A stress-induced rice (Oryza sativa L.) beta-glucosidase represents a new subfamily of glycosyl hydrolase family 5 containing a fascin-like domain

GH5BG, the cDNA for a stress-induced GH5 (glycosyl hydrolase family 5) beta-glucosidase, was cloned from rice (Oryza sativa L.) seedlings. The GH5BG cDNA encodes a 510-amino-acid precursor protein that comprises 19 amino acids of prepeptide and 491 amino acids of mature protein. The protein was predicted to be extracellular. The mature protein is a member of a plant-specific subgroup of the GH5 exoglucanase subfamily that contains two major domains, a beta-1,3-exoglucanase-like domain and a fascin-like domain that is not commonly found in plant enzymes. The GH5BG mRNA is highly expressed in the shoot during germination and in leaf sheaths of mature plants. The GH5BG was up-regulated in response to salt stress, submergence stress, methyl jasmonate and abscisic acid in rice seedlings. A GUS (glucuronidase) reporter tagged at the C-terminus of GH5BG was found to be secreted to the apoplast when expressed in onion (Allium cepa) cells. A thioredoxin fusion protein produced from the GH5BG cDNA in Escherichia coli hydrolysed various pNP (p-nitrophenyl) glycosides, including beta-D-glucoside, alpha-L-arabinoside, beta-D-fucoside, beta-D-galactoside, beta-D-xyloside and beta-D-cellobioside, as well as beta-(1,4)-linked glucose oligosaccharides and beta-(1,3)-linked disaccharide (laminaribiose). The catalytic efficiency (kcat/K(m)) for hydrolysis of beta-(1,4)-linked oligosaccharides by the enzyme remained constant as the DP (degree of polymerization) increased from 3 to 5. This substrate specificity is significantly different from fungal GH5 exoglucanases, such as the exo-beta-(1,3)-glucanase of the yeast Candida albicans, which may correlate with a marked reduction in a loop that makes up the active-site wall in the Candida enzyme.     

Functional and biophysical characterization of a hyperthermostable GH51 α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase from <Emphasis Type="Italic">Thermotoga petrophila</Emphasis>

A hyperthermostable glycoside hydrolase family 51 (GH51) α-l-arabinofuranosidase from Thermotoga petrophila RKU-1 (TpAraF) was cloned, overexpressed, purified and characterized. The recombinant enzyme had optimum activity at pH 6.0 and 70°C with linear α-1,5-linked arabinoheptaose as substrate. The substrate cleavage pattern monitored by capillary zone electrophoresis showed that TpAraF is a classical exo-acting enzyme producing arabinose as its end-product. Far-UV circular dichroism analysis displayed a typical spectrum of α/β barrel proteins analogously observed for other GH51 α-l-arabinofuranosidases. Moreover, TpAraF was crystallized in two crystalline forms, which can be used to determine its crystallographic structure.     

Unique active-site and subsite features in the arabinogalactan-degrading GH43 exo-β-1,3-galactanase from Phanerochaete chrysosporium

Arabinogalactan proteins (AGPs) are plant proteoglycans with functions in growth and development. However, these functions are largely unexplored, mainly because of the complexity of the sugar moieties. These carbohydrate sequences are generally analyzed with the aid of glycoside hydrolases. The exo-β-1,3-galactanase is a glycoside hydrolase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. However, its structure is not known in relation to its mechanism bypassing side chains. In this study, we solved the apo and liganded structures of Pc1,3Gal43A, which reveal a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family 35 (CBM35) binding domain. GH43_sub24 is known to lack the catalytic base Asp conserved among other GH43 subfamilies. Our structure in combination with kinetic analyses reveals that the tautomerized imidic acid group of Gln²⁶³ serves as the catalytic base residue instead. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite −1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6–linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s, including those that bind galactomannan. Specifically, we noted a Gly → Trp substitution, which affects pyranose stacking, and an Asp → Asn substitution in the binding pocket, which recognizes β-linked rather than α-linked Gal residues. These findings should facilitate further structural analysis of AGPs and may also be helpful in engineering designer enzymes for efficient biomass utilization.     

Characterization of an exo-beta-1,3-galactanase from Clostridium thermocellum

A gene encoding an exo-beta-1,3-galactanase from Clostridium thermocellum, Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-beta-1,3-galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50 degrees C and catalyzes hydrolysis only of beta-1,3-linked galactosyl oligosaccharides and polysaccharides. High-performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from beta-1,3-galactan in an exo-acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a beta-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-beta-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including beta-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a beta-1,3-galactobiose, beta-1,4-galactosyl glucose, or beta-1,4-galactosyl N-acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward beta-1,3-galactan and alpha-l-arabinofuranosidase-treated AGP.     

Cellvibrio japonicus alpha-L-arabinanase 43A has a novel five-blade beta-propeller fold

Cellvibrio japonicus arabinanase Arb43A hydrolyzes the alpha-1,5-linked L-arabinofuranoside backbone of plant cell wall arabinans. The three-dimensional structure of Arb43A, determined at 1.9 A resolution, reveals a five-bladed beta-propeller fold. Arb43A is the first enzyme known to display this topology. A long V-shaped surface groove, partially enclosed at one end, forms a single extended substrate-binding surface across the face of the propeller. Three carboxylates deep in the active site groove provide the general acid and base components for glycosidic bond hydrolysis with inversion of anomeric configuration.     

How family 26 glycoside hydrolases orchestrate catalysis on different polysaccharides: structure and activity of a Clostridium thermocellum lichenase, CtLic26A

One of the most intriguing features of the 90 glycoside hydrolase families (GHs) is the range of specificities displayed by different members of the same family, whereas the catalytic apparatus and mechanism are often invariant. Family GH26 predominantly comprises beta-1,4 mannanases; however, a bifunctional Clostridium thermocellum GH26 member (hereafter CtLic26A) displays a markedly different specificity. We show that CtLic26A is a lichenase, specific for mixed (Glcbeta1,4Glcbeta1,4Glcbeta1,3)n oligo- and polysaccharides, and displays no activity on manno-configured substrates or beta-1,4-linked homopolymers of glucose or xylose. The three-dimensional structure of the native form of CtLic26A has been solved at 1.50-A resolution, revealing a characteristic (beta/alpha)8 barrel with Glu-109 and Glu-222 acting as the catalytic acid/base and nucleophile in a double-displacement mechanism. The complex with the competitive inhibitor, Glc-beta-1,3-isofagomine (Ki 1 microm), at 1.60 A sheds light on substrate recognition in the -2 and -1 subsites and illuminates why the enzyme is specific for lichenan-based substrates. Hydrolysis of beta-mannosides by GH26 members is thought to proceed through transition states in the B2,5 (boat) conformation in which structural distinction of glucosides versus mannosides reflects not the configuration at C2 but the recognition of the pseudoaxial O3 of the B2,5 conformation. We suggest a different conformational itinerary for the GH26 enzymes active on gluco-configured substrates.     

Characterization of an exo-beta-1,3-D-galactanase from Streptomyces avermitilis NBRC14893 acting on arabinogalactan-proteins

A gene belonging to glycoside hydrolase family 43 (GH43) was isolated from Streptomyces avermitilis NBRC14893. The gene encodes a modular protein consisting of N-terminal GH43 module and a family 13 carbohydrate-binding module at the C-terminus. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme specifically hydrolyzed only beta-1,3-linkage of two D-galactosyl residues at non-reducing ends of the substrates. The analysis of the hydrolysis products indicated that the enzyme produced galactose from beta-1,3-D-galactan in an exo-acting manner. When the enzyme catalyze hydrolysis of the arabinogalactan-protein, the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate beta-1,6-linked D-galactosyl side chains. These properties are the same as the other previously reported exo-beta-1,3-D-galactanases. Therefore, we concluded the isolated gene certainly encodes an exo-beta-1,3-D-galactanase. This is the first report of exo-beta-1,3-D-galactanase from actinomycetes.     

Crystallographic insight into the evolutionary origins of xyloglucan endotransglycosylases and endohydrolases

The xyloglucan endotransglycosylase/hydrolase (XTH) gene family encodes enzymes of central importance to plant cell wall remodeling. The evolutionary history of plant XTH gene products is incompletely understood vis‐à‐vis the larger body of bacterial endoglycanases in Glycoside Hydrolase Family 16 (GH16). To provide molecular insight into this issue, high‐resolution X‐ray crystal structures and detailed enzyme kinetics of an extant transitional plant endoglucanase (EG) were determined. Functionally intermediate between plant XTH gene products and bacterial licheninases of GH16, Vitis vinifera EG16 (VvEG16) effectively catalyzes the hydrolysis of the backbones of two dominant plant cell wall matrix glycans, xyloglucan (XyG) and β(1,3)/β(1,4)‐mixed‐linkage glucan (MLG). Crystallographic complexes with extended oligosaccharide substrates reveal the structural basis for the accommodation of both unbranched, mixed‐linked (MLG) and highly decorated, linear (XyG) polysaccharide chains in a broad, extended active‐site cleft. Structural comparison with representative bacterial licheninases, a xyloglucan endotranglycosylase (XET), and a xyloglucan endohydrolase (XEH) outline the functional ramifications of key sequence deletions and insertions across the phylogenetic landscape of GH16. Although the biological role(s) of EG16 orthologs remains to be fully resolved, the present biochemical and tertiary structural characterization provides key insight into plant cell wall enzyme evolution, which will continue to inform genomic analyses and functional studies across species.     

A novel glycosylphosphatidylinositol-anchored glycoside hydrolase from Ustilago esculenta functions in β-1,3-glucan degradation

A glycoside hydrolase responsible for laminarin degradation was partially purified to homogeneity from a Ustilago esculenta culture filtrate by weak-cation-exchange, strong-cation-exchange, and size-exclusion chromatography. Three proteins in enzymatically active fractions were digested with chymotrypsin followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis, resulting in the identification of three peptide sequences that shared significant similarity to a putative β-1,3-glucanase, a member of glucoside hydrolase family 16 (GH16) from Sporisorium reilianum SRZ2. A gene encoding a laminarin-degrading enzyme from U. esculenta, lam16A, was isolated by PCR using degenerate primers designed based on the S. reilianum SRZ2 β-1,3-glucanase gene. Lam16A possesses a GH16 catalytic domain with an N-terminal signal peptide and a C-terminal glycosylphosphatidylinositol (GPI) anchor peptide. Recombinant Lam16A fused to an N-terminal FLAG peptide (Lam16A-FLAG) overexpressed in Aspergillus oryzae exhibited hydrolytic activity toward β-1,3-glucan specifically and was localized both in the extracellular and in the membrane fractions but not in the cell wall fraction. Lam16A without a GPI anchor signal peptide was secreted extracellularly and was not detected in the membrane fraction. Membrane-anchored Lam16A-FLAG was released completely by treatment with phosphatidylinositol-specific phospholipase C. These results suggest that Lam16A is anchored in the plasma membrane in order to modify β-1,3-glucan associated with the inner cell wall and that Lam16A is also used for the catabolism of β-1,3-glucan after its release in the extracellular medium.     

N-linked oligosaccharide processing enzyme glucosidase II produces 1,5-anhydrofructose as a side product

alpha-1,4-Glucan lyase cleaves alpha-1,4-linkages of nonreducing termini of alpha-1,4-glucans to produce 1,5-anhydrofructose (1,5-AnFru). The enzymes isolated from fungi and algae show high homology with glycoside hydrolase family 31. Purification of alpha-1,4-glucan lyase from rat liver using DEAE Cellulose chromatography resulted in separation of two enzymatic active fractions, one was bound to the column and the other was in the flow-through. Partial amino acid sequence determined from the lyase, retained on the anion exchange column, were identical with that of the N:-linked oligosaccharide processing enzyme glucosidase II. The lyase showed similar enzymatic properties as the microsomal glucosidase such as inhibition by 1-deoxynojirimycin and castanospermine. On the other hand, glucosidase II purified from rat liver microsomes produced not only glucose but also a small amount of 1,5-AnFru using maltose as substrate. Furthermore, CHO cells overexpressing pig liver glucosidase II showed a 1.5- to 2-fold higher lyase activity compared to the nontransfected CHO cells. Conversely, no lyase activity was detectable either in PHAR2.7, the glucosidase II-deficient mutant from a mouse lymphoma cell line, or in Saccharomyces cerevisiae strain YG427 having the glucosidase II gene disrupted. These data demonstrate that glucosidase II possesses an additional enzymatic activity of releasing 1,5-AnFru from maltose.     

Structural and functional analysis of a glycoside hydrolase family 97 enzyme from Bacteroides thetaiotaomicron

SusB, an 84-kDa alpha-glucoside hydrolase involved in the starch utilization system (sus) of Bacteroides thetaiotaomicron, belongs to glycoside hydrolase (GH) family 97. We have determined the enzymatic characteristics and the crystal structures in free and acarbose-bound form at 1.6A resolution. SusB hydrolyzes the alpha-glucosidic linkage, with inversion of anomeric configuration liberating the beta-anomer of glucose as the reaction product. The substrate specificity of SusB, hydrolyzing not only alpha-1,4-glucosidic linkages but also alpha-1,6-, alpha-1,3-, and alpha-1,2-glucosidic linkages, is clearly different from other well known glucoamylases belonging to GH15. The structure of SusB was solved by the single-wavelength anomalous diffraction method with sulfur atoms as anomalous scatterers using an in-house x-ray source. SusB includes three domains as follows: the N-terminal, catalytic, and C-terminal domains. The structure of the SusB-acarbose complex shows a constellation of carboxyl groups at the catalytic center; Glu532 is positioned to provide protonic assistance to leaving group departure, with Glu439 and Glu508 both positioned to provide base-catalyzed assistance for inverting nucleophilic attack by water. A structural comparison with other glycoside hydrolases revealed significant similarity between the catalytic domain of SusB and those of alpha-retaining glycoside hydrolases belonging to GH27, -36, and -31 despite the differences in catalytic mechanism. SusB and the other retaining enzymes appear to have diverged from a common ancestor and individually acquired the functional carboxyl groups during the process of evolution. Furthermore, sequence comparison of the active site based on the structure of SusB indicated that GH97 included both retaining and inverting enzymes.     

Structural basis for inhibition of xyloglucan-specific endo-β-1,4-glucanase (XEG) by XEG-protein inhibitor

Microorganisms such as plant pathogens secrete glycoside hydrolases (GHs) to digest the polysaccharide chains of plant cell walls. The degradation of cell walls by these enzymes is a crucial step for nutrition and invasion. To protect the cell wall from these enzymes, plants secrete glycoside hydrolase inhibitor proteins (GHIPs). Xyloglucan-specific endo-β-1,4-glucanase (XEG), a member of GH family 12 (GH12), could be a great threat to many plants because xyloglucan is a major component of the cell wall in most plants. Understanding the inhibition mechanism of XEG by GHIP is therefore of great importance in the field of plant defense, but to date the mechanism and specificity of GHIPs remain unclear. We have determined the crystal structure of XEG in complex with extracellular dermal glycoprotein (EDGP), a carrot GHIP that inhibits XEG. The structure reveals that the conserved arginines of EDGP intrude into the active site of XEG and interact with the catalytic glutamates of the enzyme. We have also determined the crystal structure of the XEG-xyloglucan complex. These structures show that EDGP closely mimics the XEG-xyloglucan interaction. Although EDGP shares structural similarity to a wheat GHIP (Triticum aestivum xylanase inhibitor-IA (TAXI-IA)) that inhibits GH11 family xylanases, the arrangement of GH and GHIP in the XEG-EDGP complex is distinct from that in the xylanase-TAXI-IA complex. Our findings imply that plants have evolved structures of GHIPs to inhibit different GH family members that attack their cell walls.     

Endo-β-1,3-glucanase GLU1, from the fruiting body of Lentinula edodes, belongs to a new glycoside hydrolase family

The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).     

Evidence That GH115 α-Glucuronidase Activity,Which Is Required to Degrade Plant Biomass,Is Dependent on Conformational Flexibility

The microbial degradation of the plant cell wall is an important biological process that is highly relevant to environmentally significant industries such as the bioenergy and biorefining sectors. A major component of the wall is glucuronoxylan, a β1,4-linked xylose polysaccharide that is decorated with α-linked glucuronic and/or methylglucuronic acid (GlcA/MeGlcA). Recently three members of a glycoside hydrolase family, GH115, were shown to hydrolyze MeGlcA side chains from the internal regions of xylan, an activity that has not previously been described. Here we show that a dominant member of the human microbiota, Bacteroides ovatus, contains a GH115 enzyme, BoAgu115A, which displays glucuronoxylan α-(4-O-methyl)-glucuronidase activity. The enzyme is significantly more active against substrates in which the xylose decorated with GlcA/MeGlcA is flanked by one or more xylose residues. The crystal structure of BoAgu115A revealed a four-domain protein in which the active site, comprising a pocket that abuts a cleft-like structure, is housed in the second domain that adopts a TIM barrel-fold. The third domain, a five-helical bundle, and the C-terminal β-sandwich domain make inter-chain contacts leading to protein dimerization. Informed by the structure of the enzyme in complex with GlcA in its open ring form, in conjunction with mutagenesis studies, the potential substrate binding and catalytically significant amino acids were identified. Based on the catalytic importance of residues located on a highly flexible loop, the enzyme is required to undergo a substantial conformational change to form a productive Michaelis complex with glucuronoxylan.     

Improvement of corn stover bioconversion efficiency by using plant glycoside hydrolase

Plant cell wall is the most abundant substrate for bioethanol production, and plants also represent a key resource for glycoside hydrolase (GH). To exploit efficient way for bioethanol production with lower cellulase loading, the potential of plant GH for lignocellulose bioconversion was evaluated. The GH activity for cell wall proteins (CWPs) was detected from fresh corn stover (FCS), and the synergism of which with Trichoderma reesei cellulase was also observed. The properties for the GH of FCS make it a promising enzyme additive for lignocellulose biodegradation. To make use of the plant GH, novel technology for hydrolysis and ethanol fermentation was developed with corn stover as substrate. Taking steam-exploded corn stover as substrate for hydrolysis and ethanol fermentation, compared with T. reesei cellulase loaded alone, the final glucose and ethanol accumulation increased by 60% and 63% respectively with GH of FCS as an addition.     

Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH12

The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed "hemicellulose." One such hemicellulose is xyloglucan, which displays a beta-1,4-linked d-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand-free and xyloglucan-oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (beta/alpha)(8) and beta-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the beta-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzyme is similarly competent in the degradation of unbranched glucans. In the case of the (beta/alpha)(8) GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.     

Molecular modeling of family GH16 glycoside hydrolases: potential roles for xyloglucan transglucosylases/hydrolases in cell wall modification in the poaceae

Family GH16 glycoside hydrolases can be assigned to five subgroups according to their substrate specificities, including xyloglucan transglucosylases/hydrolases (XTHs), (1,3)-beta-galactanases, (1,4)-beta-galactanases/kappa-carrageenases, "nonspecific" (1,3/1,3;1,4)-beta-D-glucan endohydrolases, and (1,3;1,4)-beta-D-glucan endohydrolases. A structured family GH16 glycoside hydrolase database has been constructed (http://www.ghdb.uni-stuttgart.de) and provides multiple sequence alignments with functionally annotated amino acid residues and phylogenetic trees. The database has been used for homology modeling of seven glycoside hydrolases from the GH16 family with various substrate specificities, based on structural coordinates for (1,3;1,4)-beta-D-glucan endohydrolases and a kappa-carrageenase. In combination with multiple sequence alignments, the models predict the three-dimensional (3D) dispositions of amino acid residues in the substrate-binding and catalytic sites of XTHs and (1,3/1,3;1,4)-beta-d-glucan endohydrolases; there is no structural information available in the databases for the latter group of enzymes. Models of the XTHs, compared with the recently determined structure of a Populus tremulos x tremuloides XTH, reveal similarities with the active sites of family GH11 (1,4)-beta-D-xylan endohydrolases. From a biological viewpoint, the classification, molecular modeling and a new 3D structure of the P. tremulos x tremuloides XTH establish structural and evolutionary connections between XTHs, (1,3;1,4)-beta-D-glucan endohydrolases and xylan endohydrolases. These findings raise the possibility that XTHs from higher plants could be active not only on cell wall xyloglucans, but also on (1,3;1,4)-beta-D-glucans and arabinoxylans, which are major components of walls in grasses. A role for XTHs in (1,3;1,4)-beta-D-glucan and arabinoxylan modification would be consistent with the apparent overrepresentation of XTH sequences in cereal expressed sequence tags databases.     

Crystal structures of a poplar xyloglucan endotransglycosylase reveal details of transglycosylation acceptor binding

Xyloglucan endotransglycosylases (XETs) cleave and religate xyloglucan polymers in plant cell walls via a transglycosylation mechanism. Thus, XET is a key enzyme in all plant processes that require cell wall remodeling. To provide a basis for detailed structure-function studies, the crystal structure of Populus tremula x tremuloides XET16A (PttXET16A), heterologously expressed in Pichia pastoris, has been determined at 1.8-A resolution. Even though the overall structure of PttXET16A is a curved beta-sandwich similar to other enzymes in the glycoside hydrolase family GH16, parts of its substrate binding cleft are more reminiscent of the distantly related family GH7. In addition, XET has a C-terminal extension that packs against the conserved core, providing an additional beta-strand and a short alpha-helix. The structure of XET in complex with a xyloglucan nonasaccharide, XLLG, reveals a very favorable acceptor binding site, which is a necessary but not sufficient prerequisite for transglycosylation. Biochemical data imply that the enzyme requires sugar residues in both acceptor and donor sites to properly orient the glycosidic bond relative to the catalytic residues.     

Structural insights into the substrate specificity and function of Escherichia coli K12 YgjK, a glucosidase belonging to the glycoside hydrolase family 63

Proteins belonging to the glycoside hydrolase family 63 (GH63) are found in bacteria, archaea, and eukaryotes. Eukaryotic GH63 proteins are processing α-glucosidase I enzymes that hydrolyze an oligosaccharide precursor of eukaryotic N-linked glycoproteins. In contrast, the functions of the bacterial and archaeal GH63 proteins are unclear. Here we determined the crystal structure of a bacterial GH63 enzyme, Escherichia coli K12 YgjK, at 1.78 Å resolution and investigated some properties of the enzyme. YgjK consists of the N-domain and the A-domain, joined by a linker region. The N-domain is composed of 18 antiparallel β-strands and is classified as a super-β-sandwich. The A-domain contains 16 α-helices, 12 of which form an (α/α)₆-barrel; the remaining 4 α-helices are found in an extra structural unit that we designated as the A′-region. YgjK, a member of the glycoside hydrolase clan GH-G, shares structural similarity with glucoamylase (GH15) and chitobiose phosphorylase (GH65), both of which belong to clan GH-L. In crystal structures of YgjK in complex with glucose, mannose, and galactose, all of the glucose, mannose, and galactose units were located in the catalytic cleft. YgjK showed the highest activity for the α-1,3-glucosidic linkage of nigerose, but also hydrolyzed trehalose, kojibiose, and maltooligosaccharides from maltose to maltoheptaose, although the activities were low. These findings suggest that YgjK is a glucosidase with relaxed specificity for sugars.