Molecular modeling and site-directed mutagenesis reveal the benzylisoquinoline binding site of the short-chain dehydrogenase/reductase salutaridine reductase

Recently, the NADPH-dependent short-chain dehydrogenase/reductase (SDR) salutaridine reductase (E.C. 1.1.1.248) implicated in morphine biosynthesis was cloned from Papaver somniferum. In this report, a homology model of the Papaver bracteatum homolog was created based on the x-ray structure of human carbonyl reductase 1. The model shows the typical alpha/beta-folding pattern of SDRs, including the four additional helices alphaF'-1 to alphaF'-4 assumed to prevent the dimerization of the monomeric short-chain dehydrogenases/reductases. Site-directed mutagenesis of asparagine-152, serine-180, tyrosine-236, and lysine-240 resulted in enzyme variants with strongly reduced performance or inactive enzymes, showing the involvement of these residues in the proton transfer system for the reduction of salutaridine. The strong preference for NADPH over NADH could be abolished by replacement of arginine residues 44 and 48 by glutamic acid, confirming the interaction between the arginines and the 2'-phosphate group. Docking of salutaridine into the active site revealed nine amino acids presumably responsible for the high substrate specificity of salutaridine reductase. Some of these residues are arranged in the right position by an additional alphaE' helix, which is not present in SDRs analyzed so far. Enzyme kinetic data from mutagenic replacement emphasize the critical role of these residues in salutaridine binding and provide the first data on the molecular interaction of benzylisoquinoline alkaloids with enzymes.     

Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis

Plants of the order Ranunculales, especially members of the species Papaver, accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the antitussive codeine. In this study, we investigated the molecular basis for this exceptional biosynthetic capability by comparison of alkaloid profiles with gene expression profiles between 16 different Papaver species. Out of 2000 expressed sequence tags obtained from P. somniferum, 69 show increased expression in morphinan alkaloid-containing species. One of these cDNAs, exhibiting an expression pattern very similar to previously isolated cDNAs coding for enzymes in benzylisoquinoline biosynthesis, showed the highest amino acid identity to reductases in menthol biosynthesis. After overexpression, the protein encoded by this cDNA reduced the keto group of salutaridine yielding salutaridinol, an intermediate in morphine biosynthesis. The stereoisomer 7-epi-salutaridinol was not formed. Based on its similarities to a previously purified protein from P. somniferum with respect to the high substrate specificity, molecular mass and kinetic data, the recombinant protein was identified as salutaridine reductase (SalR; EC 1.1.1.248). Unlike codeinone reductase, an enzyme acting later in the pathway that catalyses the reduction of a keto group and which belongs to the family of the aldo-keto reductases, the cDNA identified in this study as SalR belongs to the family of short chain dehydrogenases/reductases and is related to reductases in monoterpene metabolism.     

Removal of Substrate Inhibition and Increase in Maximal Velocity in the Short Chain Dehydrogenase/Reductase Salutaridine Reductase Involved in Morphine Biosynthesis

Salutaridine reductase (SalR, EC 1.1.1.248) catalyzes the stereospecific reduction of salutaridine to 7(S)-salutaridinol in the biosynthesis of morphine. It belongs to a new, plant-specific class of short-chain dehydrogenases, which are characterized by their monomeric nature and increased length compared with related enzymes. Homology modeling and substrate docking suggested that additional amino acids form a novel α-helical element, which is involved in substrate binding. Site-directed mutagenesis and subsequent studies on enzyme kinetics revealed the importance of three residues in this element for substrate binding. Further replacement of eight additional residues led to the characterization of the entire substrate binding pocket. In addition, a specific role in salutaridine binding by either hydrogen bond formation or hydrophobic interactions was assigned to each amino acid. Substrate docking also revealed an alternative mode for salutaridine binding, which could explain the strong substrate inhibition of SalR. An alternate arrangement of salutaridine in the enzyme was corroborated by the effect of various amino acid substitutions on substrate inhibition. In most cases, the complete removal of substrate inhibition was accompanied by a substantial loss in enzyme activity. However, some mutations greatly reduced substrate inhibition while maintaining or even increasing the maximal velocity. Based on these results, a double mutant of SalR was created that exhibited the complete absence of substrate inhibition and higher activity compared with wild-type SalR.The benzylisoquinoline alkaloids (BIAs)³ comprise a large and diverse group of nitrogen-containing secondary metabolites with about 2500 compounds identified in plants (1). Among them are several important pharmaceuticals, such as the antimicrobials berberine and sanguinarine, and the vasodilator papaverine. The most prominent compounds of this class are the antitussive codeine, the analgesic morphine, and their biosynthetic precursor thebaine. The latter is used as the starting molecule for the production of a variety of semi-synthetic analgesics including oxycontin and buprenorphine. Pentacyclic morphinan alkaloids possess several chiral centers, which preclude chemical synthesis as an option for the efficient production of these widely used pharmaceuticals. Therefore, the worldwide supply of these narcotic compounds is still achieved by their isolation mainly from the opium poppy, Papaver somniferum L. With the availability of an increasing number of isolated genes encoding several pathway enzymes, recent interest has focused on the qualitative and quantitative modulation of the alkaloid profile in transgenic opium poppy plants (2–6), the production of BIAs in microbes (7, 8), and de novo synthesis by a combination of chemical and biochemical conversions. BIA biosynthesis begins with the condensation of the tyrosine-derived precursors dopamine and p-hydroxyphenylacetaldehyde to (S)-norcoclaurine (see Fig. 1) (1). Subsequent regiospecific O- and N-methylations and aromatic ring hydroxylation lead to (S)-reticuline, which is the central intermediate for almost all BIAs. For morphinan alkaloid biosynthesis, (S)-reticuline undergoes an inversion of stereochemistry to (R)-reticuline, followed by C-C phenol coupling catalyzed by a unique cytochrome P450-dependent monooxygenase to yield salutaridine. Subsequent stereospecific reduction to 7(S)-salutaridinol is required for the attachment of an acetyl moiety to produce salutaridinol-7-O-acetate, which spontaneously rearranges to thebaine (9). The O-demethylation of thebaine and the reduction of codeinone to codeine represent the penultimate steps in morphine biosynthesis. Cognate cDNAs have been isolated for all of the enzymes leading to (S)-reticuline, as well as those involved in the conversion of (R)-reticuline to salutaridine-7-O-acetate (1). Salutaridine reductase (SalR, EC 1.1.1.248) catalyzes the stereospecific, NADPH-dependent reduction of salutaridine to 7(S)-salutaridinol and is a member of the classical subgroup of the short chain dehydrogenase/reductase (SDR) protein family (10, 11). The main characteristics of this category of SDRs are the largely conserved TGXXXGhG motif for cofactor binding and the YXXXK motif, which together with an upstream Ser residue represent the catalytic center (12). In this catalytic triad, Lys forms hydrogen bonds with the ribose moiety of the cofactor, which itself is hydrogen bonded to Tyr. This hydrogen bond network is presumed to lower the pK_a of the Tyr hydroxyl group, which functions as the catalytic base. Ser has been suggested to either stabilize the substrate (13, 14) or to interact with Tyr (15). Additionally, an Asn residue has been proposed to stabilize the position of the Lys residue, thereby forming a proton relay system involving water (16). Most other members of the SDR protein family are categorized into three additional subgroups (i.e. divergent, intermediate, or complex) exhibiting different overall sizes and slight amino acid sequence variations in conserved regions (17). Non-classical SDRs predominantly consist of isomerases (EC 5.-.-.-), such as galactose epimerase, and lyases (EC 4.-.-.-), such as glucose dehydratase, whereas classical SDRs encompass oxidoreductases (EC 1.-.-.-), such as SalR. Although classical SDRs are typically multimeric, SalR is a monomer because of an additional stretch of 40 amino acids preceding the YXXXK catalytic motif. In porcine testicular carbonyl reductase, these amino acids form a helix blocking the dimer interface (18) and homology modeling revealed a similar feature in SalR (19). In contrast with porcine testicular carbonyl reductase, SalR exhibits an additional stretch of 40 amino acids that have only been detected in some SDRs from plants (11, 20–22). Although attempts to obtain a crystal structure for SalR have so far been unsuccessful, homology modeling using porcine testicular carbonyl reductase as a template produced a tertiary structure in which the additional amino acids form an additional helix (19). The subsequent docking of salutaridine into the active site of this model suggested the involvement of this structural element in substrate binding, which was supported by preliminary site-directed mutagenesis.Open in a separate windowFIGURE 1.Selected steps in morphine biosynthesis. Double arrows indicate the involvement of more than one enzyme. The SalR reaction is highlighted. HPAA, p-hydroxyphenylacetaldehyde; SalR, salutaridine reductase.SalR from the Persian poppy Papaver bracteatum L. shows strong substrate inhibition with a K_i around 150 μm (19). Substrate inhibition can substantially and negatively impact chemical engineering strategies by limiting the quantity of substrate that can be fed into a system, which reduces overall efficiency. The strong substrate inhibition exhibited by SalR could limit its biotechnological application in plant, microbial, or enzyme-based systems. To investigate the structural basis of substrate inhibition, we substituted all amino acids putatively involved in salutaridine binding and analyzed various kinetic parameters. Over the course of these experiments, substrate docking required modification because some mutations had unexpected consequences that did not fully agree with the original docking. The discrepancy was mainly due to the side chain arrangements of amino acids residing in the new helix. Precise prediction of this domain is difficult because of the lack of an equivalent crystal structure. In this report, we present a revised substrate docking for salutaridine into SalR, supported by comprehensive site-directed mutagenesis, which facilitated the creation of an enzyme variant devoid of substrate inhibition.     

Crystal structure of meso-2,3-butanediol dehydrogenase in a complex with NAD+ and inhibitor mercaptoethanol at 1.7 A resolution for understanding of chiral substrate recognition mechanisms

The crystal structure of a ternary complex of meso-2,3-butanediol dehydrogenase with NAD+ and a competitive inhibitor, mercaptoethanol, has been determined at 1.7 A resolution by means of molecular replacement and refined to a final R-factor of 0.194. The overall structure is similar to those of the other short chain dehydrogenase/reductase enzymes. The NAD+ binding site, and the positions of catalytic residues Ser139, Tyr152, and Lys156 are also conserved. The crystal structure revealed that mercaptoethanol bound specifically to meso-2,3-butanediol dehydrogenase. Two residues around the active site, Gln140 and Gly183, forming hydrogen bonds with the inhibitor, are important but not sufficient for distinguishing stereoisomerism of a chiral substrate.     

Stereochemical course and steady state mechanism of the reaction catalyzed by the GDP-fucose synthetase from Escherichia coli.

Recently the genes encoding the human and Escherichia coli GDP-mannose dehydratase and GDP-fucose synthetase (GFS) protein have been cloned and it has been shown that these two proteins alone are sufficient to convert GDP mannose to GDP fucose in vitro. GDP-fucose synthetase from E. coli is a novel dual function enzyme in that it catalyzes epimerizations and a reduction reaction at the same active site. This aspect separates fucose biosynthesis from that of other deoxy and dideoxy sugars in which the epimerase and reductase activities are present on separate enzymes encoded by separate genes. By NMR spectroscopy we have shown that GFS catalyzes the stereospecific hydride transfer of the ProS hydrogen from NADPH to carbon 4 of the mannose sugar. This is consistent with the stereospecificity observed for other members of the short chain dehydrogenase reductase family of enzymes of which GFS is a member. Additionally the enzyme is able to catalyze the epimerization reaction in the absence of NADP or NADPH. The kinetic mechanism of GFS as determined by product inhibition and fluorescence binding studies is consistent with a random mechanism. The dissociation constants determined from fluorescence studies indicate that the enzyme displays a 40-fold stronger affinity for the substrate NADPH as compared with the product NADP and utilizes NADPH preferentially as compared with NADH. This study on GFS, a unique member of the short chain dehydrogenase reductase family, coupled with that of its recently published crystal structure should aid in the development of antimicrobial or anti-inflammatory compounds that act by blocking selectin-mediated cell adhesion.     

Identification of lysine-78 as an essential residue in the Saccharomyces cerevisiae xylose reductase

Yeast xylose reductases are hypothesized as hybrid enzymes as their primary sequences contain elements of both the aldo-keto reductases (AKR) and short chain dehydrogenase/reductase (SDR) enzyme families. During catalysis by members of both enzyme families, an essential Lys residue H-bonds to a Tyr residue that donates proton to the aldehyde substrate. In the Saccharomyces cerevisiae xylose reductase, Tyr49 has been identified as the proton donor. However, the primary sequence of the enzyme contains two Lys residues, Lys53 and Lys78, corresponding to the conserved motifs for SDR and AKR enzyme families, respectively, that may H-bond to Tyr49. We used site-directed mutagenesis to substitute each of these Lys residues with Met. The activity of the K53M variant was slightly decreased as compared to the wild-type, while that of the K78M variant was negligible. The results suggest that Lys78 is the essential residue that H-bonds to Tyr49 during catalysis and indicate that the active site residues of yeast xylose reductases match those of the AKR, rather than SDR, enzymes. Intrinsic enzyme fluorescence spectroscopic analysis suggests that Lys78 may also contribute to the efficient binding of NADPH to the enzyme.     

Insights into diterpene cyclization from structure of bifunctional abietadiene synthase from Abies grandis

Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 Å resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains (α, β, and γ). The class I active site is within the C-terminal α domain, and the class II active site is between the N-terminal γ and β domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of terpene synthases. Molecular dynamics simulations were carried out to determine the effect of substrate binding on enzymatic structure. Although such studies of the class I active site do lead to an enclosed substrate-Mg²⁺ complex similar to that observed in crystal structures of related plant enzymes, it does not enforce a single substrate conformation consistent with the known product stereochemistry. Simulations of the class II active site were more informative, with observation of a well ordered external loop migration. This “loop-in” conformation not only limits solvent access but also greatly increases the number of conformational states accessible to the substrate while destabilizing the nonproductive substrate conformation present in the “loop-out” conformation. Moreover, these conformational changes at the class II active site drive the substrate toward the proposed transition state. Docked substrate complexes were further assessed with regard to the effects of site-directed mutations on class I and II activities.     

Structure and reaction mechanism of phosphoethanolamine methyltransferase from the malaria parasite Plasmodium falciparum: an antiparasitic drug target

In the malarial parasite Plasmodium falciparum, a multifunctional phosphoethanolamine methyltransferase (PfPMT) catalyzes the methylation of phosphoethanolamine (pEA) to phosphocholine for membrane biogenesis. This pathway is also found in plant and nematodes, but PMT from these organisms use multiple methyltransferase domains for the S-adenosylmethionine (AdoMet) reactions. Because PfPMT is essential for normal growth and survival of Plasmodium and is not found in humans, it is an antiparasitic target. Here we describe the 1.55 Å resolution crystal structure of PfPMT in complex with AdoMet by single-wavelength anomalous dispersion phasing. In addition, 1.19–1.52 Å resolution structures of PfPMT with pEA (substrate), phosphocholine (product), sinefungin (inhibitor), and both pEA and S-adenosylhomocysteine bound were determined. These structures suggest that domain rearrangements occur upon ligand binding and provide insight on active site architecture defining the AdoMet and phosphobase binding sites. Functional characterization of 27 site-directed mutants identifies critical active site residues and suggests that Tyr-19 and His-132 form a catalytic dyad. Kinetic analysis, isothermal titration calorimetry, and protein crystallography of the Y19F and H132A mutants suggest a reaction mechanism for the PMT. Not only are Tyr-19 and His-132 required for phosphobase methylation, but they also form a “catalytic” latch that locks ligands in the active site and orders the site for catalysis. This study provides the first insight on this antiparasitic target enzyme essential for survival of the malaria parasite; however, further studies of the multidomain PMT from plants and nematodes are needed to understand the evolutionary division of metabolic function in the phosphobase pathway of these organisms.     

Crystal structure of human L-xylulose reductase holoenzyme: probing the role of Asn107 with site-directed mutagenesis

L-Xylulose reductase (XR), an enzyme in the uronate cycle of glucose metabolism, belongs to the short-chain dehydrogenase/reductase (SDR) superfamily. Among the SDR enzymes, XR shows the highest sequence identity (67%) with mouse lung carbonyl reductase (MLCR), but the two enzymes show different substrate specificities. The crystal structure of human XR in complex with reduced nicotinamide adenine dinucleotide phosphate (NADPH) was determined at 1.96 A resolution by using the molecular replacement method and the structure of MLCR as the search model. Features unique to human XR include electrostatic interactions between the N-terminal residues of subunits related by the P-axis, termed according to SDR convention, and an interaction between the hydroxy group of Ser185 and the pyrophosphate of NADPH. Furthermore, identification of the residues lining the active site of XR (Cys138, Val143, His146, Trp191, and Met200) together with a model structure of XR in complex with L-xylulose, revealed structural differences with other members of the SDR family, which may account for the distinct substrate specificity of XR. The residues comprising a recently proposed catalytic tetrad in the SDR enzymes are conserved in human XR (Asn107, Ser136, Tyr149, and Lys153). To examine the role of Asn107 in the catalytic mechanism of human XR, mutant forms (N107D and N107L) were prepared. The two mutations increased K(m) for the substrate (>26-fold) and K(d) for NADPH (95-fold), but only the N107L mutation significantly decreased k(cat) value. These results suggest that Asn107 plays a critical role in coenzyme binding rather than in the catalytic mechanism.     

Characterization of member of DUF1888 protein family, self-cleaving and self-assembling endopeptidase

The crystal structure of SO1698 protein from Shewanella oneidensis was determined by a SAD method and refined to 1.57 Å. The structure is a β sandwich that unexpectedly consists of two polypeptides; the N-terminal fragment includes residues 1–116, and the C-terminal one includes residues 117–125. Electron density also displayed the Lys-98 side chain covalently linked to Asp-116. The putative active site residues involved in self-cleavage were identified; point mutants were produced and characterized structurally and in a biochemical assay. Numerical simulations utilizing molecular dynamics and hybrid quantum/classical calculations suggest a mechanism involving activation of a water molecule coordinated by a catalytic aspartic acid.     

Structural basis for three-step sequential catalysis by the cholesterol side chain cleavage enzyme CYP11A1

Mitochondrial cytochrome P450 11A1 (CYP11A1 or P450 11A1) is the only known enzyme that cleaves the side chain of cholesterol, yielding pregnenolone, the precursor of all steroid hormones. Pregnenolone is formed via three sequential monooxygenation reactions that involve the progressive production of 22R-hydroxycholesterol (22HC) and 20α,22R-dihydroxycholesterol, followed by the cleavage of the C20-C22 bond. Herein, we present the 2.5-Å crystal structure of CYP11A1 in complex with the first reaction intermediate, 22HC. The active site cavity in CYP11A1 represents a long curved tube that extends from the protein surface to the heme group, the site of catalysis. 22HC occupies two-thirds of the cavity with the 22R-hydroxyl group nearest the heme, 2.56 Å from the iron. The space at the entrance to the active site is not taken up by 22HC but filled with ordered water molecules. The network formed by these water molecules allows the “soft” recognition of the 22HC 3β-hydroxyl. Such a mode of 22HC binding suggests shuttling of the sterol intermediates between the active site entrance and the heme group during the three-step reaction. Translational freedom of 22HC and torsional motion of its aliphatic tail are supported by solution studies. The CYP11A1–22HC co-complex also provides insight into the structural basis of the strict substrate specificity and high catalytic efficiency of the enzyme and highlights conserved structural motifs involved in redox partner interactions by mitochondrial P450s.     

Structural basis for CO2 fixation by a novel member of the disulfide oxidoreductase family of enzymes, 2-ketopropyl-coenzyme M oxidoreductase/carboxylase

The NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is the terminal enzyme in a metabolic pathway that results in the conversion of propylene to the central metabolite acetoacetate in Xanthobacter autotrophicus Py2. This enzyme is an FAD-containing enzyme that is a member of the NADPH:disulfide oxidoreductase (DSOR) family of enzymes that include glutathione reductase, dihydrolipoamide dehydrogenase, trypanothione reductase, thioredoxin reductase, and mercuric reductase. In contrast to the prototypical reactions catalyzed by members of the DSOR family, the NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase catalyzes the reductive cleavage of the thioether linkage of 2-ketopropyl-coenzyme M, and the subsequent carboxylation of the ketopropyl cleavage product, yielding the products acetoacetate and free coenzyme M. The structure of 2-KPCC reveals a unique active site in comparison to those of other members of the DSOR family of enzymes and demonstrates how the enzyme architecture has been adapted for the more sophisticated biochemical reaction. In addition, comparison of the structures in the native state and in the presence of bound substrate indicates the binding of the substrate 2-ketopropyl-coenzyme M induces a conformational change resulting in the collapse of the substrate access channel. The encapsulation of the substrate in this manner is reminiscent of the conformational changes observed in the well-characterized CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxidase (Rubisco).     

Biochemical and structural studies of uncharacterized protein PA0743 from Pseudomonas aeruginosa revealed NAD+-dependent L-serine dehydrogenase

The β-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various β-hydroxy acid substrates to corresponding semialdehydes. Several known enzymes include β-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of β-hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted β-hydroxyisobutyrate dehydrogenase PA0743 from Pseudomonas aeruginosa catalyzes an NAD⁺-dependent oxidation of l-serine and methyl-l-serine but exhibits low activity against β-hydroxyisobutyrate. Two crystal structures of PA0743 were solved at 2.2–2.3-Å resolution and revealed an N-terminal Rossmann fold domain connected by a long α-helix to the C-terminal all-α domain. The PA0743 apostructure showed the presence of additional density modeled as HEPES bound in the interdomain cleft close to the predicted catalytic Lys-171, revealing the molecular details of the PA0743 substrate-binding site. The structure of the PA0743-NAD⁺ complex demonstrated that the opposite side of the enzyme active site accommodates the cofactor, which is also bound near Lys-171. Site-directed mutagenesis of PA0743 emphasized the critical role of four amino acid residues in catalysis including the primary catalytic residue Lys-171. Our results provide further insight into the molecular mechanisms of substrate selectivity and activity of β-hydroxyacid dehydrogenases.     

Structural basis of substrate specificity in human glyoxylate reductase/hydroxypyruvate reductase

Human glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is a D-2-hydroxy-acid dehydrogenase that plays a critical role in the removal of the metabolic by-product glyoxylate from within the liver. Deficiency of this enzyme is the underlying cause of primary hyperoxaluria type 2 (PH2) and leads to increased urinary oxalate levels, formation of kidney stones and renal failure. Here we describe the crystal structure of human GRHPR at 2.2 A resolution. There are four copies of GRHPR in the crystallographic asymmetric unit: in each homodimer, one subunit forms a ternary (enzyme+NADPH+reduced substrate) complex, and the other a binary (enzyme+NADPH) form. The spatial arrangement of the two enzyme domains is the same in binary and ternary forms. This first crystal structure of a true ternary complex of an enzyme from this family demonstrates the relationship of substrate and catalytic residues within the active site, confirming earlier proposals of the mode of substrate binding, stereospecificity and likely catalytic mechanism for these enzymes. GRHPR has an unusual substrate specificity, preferring glyoxylate and hydroxypyruvate, but not pyruvate. A tryptophan residue (Trp141) from the neighbouring subunit of the dimer is projected into the active site region and appears to contribute to the selectivity for hydroxypyruvate. This first crystal structure of a human GRHPR enzyme also explains the deleterious effects of naturally occurring missense mutations of this enzyme that lead to PH2.     

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.     

Structure and function of APH(4)-Ia, a hygromycin B resistance enzyme

The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 Å resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2″) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.     

Crystal structure of human guanosine monophosphate reductase 2 (GMPR2) in complex with GMP

Guanosine monophosphate reductase (GMPR) catalyzes the irreversible and NADPH-dependent reductive deamination of GMP to IMP, and plays a critical role in re-utilization of free intracellular bases and purine nucleosides. Here, we report the first crystal structure of human GMP reductase 2 (hGMPR2) in complex with GMP at 3.0 A resolution. The protein forms a tetramer composed of subunits adopting the ubiquitous (alpha/beta)8 barrel fold. Interestingly, the substrate GMP is bound to hGMPR2 through interactions with Met269, Ser270, Arg286, Ser288, and Gly290; this makes the conformation of the adjacent flexible binding region (residues 268-289) fixed, much like a door on a hinge. Structure comparison and sequence alignment analyses show that the conformation of the active site loop (residues 179-187) is similar to those of hGMPR1 and inosine monophosphate dehydrogenases (IMPDHs). We propose that Cys186 is the potential active site, and that the conformation of the loop (residues 129-133) suggests a preference for the coenzyme NADPH over NADH. This structure provides important information towards understanding the functions of members of the GMPR family.