Crystal structure of S-adenosylhomocysteine hydrolase from rat liver.

The crystal structure of rat liver S-adenosyl-L-homocysteine hydrolase (AdoHcyase, EC 3.3.1.1) which catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) has been determined at 2.8 A resolution. AdoHcyase from rat liver is a tetrameric enzyme with 431 amino acid residues in each identical subunit. The subunit is composed of the catalytic domain, the NAD+-binding domain, and the small C-terminal domain. Both catalytic and NAD+-binding domains are folded into an ellipsoid with a typical alpha/beta twisted open sheet structure. The C-terminal section is far from the main body of the subunit and extends into the opposite subunit. An NAD+ molecule binds to the consensus NAD+-binding cleft of the NAD+-binding domain. The peptide folding pattern of the catalytic domain is quite similar to the patterns observed in many methyltransferases. Although the crystal structure does not contain AdoHcy or its analogue, there is a well-formed AdoHcy-binding crevice in the catalytic domain. Without introducing any major structural changes, an AdoHcy molecule can be placed in the catalytic domain. In the structure described here, the catalytic and NAD+-binding domains are quite far apart from each other. Thus, the enzyme appears to have an "open" conformation in the absence of substrate. It is likely that binding of AdoHcy induces a large conformational change so as to place the ribose moiety of AdoHcy in close proximity to the nicotinamide moiety of NAD+. A catalytic mechanism of AdoHcyase has been proposed on the basis of this crystal structure. Glu155 acts as a proton acceptor from the O3'-H when the proton of C3'-H is abstracted by NAD+. His54 or Asp130 acts as a general acid-base catalyst, while Cys194 modulates the oxidation state of the bound NAD+. The polypeptide folding pattern of the catalytic domain suggests that AdoHcy molecules can travel freely to and from AdoHcyase and methyltransferases to properly regulate methyltransferase activities. We believe that the crystal structure described here can provide insight into the molecular architecture of this important regulatory enzyme.     

Structure of L-aspartate oxidase: implications for the succinate dehydrogenase/fumarate reductase oxidoreductase family.

BACKGROUND: Given the vital role of NAD+ in cell metabolism, the enzymes involved in bacterial de novo NAD+ biosynthesis are possible targets for drug design against pathogenic bacteria. The first reaction in the pathway is catalysed by L-aspartate oxidase (LASPO), a flavoenzyme that converts aspartate to iminoaspartate using either molecular oxygen or fumarate as electron acceptors. LASPO has considerable sequence homology with the flavoprotein subunits of succinate dehydrogenase (SDH) and fumarate reductase (FRD). RESULTS: The crystal structure of the apoform of LASPO from Escherichia coli has been determined to 2.2 A resolution. The enzyme shows a novel fold for an FAD-dependent protein, comprising a three-domain structure: an FAD-binding domain with the dinucleotide-binding fold, a C-terminal three-helical bundle domain, and an alpha + beta capping domain, which is topologically similar to the small subunit of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase. The interface between the FAD-binding and capping domains defines a cleft in which the active site is located. CONCLUSIONS: A number of strictly conserved residues present in all three domains indicate that LASPO, SDH and FRD share the same overall folding topology. Many of these conserved residues are in the FAD-binding site and active centre, suggesting a similar catalytic mechanism. Thus, LASPO, SDH and FRD form a class of functionally and structurally related oxidoreductases that are all able to reduce fumarate and to oxidise a dicarboxylate substrate.     

The 2.3-A crystal structure of the shikimate 5-dehydrogenase orthologue YdiB from Escherichia coli suggests a novel catalytic environment for an NAD-dependent dehydrogenase

We present here the 2.3-A crystal structure of the Escherichia coli YdiB protein, an orthologue of shikimate 5-dehydrogenase. This enzyme catalyzes the reduction of 3-dehydroshikimate to shikimate as part of the shikimate pathway, which is absent in mammals but required for the de novo synthesis of aromatic amino acids, quinones, and folate in many other organisms. In this context, the shikimate pathway has been promoted as a target for the development of antimicrobial agents. The crystal structure of YdiB shows that the protomer contains two alpha/beta domains connected by two alpha-helices, with the N-terminal domain being novel and the C-terminal domain being a Rossmann fold. The NAD+ cofactor, which co-purified with the enzyme, is bound to the Rossmann domain in an elongated fashion with the nicotinamide ring in the pro-R conformation. Its binding site contains several unusual features, including a cysteine residue in close apposition to the nicotinamide ring and a clamp over the ribose of the adenosine moiety formed by phenylalanine and lysine residues. The structure explains the specificity for NAD versus NADP in different members of the shikimate dehydrogenase family on the basis of variations in the amino acid identity of several other residues in the vicinity of this ribose group. A cavity lined by residues that are 100% conserved among all shikimate dehydrogenases is found between the two domains of YdiB, in close proximity to the hydride acceptor site on the nicotinamide ring. Shikimate was modeled into this site in a geometry such that all of its heteroatoms form high quality hydrogen bonds with these invariant residues. Their strong conservation in all orthologues supports the possibility of developing broad spectrum inhibitors of this enzyme. The nature and disposition of the active site residues suggest a novel reaction mechanism in which an aspartate acts as the general acid/base catalyst during the hydride transfer reaction.     

Structure of the Methanococcus jannaschii mevalonate kinase, a member of the GHMP kinase superfamily.

The mevalonate-dependent pathway is used by many organisms to synthesize isopentenyl pyrophosphate, the building block for the biosynthesis of many biologically important compounds, including farnesyl pyrophosphate, dolichol, and many sterols. Mevalonate kinase (MVK) catalyzes a critical phosphoryl transfer step, producing mevalonate 5'-phosphate. The crystal structure of thermostable MVK from Methanococcus jannaschii has been determined at 2.4 A, revealing an overall fold similar to the homoserine kinase from M. jannaschii. In addition, the enzyme shows structural similarity with mevalonate 5-diphosphate decarboxylase and domain IV of elongation factor G. The active site of MVK is in the cleft between its N- and C-terminal domains. Several structural motifs conserved among species, including a phosphate-binding loop, have been found in this cavity. Asp(155), an invariant residue among MVK sequences, is located close to the putative phosphate-binding site and has been assumed to play the catalytic role. Analysis of the MVK model in the context of the other members of the GHMP kinase family offers the opportunity to understand both the mechanism of these enzymes and the structural details that may lead to the design of novel drugs.     

Sequestration of the active site by interdomain shifting. Crystallographic and spectroscopic evidence for distinct conformations of L-3-hydroxyacyl-CoA dehydrogenase

l-3-Hydroxyacyl-CoA dehydrogenase reversibly catalyzes the conversion of l-3-hydroxyacyl-CoA to 3-ketoacyl-CoA concomitant with the reduction of NAD(+) to NADH as part of the beta-oxidation spiral. In this report, crystal structures have been solved for the apoenzyme, binary complexes of the enzyme with reduced cofactor or 3-hydroxybutyryl-CoA substrate, and an abortive ternary complex of the enzyme with NAD(+) and acetoacetyl-CoA. The models illustrate positioning of cofactor and substrate within the active site of the enzyme. Comparison of these structures with the previous model of the enzyme-NAD(+) complex reveals that although significant shifting of the NAD(+)-binding domain relative to the C-terminal domain occurs in the ternary and substrate-bound complexes, there are few differences between the apoenzyme and cofactor-bound complexes. Analysis of these models clarifies the role of key amino acids implicated in catalysis and highlights additional critical residues. Furthermore, a novel charge transfer complex has been identified in the course of abortive ternary complex formation, and its characterization provides additional insight into aspects of the catalytic mechanism of l-3-hydroxyacyl-CoA dehydrogenase.     

Bifunctional NMN adenylyltransferase/ADP-ribose pyrophosphatase: structure and function in bacterial NAD metabolism

Bacterial NadM-Nudix is a bifunctional enzyme containing a nicotinamide mononucleotide (NMN) adenylyltransferase and an ADP-ribose (ADPR) pyrophosphatase domain. While most members of this enzyme family, such as that from a model cyanobacterium Synechocystis sp., are involved primarily in nicotinamide adenine dinucleotide (NAD) salvage/recycling pathways, its close homolog in a category-A biodefense pathogen, Francisella tularensis, likely plays a central role in a recently discovered novel pathway of NAD de novo synthesis. The crystal structures of NadM-Nudix from both species, including their complexes with various ligands and catalytic metal ions, revealed detailed configurations of the substrate binding and catalytic sites in both domains. The structure of the N-terminal NadM domain may be exploited for designing new antitularemia therapeutics. The ADPR binding site in the C-terminal Nudix domain is substantially different from that of Escherichia coli ADPR pyrophosphatase, and is more similar to human NUDT9. The latter observation provided new insights into the ligand binding mode of ADPR-gated Ca2+ channel TRPM2.     

Crystal structure of muconate lactonizing enzyme at 3 A resolution

The crystal structure of muconate lactonizing enzyme has been solved at 3 A resolution, and an unambiguous alpha-carbon backbone chain trace made. The enzyme contains three domains; the central domain is a parallel-stranded alpha-beta barrel, which has previously been reported in six other enzymes, including triose phosphate isomerase and pyruvate kinase. One novel feature of this enzyme is that its alpha-beta barrel has only seven parallel alpha-helices around the central core of eight parallel beta-strands; all other known alpha-beta barrels contain eight such helices. The N-terminal (alpha + beta) and C-terminal domains cover the cleft where the eighth helix would be. The active site of muconate lactonizing enzyme has been found by locating the manganese ion that is essential for catalytic activity, and by binding and locating an inhibitor, alpha-ketoglutarate. The active site lies in a cleft between the N-terminal and barrel domains; when the active sites of muconate lactonizing enzyme and triose phosphate isomerase are superimposed, barrel-strand 1 of triose phosphate isomerase is aligned with barrel-strand 3 of muconate lactonizing enzyme. This implies that structurally homologous active-site residues in the two enzymes are carried on different parts of the primary sequence; the ancestral gene would had to have been transposed during its evolution to the modern proteins, which seems unlikely. Therefore, these two enzymes may be related by convergent, rather than divergent, evolution.     

Three dimensional structure of porcine pancreatic alpha-amylase at 2.9 A resolution. Role of calcium in structure and activity.

The crystal structure of porcine pancreatic alpha-amylase (PPA) has been solved at 2.9 A resolution by X-ray crystallographic methods. The enzyme contains three domains. The larger, in the N-terminal part, consists of 330 amino acid residues. This central domain has the typical parallel-stranded alpha-beta barrel structure (alpha beta)8, already found in a number of other enzymes like triose phosphate isomerase and pyruvate kinase. The C-terminal domain forms a distinct globular unit where the chain folds into an eight-stranded antiparallel beta-barrel. The third domain lies between a beta-strand and a alpha-helix of the central domain, in a position similar to those found for domain B in triose phosphate isomerase and pyruvate kinase. It is essentially composed of antiparallel beta-sheets. The active site is located in a cleft within the N-terminal central domain, at the carboxy-end of the beta-strands of the (alpha beta)8 barrel. Binding of various substrate analogues to the enzyme suggests that the amino acid residues involved in the catalytic reaction are a pair of aspartic acids. A number of other residues surround the substrate and seem to participate in its binding via hydrogen bonds and hydrophobic interactions. The 'essential' calcium ion has been located near the active site region and between two domains, each of them providing two calcium ligands. On the basis of sequence comparisons this calcium binding site is suggested to be a common structural feature of all alpha-amylases. It represents a new type of calcium-protein interaction pattern.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of Haemophilus influenzae NadR protein. A bifunctional enzyme endowed with NMN adenyltransferase and ribosylnicotinimide kinase activities

Haemophilus influenzae NadR protein (hiNadR) has been shown to be a bifunctional enzyme possessing both NMN adenylytransferase (NMNAT; EC ) and ribosylnicotinamide kinase (RNK; EC ) activities. Its function is essential for the growth and survival of H. influenzae and thus may present a new highly specific anti-infectious drug target. We have solved the crystal structure of hiNadR complexed with NAD using the selenomethionine MAD phasing method. The structure reveals the presence of two distinct domains. The N-terminal domain that hosts the NMNAT activity is closely related to archaeal NMNAT, whereas the C-terminal domain, which has been experimentally demonstrated to possess ribosylnicotinamide kinase activity, is structurally similar to yeast thymidylate kinase and several other P-loop-containing kinases. There appears to be no cross-talk between the two active sites. The bound NAD at the active site of the NMNAT domain reveals several critical interactions between NAD and the protein. There is also a second non-active-site NAD molecule associated with the C-terminal RNK domain that adopts a highly folded conformation with the nicotinamide ring stacking over the adenine base. Whereas the RNK domain of the hiNadR structure presented here is the first structural characterization of a ribosylnicotinamide kinase from any organism, the NMNAT domain of hiNadR defines yet another member of the pyridine nucleotide adenylyltransferase family.     

Crystal structure of prephenate dehydrogenase from Aquifex aeolicus. Insights into the catalytic mechanism

The enzyme prephenate dehydrogenase catalyzes the oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate for the biosynthesis of tyrosine. Prephenate dehydrogenases exist as either monofunctional or bifunctional enzymes. The bifunctional enzymes are diverse, since the prephenate dehydrogenase domain is associated with other enzymes, such as chorismate mutase and 3-phosphoskimate 1-carboxyvinyltransferase. We report the first crystal structure of a monofunctional prephenate dehydrogenase enzyme from the hyper-thermophile Aquifex aeolicus in complex with NAD+. This protein consists of two structural domains, a modified nucleotide-binding domain and a novel helical prephenate binding domain. The active site of prephenate dehydrogenase is formed at the domain interface and is shared between the subunits of the dimer. We infer from the structure that access to the active site is regulated via a gated mechanism, which is modulated by an ionic network involving a conserved arginine, Arg250. In addition, the crystal structure reveals for the first time the positions of a number of key catalytic residues and the identity of other active site residues that may participate in the reaction mechanism; these residues include Ser126 and Lys246 and the catalytic histidine, His147. Analysis of the structure further reveals that two secondary structure elements, beta3 and beta7, are missing in the prephenate dehydrogenase domain of the bifunctional chorismate mutase-prephenate dehydrogenase enzymes. This observation suggests that the two functional domains of chorismate mutase-prephenate dehydrogenase are interdependent and explains why these domains cannot be separated.     

Structure of pyruvate dehydrogenase kinase. Novel folding pattern for a serine protein kinase

The structure of mitochondrial pyruvate dehydrogenase kinase isozyme 2 is of interest because it represents a family of serine-specific protein kinases that lack sequence similarity with all other eukaryotic protein kinases. Similarity exists instead with key motifs of prokaryotic histidine protein kinases and a family of eukaryotic ATPases. The 2.5-A crystal structure reported here reveals that pyruvate dehydrogenase kinase isozyme 2 has two domains of about the same size. The N-terminal half is dominated by a bundle of four amphipathic alpha-helices, whereas the C-terminal half is folded into an alpha/beta sandwich that contains the nucleotide-binding site. Analysis of the structure reveals this C-terminal domain to be very similar to the nucleotide-binding domain of bacterial histidine kinases, but the catalytic mechanism appears similar to that of the eukaryotic serine kinases and ATPases.     

Crystal structure of YegS, a homologue to the mammalian diacylglycerol kinases, reveals a novel regulatory metal binding site

The human lipid kinase family controls cell proliferation, differentiation, and tumorigenesis and includes diacylglycerol kinases, sphingosine kinases, and ceramide kinases. YegS is an Escherichia coli protein with significant sequence homology to the catalytic domain of the human lipid kinases. We have solved the crystal structure of YegS and shown that it is a lipid kinase with phosphatidylglycerol kinase activity. The crystal structure reveals a two-domain protein with significant structural similarity to a family of NAD kinases. The active site is located in the interdomain cleft formed by four conserved sequence motifs. Surprisingly, the structure reveals a novel metal binding site composed of residues conserved in most lipid kinases.     

Crystal structure and enzyme kinetics of the (S)-specific 1-phenylethanol dehydrogenase of the denitrifying bacterium strain EbN1

(S)-1-Phenylethanol dehydrogenase (PED) from the denitrifying bacterium strain EbN1 catalyzes the NAD+-dependent, stereospecific oxidation of (S)-1-phenylethanol to acetophenone and the biotechnologically interesting reverse reaction. This novel enzyme belongs to the short-chain alcohol dehydrogenase/aldehyde reductase family. The coding gene (ped) was heterologously expressed in Escherichia coli and the purified protein was crystallized. The X-ray structures of the apo-form and the NAD+-bound form were solved at a resolution of 2.1 and 2.4 A, respectively, revealing that the enzyme is a tetramer with two types of hydrophobic dimerization interfaces, similar to beta-oxoacyl-[acyl carrier protein] reductase (FabG) from E. coli. NAD+-binding is associated with a conformational shift of the substrate binding loop of PED from a crystallographically unordered "open" to a more ordered "closed" form. Modeling the substrate acetophenone into the active site revealed the structural prerequisites for the strong enantioselectivity of the enzyme and for the catalytic mechanism. Studies on the steady-state kinetics of PED indicated a highly positive cooperativity of both catalytic directions with respect to the substrates. This is contrasted by the behavior of FabG. Moreover, PED exhibits extensive regulation on the enzyme level, being inhibited by elevated concentrations of substrates and products, as well as the wrong enantiomer of 1-phenylethanol. These regulatory properties of PED are consistent with the presence of a putative "transmission module" between the subunits. This module consists of the C-terminal loops of all four subunits, which form a special interconnected structural domain and mediate close contact of the subunits, and of a phenylalanine residue in each subunit that reaches out between substrate-binding loop and C-terminal domain of an adjacent subunit. These elements may transmit the substrate-induced conformational change of the substrate binding loop from one subunit to the others in the tetrameric complex and thus mediate the cooperative behavior of PED.     

Characterization of Salmonella typhimurium YegS, a putative lipid kinase homologous to eukaryotic sphingosine and diacylglycerol kinases

Salmonella typhimurium YegS is a protein conserved in many prokaryotes. Although the function of YegS is not definitively known, it has been annotated as a potential diacylglycerol or sphingosine kinase based on sequence similarity with eukaryotic enzymes of known function. To further characterize YegS, we report its purification, biochemical analysis, crystallization, and structure determination. The crystal structure of YegS reveals a two-domain fold related to bacterial polyphosphate/ATP NAD kinases, comprising a central cleft between an N-terminal alpha/beta domain and a C-terminal two-layer beta-sandwich domain; conserved structural features are consistent with nucleotide binding within the cleft. The N-terminal and C-terminal domains of YegS are however counter-rotated, relative to the polyphosphate/ATP NAD kinase archetype, such that the potential nucleotide binding site is blocked. There are also two Ca2+ binding sites and two hydrophobic clefts, one in each domain of YegS. Analysis of mutagenesis data from eukaryotic homologues of YegS suggest that the N-terminal cleft may bind activating lipids while the C-terminal cleft may bind the lipid substrate. Microcalorimetry experiments showed interaction between recombinant YegS and Mg2+, Ca2+, and Mn2+ ions, with a weaker interaction also observed with polyphosphates and ATP. However, biochemical assays showed that recombinant YegS is endogenously neither an active diacylglycerol nor sphingosine kinase. Thus although the bioinformatics analysis and structure of YegS indicate that many of the ligand recognition determinants for lipid kinase activity are present, the absence of such activity may be due to specificity for a different lipid substrate or the requirement for activation by an, as yet, undetermined mechanism. In this regard the specific interaction of YegS with the periplasmic chaperone OmpH, which we demonstrate from pulldown experiments, may be of significance. Such an interaction suggests that YegS can be translocated to the periplasm and directed to the outer-membrane, an environment that may be required for enzyme activity.     

Biochemical characterization and crystal structure determination of human heart short chain L-3-hydroxyacyl-CoA dehydrogenase provide insights into catalytic mechanism

Human heart short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) catalyzes the oxidation of the hydroxyl group of L-3-hydroxyacyl-CoA to a keto group, concomitant with the reduction of NAD+ to NADH, as part of the beta-oxidation pathway. The homodimeric enzyme has been overexpressed in Escherichia coli, purified to homogeneity, and studied using biochemical and crystallographic techniques. The dissociation constants of NAD+ and NADH have been determined over a broad pH range and indicate that SCHAD binds reduced cofactor preferentially. Examination of apparent catalytic constants reveals that SCHAD displays optimal enzymatic activity near neutral pH, with catalytic efficiency diminishing rapidly toward pH extremes. The crystal structure of SCHAD complexed with NAD+ has been solved using multiwavelength anomalous diffraction techniques and a selenomethionine-substituted analogue of the enzyme. The subunit structure is comprised of two domains. The first domain is similar to other alpha/beta dinucleotide folds but includes an unusual helix-turn-helix motif which extends from the central beta-sheet. The second, or C-terminal, domain is primarily alpha-helical and mediates subunit dimerization and, presumably, L-3-hydroxyacyl-CoA binding. Molecular modeling studies in which L-3-hydroxybutyryl-CoA was docked into the enzyme-NAD+ complex suggest that His 158 serves as a general base, abstracting a proton from the 3-OH group of the substrate. Furthermore, the ability of His 158 to perform such a function may be enhanced by an electrostatic interaction with Glu 170, consistent with previous biochemical observations. These studies provide further understanding of the molecular basis of several inherited metabolic disease states correlated with L-3-hydroxyacyl-CoA dehydrogenase deficiencies.     

Regulatory roles of the N-terminal domain based on crystal structures of human pyruvate dehydrogenase kinase 2 containing physiological and synthetic ligands

Pyruvate dehydrogenase kinase (PDHK) regulates the activity of the pyruvate dehydrogenase multienzyme complex. PDHK inhibition provides a route for therapeutic intervention in diabetes and cardiovascular disorders. We report crystal structures of human PDHK isozyme 2 complexed with physiological and synthetic ligands. Several of the PDHK2 structures disclosed have C-terminal cross arms that span a large trough region between the N-terminal regulatory (R) domains of the PDHK2 dimers. The structures containing bound ATP and ADP demonstrate variation in the conformation of the active site lid, residues 316-321, which enclose the nucleotide beta and gamma phosphates at the active site in the C-terminal catalytic domain. We have identified three novel ligand binding sites located in the R domain of PDHK2. Dichloroacetate (DCA) binds at the pyruvate binding site in the center of the R domain, which together with ADP, induces significant changes at the active site. Nov3r and AZ12 inhibitors bind at the lipoamide binding site that is located at one end of the R domain. Pfz3 (an allosteric inhibitor) binds in an extended site at the other end of the R domain. We conclude that the N-terminal domain of PDHK has a key regulatory function and propose that the different inhibitor classes act by discrete mechanisms. The structures we describe provide insights that can be used for structure-based design of PDHK inhibitors.     

Binary structure of the two-domain (3R)-hydroxyacyl-CoA dehydrogenase from rat peroxisomal multifunctional enzyme type 2 at 2.38 A resolution

The crystal structure of (3R)-hydroxyacyl-CoA dehydrogenase of rat peroxisomal multifunctional enzyme type 2 (MFE-2) was solved at 2.38 A resolution. The catalytic entity reveals an alpha/beta short chain alcohol dehydrogenase/reductase (SDR) fold and the conformation of the bound nicotinamide adenine dinucleotide (NAD(+)) found in other SDR enzymes. Of great interest is the separate COOH-terminal domain, which is not seen in other SDR structures. This domain completes the active site cavity of the neighboring monomer and extends dimeric interactions. Peroxisomal diseases that arise because of point mutations in the dehydrogenase-coding region of the MFE-2 gene can be mapped to changes in amino acids involved in NAD(+) binding and protein dimerization.     

Structure and mechanism of T4 polynucleotide kinase: an RNA repair enzyme

T4 polynucleotide kinase (Pnk), in addition to being an invaluable research tool, exemplifies a family of bifunctional enzymes with 5'-kinase and 3'-phosphatase activities that play key roles in RNA and DNA repair. T4 Pnk is a homotetramer composed of a C-terminal phosphatase domain and an N-terminal kinase domain. The 2.0 A crystal structure of the isolated kinase domain highlights a tunnel-like active site through the heart of the enzyme, with an entrance on the 5' OH acceptor side that can accommodate a single-stranded polynucleotide. The active site is composed of essential side chains that coordinate the beta phosphate of the NTP donor and the 3' phosphate of the 5' OH acceptor, plus a putative general acid that activates the 5' OH. The structure rationalizes the different specificities of T4 and eukaryotic Pnk and suggests a model for the assembly of the tetramer.     

Crystal structure of alpha-galactosidase from Trichoderma reesei and its complex with galactose: implications for catalytic mechanism

The crystal structures of alpha-galactosidase from the mesophilic fungus Trichoderma reesei and its complex with the competitive inhibitor, beta-d-galactose, have been determined at 1.54 A and 2.0 A resolution, respectively. The alpha-galactosidase structure was solved by the quick cryo-soaking method using a single Cs derivative. The refined crystallographic model of the alpha-galactosidase consists of two domains, an N-terminal catalytic domain of the (beta/alpha)8 barrel topology and a C-terminal domain which is formed by an antiparallel beta-structure. The protein contains four N-glycosylation sites located in the catalytic domain. Some of the oligosaccharides were found to participate in inter-domain contacts. The galactose molecule binds to the active site pocket located in the center of the barrel of the catalytic domain. Analysis of the alpha-galactosidase- galactose complex reveals the residues of the active site and offers a structural basis for identification of the putative mechanism of the enzymatic reaction. The structure of the alpha-galactosidase closely resembles those of the glycoside hydrolase family 27. The conservation of two catalytic Asp residues, identified for this family, is consistent with a double-displacement reaction mechanism for the alpha-galactosidase. Modeling of possible substrates into the active site reveals specific hydrogen bonds and hydrophobic interactions that could explain peculiarities of the enzyme kinetics.