex with isopropylmalate, NADP, and Mg(2+) to 2.0 A resolution. On the basis of a comparison with previously determined structures, we identify distinct changes caused by the amino acid substitution and by the binding of substrates. The S113E complex exhibits alterations in global and active site conformations compared with other IDH structures that include loop and helix conformational changes near the active site. In addition, the angle of the hinge that relates the two domains was altered in this structure, which suggests that the S113E substitution and the binding of substrates act together to promote catalysis of isopropylmalate. Ligand binding results in reorientation of the active site helix that contains residues 113 through 116. E113 exhibits new interactions, including van der Waals contacts with the isopropyl group of isopropylmalate and a hydrogen bond with N115, which in turn forms a hydrogen bond with NADP. In addition, the loop and helix regions that bind NADP are altered, as is the loop that connects the NADP binding region to the active site helix, changing the relationship between substrates and enzyme. In combination, these interactions appear to provide the basis for the switch in substrate specificity.     

Structural studies of Saccharomyces cerevesiae mitochondrial NADP-dependent isocitrate dehydrogenase in different enzymatic states reveal substantial conformational changes during the catalytic reaction

Isocitrate dehydrogenases (IDHs) catalyze oxidative decarboxylation of isocitrate (ICT) into alpha-ketoglutarate (AKG). We report here the crystal structures of Saccharomyces cerevesiae mitochondrial NADP-IDH Idp1p in binary complexes with coenzyme NADP, or substrate ICT, or product AKG, and in a quaternary complex with NADPH, AKG, and Ca(2+), which represent different enzymatic states during the catalytic reaction. Analyses of these structures identify key residues involved in the binding of these ligands. Comparisons among these structures and with the previously reported structures of other NADP-IDHs reveal that eukaryotic NADP-IDHs undergo substantial conformational changes during the catalytic reaction. Binding or release of the ligands can cause significant conformational changes of the structural elements composing the active site, leading to rotation of the large domain relative to the small and clasp domains along two hinge regions (residues 118-124 and residues 284-287) while maintaining the integrity of its secondary structural elements, and thus, formation of at least three distinct overall conformations. Specifically, the enzyme adopts an open conformation when bound to NADP, a quasi-closed conformation when bound to ICT or AKG, and a fully closed conformation when bound to NADP, ICT, and Ca(2+) in the pseudo-Michaelis complex or with NADPH, AKG, and Ca(2+) in the product state. The conformational changes of eukaryotic NADP-IDHs are quite different from those of Escherichia coli NADP-IDH, for which significant conformational changes are observed only between two forms of the apo enzyme, suggesting that the catalytic mechanism of eukaryotic NADP-IDHs is more complex than that of EcIDH, and involves more fine-tuned conformational changes.     

Inactivation of soluble 17 beta-hydroxysteroid dehydrogenase of human placenta by fatty acids

The sensitivity of soluble, 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) of human placenta to inactivation by fatty acids was examined. Exposure to the unsaturated fatty acids oleic, arachidonic, linoleic and linolenic acid resulted in the loss of activity. Methyl and ethyl esters of oleic acid, the saturated fatty acid, stearic acid and prostaglandins E2 and F2 alpha were without effect. Inactivation by oleic acid required the fatty acid at levels above its critical micelle concentration, 50 microM, as estimated by light-scattering. Steroid substrates and inhibitors did not protect against inactivation. NAD+, NADH, NADP+ and NADPH did protect. The concentrations of NADP+, 50 microM, and NAD, 1.5 mM, necessary for complete protection were significantly greater than their respective Michaelis constants, 0.16 microM and 15.2 microM. The data suggest that soluble 17 beta-HSD can bind to fatty acid micelles and that the binding site(s) on the enzyme are at or near pyridine nucleotide binding sites.     

Structure and mechanism of Escherichia coli pyridoxine 5'-phosphate oxidase

Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes the oxidation of either pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP), forming pyridoxal 5'-phosphate (PLP). This reaction serves as the terminal step in the de novo biosynthesis of PLP in E. coli and as a part of the salvage pathway of this coenzyme in both E. coli and mammalian cells. Recent studies have shown that in addition to the active site, PNPOx contains a noncatalytic site that binds PLP tightly. The crystal structures of PNPOx with one and two molecules of PLP bound have been determined. In the active site, the PLP pyridine ring is stacked almost parallel against the re-face of the middle ring of flavin mononucleotide (FMN). A large protein conformational change occurs upon binding of PLP. When the protein is soaked with excess PLP an additional molecule of this cofactor is bound about 11 A from the active site. A possible tunnel exists between the two sites. Site mutants were made of all residues at the active site that make interactions with the substrate. Stereospecificity studies showed that the enzyme is specific for removal of the proR hydrogen atom from the prochiral C4' carbon of PMP. The crystal structure and the stereospecificity studies suggest that the pair of electrons on C4' of the substrate are transferred to FMN as a hydride ion.     

Crystal structure of human type III 3alpha-hydroxysteroid dehydrogenase/bile acid binding protein complexed with NADP(+) and ursodeoxycholate

The crystal structure of human type III 3alpha-hydroxysteroid dehydrogenase (HSD)/bile acid binding protein (AKR1C2) complexed with NADP(+) and 3alpha,7beta-dihydroxy-5beta-cholanic acid (ursodeoxycholate) at 3.0 A resolution is presented. Thus, the three-dimensional structure has now been solved for a human HSD member of the aldo-keto reductase superfamily. AKR1C2 is implicated in the prostatic production of the potent androgen 5alpha-dihydrotestosterone and the hepatic transport of bile acids. It also catalyzes the formation of the neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one in the central nervous system, and its allosteric modulation by fluoxetine has been linked to the use of this drug for premenstrual dsyphoria. Like other members of the superfamily, AKR1C2 folds into an alpha/beta-barrel and binds NADP(+) in an extended conformation. The carboxylate of ursodeoxycholate binds to AKR1C2 in the oxyanion hole at the active site. More interestingly, the orientation of ursodeoxycholate is essentially "backwards" and "upside-down" from that observed for testosterone in the related rat 3alpha-HSD.NADP(+).testosterone ternary complex, where testosterone assumes the position of a 3-ketosteroid substrate. The orientation of ursodeoxycholate is thus similar to that expected of a 17beta-HSD substrate. The ternary structure explains the ability of AKR1C2 to catalyze 3alpha-, 17beta-, and 20alpha-HSD reactions. Comparison of the steroid binding pocket of AKR1C2 with that of rat 3alpha-HSD reveals significant differences in the positions of conserved and nonconserved loop residues, providing insights into the structural basis for the functional flexibility that is observed in all the human 3alpha-HSD isoforms but not in the rat isoform.     

Crystal structure of murine 11 beta-hydroxysteroid dehydrogenase 1: an important therapeutic target for diabetes

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the conversion of 11-dehydrocorticosterone to its active form corticosterone in rodents (or cortisone to cortisol in humans). The reductive reaction of the 11-keto to 11-hydroxyl is the pivotal switch in the activation of glucocorticoids. An excess of active glucocorticoids has been shown to play a key role in metabolic disorders such as diabetes and obesity. Therefore, 11beta-HSD1 represents an important therapeutic target for the treatment of these diseases. To facilitate the iterative design of inhibitors, we have crystallized and determined the three-dimensional structures of a binary complex of murine 11beta-HSD1 with NADP(H) to a resolution of 2.3 A and of a ternary complex with corticosterone and NADP(H) to a resolution of 3.0 A by X-ray crystallography. The enzyme forms a homodimer in the crystal and has a fold similar to those of other members of the family of short chain steroid dehydrogenases/reductases (SDRs). The structure shows a novel folding feature at the C-terminus of the enzyme. The C-terminal helix insertions provide additional dimer contacts, exert an influence on the conformations of the substrate binding loops, and present hydrophobic regions for potential membrane attachment. The structure also reveals how 11beta-HSD1 achieves its selectivity for its substrate.     

Crystal structure of human liver Delta4-3-ketosteroid 5beta-reductase (AKR1D1) and implications for substrate binding and catalysis

AKR1D1 (steroid 5beta-reductase) reduces all Delta(4)-3-ketosteroids to form 5beta-dihydrosteroids, a first step in the clearance of steroid hormones and an essential step in the synthesis of all bile acids. The reduction of the carbon-carbon double bond in an alpha,beta-unsaturated ketone by 5beta-reductase is a unique reaction in steroid enzymology because hydride transfer from NADPH to the beta-face of a Delta(4)-3-ketosteroid yields a cis-A/B-ring configuration with an approximately 90 degrees bend in steroid structure. Here, we report the first x-ray crystal structure of a mammalian steroid hormone carbon-carbon double bond reductase, human Delta(4)-3-ketosteroid 5beta-reductase (AKR1D1), and its complexes with intact substrates. We have determined the structures of AKR1D1 complexes with NADP(+) at 1.79- and 1.35-A resolution (HEPES bound in the active site), NADP(+) and cortisone at 1.90-A resolution, NADP(+) and progesterone at 2.03-A resolution, and NADP(+) and testosterone at 1.62-A resolution. Complexes with cortisone and progesterone reveal productive substrate binding orientations based on the proximity of each steroid carbon-carbon double bond to the re-face of the nicotinamide ring of NADP(+). This orientation would permit 4-pro-(R)-hydride transfer from NADPH. Each steroid carbonyl accepts hydrogen bonds from catalytic residues Tyr(58) and Glu(120). The Y58F and E120A mutants are devoid of activity, supporting a role for this dyad in the catalytic mechanism. Intriguingly, testosterone binds nonproductively, thereby rationalizing the substrate inhibition observed with this particular steroid. The locations of disease-linked mutations thought to be responsible for bile acid deficiency are also revealed.     

Identification of covalent modifications in P450 2E1 by 1,2-epoxy-3-butene in vitro

1,3-Butadiene is metabolized mainly by cytochrome P450 2E1 to several epoxides that are considered toxic and carcinogenic. The first step of BD metabolism is oxidation to 1,2-epoxy-3-butene (EB), a reactive metabolite. It has been shown that P450s can be inactivated by covalent binding of reactive metabolites to protein or heme. Molecular dosimetry studies have clearly shown that BD metabolism follows a supralinear dose response, suggestive of saturation of metabolic activation. In this study, potential binding sites of EB in human P450 2E1 were identified and modeled to test whether EB covalently binds to residues important for enzyme activity. Commercially available human P450 2E1 was reacted with EB, digested with trypsin and the resulting peptides were analyzed by Matrix-Assisted Laser Desorption/Ionization tandem Time-of-Flight mass spectrometry (MALDI-MS). The identity of EB modified peptides was confirmed by Matrix-Assisted Laser Desorption/Ionization tandem mass spectrometry (MALDI-MS/MS) sequencing. It was shown that EB binds to four histidine and two tyrosine residues. All modification sites were assigned by at least two adjacent and a minimum of eight peptide specific fragments. Protein modeling revealed that two of these covalent modifications (His(109), His(370)) are clearly associated with the active site, and that their Calpha atoms are located less than 9A from a known inhibitor binding site. In addition, the side chain of His(370) is within 4A of the heme group and its modification is expected to influence the orientation of the heme. The Calpha atom of Tyr(71) is within 14A of the potential inhibitor binding site and within 7A of the flap undergoing conformational change upon ligand binding, potentially placing Tyr(71) near the substrate as it enters and leaves the active site. The data support the hypothesis that EB can inactivate P450 2E1 by covalent modifications and thus add an additional regulatory mechanism for BD metabolism.     

5Alpha-androstane-3beta,7alpha,17beta-triol and 5alpha-androstane-3beta,7beta,17beta-triol as substrates for the human 11beta-hydroxysteroid dehydrogenase type 1

Several studies have shown that the native 7alpha-hydroxy-dehydroepiandrosterone (7alpha-hydroxy-DHEA) is a substrate for the human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) which converts the 7alpha- into the 7beta-epimer through an oxido-reduction process. Research on the 11beta-HSD1 has investigated its function and structure through using native glucocorticoid substrates and known inhibitors. Other steroid substrates are also of interest. Among testosterone metabolites, 5alpha-androstane-3beta,17beta-diol (Adiol) is a substrate for the cytochrome P450 7B1 which produces 5alpha-androstane-3beta,7alpha,17beta-triol (7alpha-Adiol). This steroid may be a substrate for the 11beta-HSD1. We used recombinant yeast-expressed 11beta-HSD1 with NADP(H)-regenerating systems for examining the products obtained after incubation with 7alpha-Adiol, 7beta-Adiol or 7-oxo-Adiol. Oxidative conditions for the 11beta-HSD1 provided no trace of 7-oxo-Adiol but the inter-conversion of 7alpha- and 7beta-hydroxy-Adiol with V(max)/K(M) (pmol min(-1) microg(-1)/microM) values of 2 and 0.5, respectively. This state was maintained under reductive conditions. The use of a 7-oxo-Adiol substrate under reductive conditions led to the production of both 7alpha- and 7beta-hydroxy-Adiol with V(max)/K(M) values of 3.43 and 0.22, respectively. These findings support the hypothesis that the oxido-reductase and epimerase activities of 11beta-HSD1 depend on the positioning of the steroid substrates within the active site and may provide insight into its fine structure and mechanism of action.     

Kinetic and stereochemical studies on reaction mechanism of mouse liver 17 beta-hydroxysteroid dehydrogenases

The kinetic mechanism of two major monomeric 17 beta-hydroxysteroid dehydrogenases from mouse liver cytosol was studied at pH 7 in both directions with NADP(H) and three steroid substrates: testosterone, 5 beta-androstane-3 alpha, 17 17 beta-diol, and estradiol-17 beta. In each case the reaction mechanism of the two enzymes was sequential, and inhibition patterns by-products and dead-end inhibitors were consisted with an ordered bi bi mechanism with the coenzyme binding to the free enzyme, although there was difference in affinity and maximum velocity for the steroidal substrates between the two enzymes. Binding studies of the coenzyme and substrate indicate the binding of coenzyme to the free enzyme, in which 1 mol of NADPH binds to 1 mol of each monomeric enzyme. The 4-pro-R-hydrogen atom of NADPH was transferred to the alpha-face of the steroid molecule by the two enzymes.     

Affinity labeling of arginyl residues at the catalytic region of estradiol 17 beta-dehydrogenase from human placenta by 16-oxoestrone

16-Oxoestrone inhibited competitively the activity of estradiol 17 beta-dehydrogenase from human placenta against estradiol in phosphate buffer (pH 7.2), suggesting reversible binding of 16-oxoestrone to the substrate-binding site. 16-Oxoestrone irreversible inactivated the estradiol 17 beta-dehydrogenase in borate buffer (pH 8.5) in a time-dependent manner, following pseudo-first-order kinetics. The rate constant (k3) obtained for the inactivation by 16-oxoestrone was 8.3 x 10(-4) s-1. The rate of inactivation was significantly decreased by addition of estrone, estradiol, estriol, NAD(H) and NADP+. Also, the rate was reduced markedly by 2'AMP, 5'ATP and 2',5' ADP, but not by NMN(H) and 3-pyridinealdehyde adeninediphospho nucleotide. The inactivation by 16-oxoestrone was neither prevented by sodium azide nor influenced by light. From these data, 16-oxoestrone, an alpha-dicarbonyl steroid, was suggested to inactive estradiol 17 beta-dehydrogenase by modification of arginyl residues located around the substrate-binding site of the enzyme. Biphasic inactivation of the enzyme by 16-oxoestrone was observed with an increase of modified arginyl residues. The first phase of the inactivation was regarded as an affinity labeling of the arginyl residues at or near the substrate-binding site of the enzyme. Stoichiometry of the inactivation indicated that two arginyl residues were essential for maintenance of the enzyme activity. The second phase was considered as chemical modification of the arginyl residues outside of the catalytic region of the enzyme.     

Molecular determinants of steroid recognition and catalysis in aldo-keto reductases. Lessons from 3alpha-hydroxysteroid dehydrogenase.

Hydroxysteroid Dehydrogenases (HSDs) regulate the occupancy of steroid hormone receptors by converting active steroid hormones into their cognate inactive metabolites. HSDs belong to either the Short-chain Dehydrogenase/Reductases (SDRs) or the Aldo-Keto Reductases (AKRs). The AKRs include virtually all mammalian 3alpha-HSDs, Type 5 17beta-HSD, ovarian 20alpha-HSDs as well as the steroid 5beta-reductases. Selective inhibitors of 3alpha-HSD isoforms could control occupancy of the androgen and GABA(A) receptors, while broader based AKR inhibitors targeting 3alpha-HSD, 20alpha-HSD and prostaglandin F2alpha synthase could maintain pregnancy. We have determined three X-ray crystal structures of rat liver 3alpha-HSD, a representative AKR. These structures are of the apoenzyme (E), the binary-complex (E.NADP-), and the ternary complex (E.NADP+.testosterone). These structures are being used with site-directed mutagenesis to define the molecular determinants of steroid recognition and catalysis as a first step in rational inhibitor design. A conserved catalytic tetrad (Tyr55, Lys84, His117 and Asp50) participates in a 'proton-relay' in which Tyr55 acts as general acid/base catalyst. Its bifunctionality relies on contributions from His117 and Lys84 which alter the pKb and pKa, respectively of this residue. Point mutation of the tetrad results in different enzymatic activities. H117E mutants display 5beta-reductase activity while Y55F and Y55S mutants retain quinone reductase activity. Our results suggest that different transition states are involved in these reaction mechanisms. The ternary complex structure shows that the mature steroid binding pocket is comprised of ten residues recruited from five loops, and that there is significant movement of a C-terminal loop on binding ligand. Mutagenesis of pocket tryptophans shows that steroid substrates and classes of nonsteroidal inhibitors exhibit different binding modes which may reflect ligand-induced loop movement. Exploitation of these findings using steroidal and nonsteroidal mechanism based inactivators may lead to selective and broad based AKR inhibitors.     

Detection and characterization of two ATP-dependent conformational changes in proteolytically inactive Escherichia coli Lon mutants by stopped flow kinetic techniques

Lon is an ATP dependent serine protease responsible for degrading denatured, oxidatively damaged and certain regulatory proteins in the cell. In this study we exploited the fluorescence properties of a dansylated peptide substrate (S4) and the intrinsic Trp residues in Lon to monitor peptide interacting with the enzyme. We generated two proteolytically inactive Lon mutants, S679A and S679W, where the active site serine is mutated to an Ala and Trp residue, respectively. Stopped-flow fluorescence spectroscopy was used to identify key enzyme intermediates generated along the reaction pathway prior to peptide hydrolysis. A two-step peptide binding event is detected in both mutants, where a conformational change occurs after a rapid equilibrium peptide binding step. The Kd for the initial peptide binding step determined by kinetic and equilibrium binding techniques is approximately 164 micromolar and 38 micromolar, respectively. The rate constants for the conformational change detected in the S679A and S679W Lon mutants are 0.74 +/- 0.10 s(-1) and 0.57 +/- 0.10 s(-1), respectively. These values are comparable to the lag rate constant determined for peptide hydrolysis (klag approximately 1 s(-1)) [Vineyard, D., et al. (2005) Biochemistry 45, 4602-4610]. Replacement of the active site Ser with Trp (S679W) allows for the detection of an ATP-dependent conformational change within the proteolytic site. The rate constant for this conformational change is 7.6 +/- 1.0 s(-1), and is essentially identical to the burst rate constant determined for ATP hydrolysis under comparable reaction conditions. Collectively, these kinetic data support a mechanism by which the binding of ATP to an allosteric site on Lon activates the proteolytic site. In this model, the energy derived from the binding of ATP minimally supports peptide cleavage by allowing peptide substrate access to the proteolytic site. However, the kinetics of peptide cleavage are enhanced by the hydrolysis of ATP.     

High-level production and optimization of monodispersity of 11beta-hydroxysteroid dehydrogenase type 1

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is an intraluminally oriented, endoplasmic reticulum (ER)-bound enzyme catalyzing the interconversion between inactive cortisone and hormonally active cortisol. Heterologous production of 11beta-HSD1, devoid of its N-terminal transmembrane segment, is possible but yields only small amounts of soluble protein. Here we show that the soluble portion of recombinant 11beta-HSD1 produced in E. coli is found mainly as multimeric aggregates in the absence of detergent, and to a large extent associated with the endogenous chaperonin GroEL and other E. coli proteins. By co-overexpressing GroEL/ES and adding an 11beta-HSD1 inhibitor during protein synthesis, we have increased the accumulation of soluble 11beta-HSD1 by more than one order of magnitude. Using monodispersity as a screening criterion, we have also optimized the purification process by evaluating various solubilizing systems for the chromatographic steps, finally obtaining stable monodisperse preparations of both human and guinea pig 11beta-HSD1. By analytical ultracentrifugation, we could demonstrate that 11beta-HSD1 mainly exists as a dimer in the solubilized state. Moreover, active site titration of human 11beta-HSD1 revealed that at least 75% of the protein in a typical preparation represents active enzyme. Equilibrium unfolding experiments indicate that addition of inhibitor and the cofactor NADP(H) can stabilize the conformational stability of this enzyme in an additive manner. The outlined procedure may provide a general method for preparing similar proteins to oligomeric homogeneity and with retained biological activity.     

Mouse 17alpha-hydroxysteroid dehydrogenase (AKR1C21) binds steroids differently from other aldo-keto reductases: identification and characterization of amino acid residues critical for substrate binding

The mouse 17alpha-hydroxysteroid dehydrogenase (m17alpha-HSD) is the unique known member of the aldo-keto reductase (AKR) superfamily able to catalyze efficiently and in a stereospecific manner the conversion of androstenedione (Delta4) into epi-testosterone (epi-T), the 17alpha-epimer of testosterone. Structural and mutagenic studies had already identified one of the residues delineating the steroid-binding cavity, A24, as the major molecular determinant for the stereospecificity of m17alpha-HSD. We report here a ternary complex crystal structure (m17alpha-HSD:NADP(+):epi-T) determined at 1.85 A resolution that confirms this and reveals a unique steroid-binding mode for an AKR enzyme. Indeed, in addition to the interactions found in all other AKRs (van der Waals contacts stabilizing the core of the steroid and the hydrogen bonds established at the catalytic site by the Y55 and H117 residues with the oxygen atom of the ketone group to be reduced), m17alpha-HSD establishes with the other extremity of the steroid nucleus an additional interaction involving K31. By combining direct mutagenesis and kinetic studies, we found that the elimination of this hydrogen bond did not affect the affinity of the enzyme for its steroid substrate but led to a slight but significant increase of its catalytic efficiency (k(cat)/K(m)), suggesting a role for K31 in the release of the steroidal product at the end of the reaction. This previously unobserved steroid-binding mode for an AKR is similar to that adopted by other steroid-binding proteins, the hydroxysteroid dehydrogenases of the short-chain dehydrogenases/reductases (SDR) family and the steroid hormone nuclear receptors. Mutagenesis and structural studies made on the human type 3 3alpha-HSD, a closely related enzyme that shares 73% amino acids identity with the m17alpha-HSD, also revealed that the residue at position 24 of these two enzymes directly affects the binding and/or the release of NADPH, in addition to its role in their 17alpha/17beta stereospecificity.