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PubMed2000

Biochemical characterization of MI-ENG1, a family 5 endoglucanase secreted by the root-knot nematode Meloidogyne incognita.

A beta-1,4-endoglucanase named MI-ENG1, homologous to the family 5 glycoside hydrolases, was previously isolated from the plant parasitic root-knot nematode Meloidogyne incognita. We describe here the detection of the enzyme in the nematode homogenate and secretion and its complete biochemical characterization. This study is the first comparison of the enzymatic properties of an animal glycoside hydrolase with plant and microbial enzymes. MI-ENG1 shares many enzymatic properties with known endoglucanases from plants, free-living or rumen-associated microorganisms and phytopathogens. In spite of the presence of a cellulose-binding domain at the C-terminus, the ability of MI-ENG1 to bind cellulose could not be demonstrated, whatever the experimental conditions used. The biochemical characterization of the enzyme is a first step towards the understanding of the molecular events taking place during the plant-nematode interaction.

Amino Acid Sequence↗

PubMed2004

X4 modules represent a new family of carbohydrate-binding modules that display novel properties.

The hydrolysis of the plant cell wall by microbial glycoside hydrolases and esterases is the primary mechanism by which stored organic carbon is utilized in the biosphere, and thus these enzymes are of considerable biological and industrial importance. Plant cell wall-degrading enzymes in general display a modular architecture comprising catalytic and non-catalytic modules. The X4 modules in glycoside hydrolases represent a large family of non-catalytic modules whose function is unknown. Here we show that the X4 modules from a Cellvibrio japonicus mannanase (Man5C) and arabinofuranosidase (Abf62A) bind to polysaccharides, and thus these proteins comprise a new family of carbohydrate-binding modules (CBMs), designated CBM35. The Man5C-CBM35 binds to galactomannan, insoluble amorphous mannan, glucomannan, and manno-oligosaccharides but does not interact with crystalline mannan, cellulose, cello-oligosaccharides, or other polysaccharides derived from the plant cell wall. Man5C-CBM35 also potentiates mannanase activity against insoluble amorphous mannan. Abf62A-CBM35 interacts with unsubstituted oat-spelt xylan but not substituted forms of the hemicellulose or xylo-oligosaccharides, and requires calcium for binding. This is in sharp contrast to other xylan-binding CBMs, which interact in a calcium-independent manner with both xylo-oligosaccharides and decorated xylans.

Amino Acid Sequence↗

PubMed2004

Crystal structure of a family 54 alpha-L-arabinofuranosidase reveals a novel carbohydrate-binding module that can bind arabinose.

As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 alpha-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a beta-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a beta-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu(221) and Asp(297), respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.

Amino Acid Motifs↗

PubMed2004

Specific xyloglucanases as a new class of polysaccharide-degrading enzymes.

Three specific xyloglucanases (XGs) were isolated from Aspergillus japonicus (32 kDa, pI 2.8), Chrysosporium lucknowense (78 kDa, pI 3.8) and Trichoderma reesei (75-105 kDa, pI 4.1-4.3). The characteristic feature of these enzymes was their high specific activity toward tamarind xyloglucan, whereas the activity against carboxymethylcellulose (CMC) and barley beta-glucan was absent or very low. Peptide mass fingerprinting using MALDI-TOF mass spectrometry showed that the T. reesei XG represents Cel74A, whose gene has been discovered recently (GenBank accession no. AY281371 ), but the enzyme has not been characterized and described elsewhere. Tryptic peptides from A. japonicus and C. lucknowense xyloglucanases did not show any identity to those from known glycoside hydrolases. All enzymes produced XXXG, XXLG/XLXG and XLLG oligosaccharides as the end products of xyloglucan hydrolysis. A. japonicus XG displayed an endo-type of attack on the polymeric substrate, while the mode of action of two other xyloglucanases was similar to the exo-type, when oligosaccharides containing four glucose residues in the main chain were split off the ends of xyloglucan molecules. These results together with growing literature data allow concluding that specific xyloglucanases may represent a new class of glycoside hydrolases, which are different from regular endo-1,4-beta-glucanases.

Amino Acid Sequence↗

PubMed2006

Bioconversion of red seaweed galactans: a focus on bacterial agarases and carrageenases.

Agars and carrageenans are 1,3-alpha-1,4-beta-galactans from the cell walls of red algae, substituted by zero (agarose), one (kappa-), two (iota-), or three (lambda-carrageenan) sulfate groups per disaccharidic monomer. Agars, kappa-, and iota-carrageenans auto-associate into crystalline fibers and are well known for their gelling properties, used in a variety of laboratory and industrial applications. These sulfated galactans constitute a crucial carbon source for a number of marine bacteria. These microorganisms secrete glycoside hydrolases specific for these polyanionic, insoluble polysaccharides, agarases and carrageenases. This article reviews the microorganisms involved in the degradation of agars and carrageenans, in their environmental and taxonomic diversity. We also present an overview on the biochemistry of the different families of galactanases. The structure-function relationships of the family GH16 beta-agarases and kappa-caraggeenases and of the family GH82 iota-carrageenases are discussed in more details. In particular, we examine how the active site topologies of these glycoside hydrolases influence their mode of action in heterogeneous phase. Finally, we discuss the next challenges in the basic and applied field of the galactans of red algae and of their related degrading microorganisms.

Agar↗

PubMed2005

Structural basis for the specificity of the reducing end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125.

Reducing end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125 (Rex) hydrolyzes xylooligosaccharides whose degree of polymerization is greater than or equal to 3, releasing the xylose unit at the reducing end. It is a unique exo-type glycoside hydrolase that recognizes the xylose unit at the reducing end in a very strict manner, even discriminating the beta-anomeric hydroxyl configuration from the alpha-anomer or 1-deoxyxylose. We have determined the crystal structures of Rex in unliganded and complex forms at 1.35-2.20-A resolution and revealed the structural aspects of its three subsites ranging from -2 to +1. The structure of Rex was compared with those of endo-type enzymes in glycoside hydrolase subfamily 8a (GH-8a). The catalytic machinery of Rex is basically conserved with other GH-8a enzymes. However, subsite +2 is blocked by a barrier formed by a kink in the loop before helix alpha10. His-319 in this loop forms a direct hydrogen bond with the beta-hydroxyl of xylose at subsite +1, contributing to the specific recognition of anomers at the reducing end.

Bacillus↗

PubMed2004

Crystal structure of exo-inulinase from Aspergillus awamori: the enzyme fold and structural determinants of substrate recognition.

Exo-inulinases hydrolyze terminal, non-reducing 2,1-linked and 2,6-linked beta-d-fructofuranose residues in inulin, levan and sucrose releasing beta-d-fructose. We present the X-ray structure at 1.55A resolution of exo-inulinase from Aspergillus awamori, a member of glycoside hydrolase family 32, solved by single isomorphous replacement with the anomalous scattering method using the heavy-atom sites derived from a quick cryo-soaking technique. The tertiary structure of this enzyme folds into two domains: the N-terminal catalytic domain of an unusual five-bladed beta-propeller fold and the C-terminal domain folded into a beta-sandwich-like structure. Its structural architecture is very similar to that of another member of glycoside hydrolase family 32, invertase (beta-fructosidase) from Thermotoga maritima, determined recently by X-ray crystallography The exo-inulinase is a glycoprotein containing five N-linked oligosaccharides. Two crystal forms obtained under similar crystallization conditions differ by the degree of protein glycosylation. The X-ray structure of the enzyme:fructose complex, at a resolution of 1.87A, reveals two catalytically important residues: Asp41 and Glu241, a nucleophile and a catalytic acid/base, respectively. The distance between the side-chains of these residues is consistent with a double displacement mechanism of reaction. Asp189, which is part of the Arg-Asp-Pro motif, provides hydrogen bonds important for substrate recognition.

Amino Acid Motifs↗

PubMed2002

Functional expression of a cDNA encoding pea (Pisum sativum L.) raffinose synthase, partial purification of the enzyme from maturing seeds, and steady-state kinetic analysis of raffinose synthesis.

Raffinose (O-alpha- D-galactopyranosyl-(1-->6)- O-alpha- D-glucopyranosyl-(1<-->2)- O-beta- D-fructofuranoside) is a widespread oligosaccharide in plant seeds and other tissues. Raffinose synthase (EC 2.4.1.82) is the key enzyme that channels sucrose into the raffinose oligosaccharide pathway. We here report on the isolation of a cDNA encoding for raffinose synthase from maturing pea ( Pisum sativum L.) seeds. The coding region of the cDNA was expressed in Spodoptera frugiperda Sf21 insect cells. The recombinant enzyme, a protein of glycoside hydrolase family 36, displayed similar kinetic properties to raffinose synthase partially purified from maturing seeds by anion-exchange and size-exclusion chromatography. Apart from the natural galactosyl donor galactinol ( O-alpha- D-galactopyranosyl-(1-->1)- L- myo-inositol), p-nitrophenyl alpha- D-galactopyranoside, an artificial substrate, was utilized as a galactosyl donor. An equilibrium constant of 4.1 was determined for the galactosyl transfer reaction from galactinol to sucrose. Steady-state kinetic analysis suggested that raffinose synthase is a transglycosidase operating by a ping-pong reaction mechanism and may also act as a glycoside hydrolase. The enzyme was strongly inhibited by 1-deoxygalactonojirimycin, a potent inhibitor for alpha-galactosidases (EC 3.2.1.22). The physiological implications of these observations are discussed.

Algorithms↗

PubMed2005

Isolation, characterization, and cloning of {alpha}-L-Arabinofuranosidase expressed during fruit ripening of Japanese pear.

alpha-L-Arabinofuranosidase (alpha-L-arafase) was purified from fruit of Japanese pear (Pyrus pyrifolia). The enzyme solubilized from the cell wall by NaCl and Triton X-100 had the homogeneity of a single 62-kD polypeptide on SDS-PAGE after purification through the steps of hydroxyapatite, anion-exchange chromatography, and size-exclusion chromatography. A related cDNA clone was isolated (PpARF2). The transcript and related protein were detected solely in the ripening fruit corresponding to the increase of alpha-L-arafase activity. Transcripts of PpARF2 were not detected in buds, leaves, roots, or shoots of the Japanese pear. The deduced amino acid sequences of PpARF2 had low identity with those of other plants or bacteria. This alpha-L-arafase belonged to glycoside hydrolase family 3, which includes some beta-xylosidases. The purified enzyme hydrolyzed mainly p-nitrophenyl alpha-L-arabinofuranoside and also reacted bifunctionally with p-nitrophenyl beta-d-xylopyranoside. However, it released only arabinose from native cell wall polysaccharides prepared from Japanese pear and from sugar beet arabinan. The enzyme did not release xylose from arabinoxylan and xylan. The only activity of the alpha-L-arafase presented here was hydrolyzing the arabinosyl residue from native polysaccharides, whereas it showed bifunctional activity against artificial substrates. According to the expression pattern and properties of the enzyme, it is a new member of the glycoside hydrolase family 3 isolated from fruit, and it may be responsible for modification of the cell wall architecture during fruit softening.

Amino Acid Sequence↗

PubMed2005

Substrate distortion in the Michaelis complex of Bacillus 1,3-1,4-beta-glucanase. Insight from first principles molecular dynamics simulations.

The structure and dynamics of the enzyme-substrate complex of Bacillus 1,3-1,4-beta-glucanase, one of the most active glycoside hydrolases, is investigated by means of Car-Parrinello molecular dynamics simulations (CPMD) combined with force field molecular dynamics (QM/MM CPMD). It is found that the substrate sugar ring located at the -1 subsite adopts a distorted 1S3 skew-boat conformation upon binding to the enzyme. With respect to the undistorted 4C1 chair conformation, the 1S3 skew-boat conformation is characterized by: (a) an increase of charge at the anomeric carbon (C1), (b) an increase of the distance between C1 and the leaving group, and (c) a decrease of the intraring O5-C1 distance. Therefore, our results clearly show that the distorted conformation resembles both structurally and electronically the transition state of the reaction in which the substrate acquires oxocarbenium ion character, and the glycosidic bond is partially broken. Together with analysis of the substrate conformational dynamics, it is concluded that the main determinants of substrate distortion have a structural origin. To fit into the binding pocket, it is necessary that the aglycon leaving group is oriented toward the beta region, and the skew-boat conformation naturally fulfills this premise. Only when the aglycon is removed from the calculation the substrate recovers the all-chair conformation, in agreement with the recent determination of the enzyme product structure. The QM/MM protocol developed here is able to predict the conformational distortion of substrate binding in glycoside hydrolases because it accounts for polarization and charge reorganization at the -1 sugar ring. It thus provides a powerful tool to model E.S complexes for which experimental information is not yet available.

Bacillus↗

PubMed2003

The structure of bovine lysosomal alpha-mannosidase suggests a novel mechanism for low-pH activation.

Lysosomal alpha-mannosidase (LAM: EC 3.2.1.24) belongs to the sequence-based glycoside hydrolase family 38 (GH38). Two other mammalian GH38 members, Golgi alpha-mannosidase II (GIIAM) and cytosolic alpha-mannosidase, are expressed in all tissues. In humans, cattle, cat and guinea pig, lack of lysosomal alpha-mannosidase activity causes the autosomal recessive disease alpha-mannosidosis. Here, we describe the three-dimensional structure of bovine lysosomal alpha-mannosidase (bLAM) at 2.7A resolution and confirm the solution state dimer by electron microscopy. We present the first structure of a mammalian GH38 enzyme that offers indications for the signal areas for mannose phosphorylation, suggests a previously undetected mechanism of low-pH activation and provides a template for further biochemical studies of the family 38 glycoside hydrolases as well as lysosomal transport. Furthermore, it provides a basis for understanding the human form of alpha-mannosidosis at the atomic level. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession codes 1o7d and r1o7dsf).

Animals↗

PubMed2004

Fiber-degrading systems of different strains of the genus Fibrobacter.

The S85 type strain of Fibrobacter succinogenes, a major ruminal fibrolytic species, was isolated 49 years ago from a bovine rumen and has been used since then as a model for extensive studies. To assess the validity of this model, we compared the cellulase- and xylanase-degrading activities of several other F. succinogenes strains originating from different ruminants, including recently isolated strains, and looked for the presence of 10 glycoside hydrolase genes previously identified in S85. The NR9 F. intestinalis type strain, representative of the second species of the genus, was also included in this study. DNA-DNA hybridization and 16S rRNA gene sequencing first classified the strains and provided the phylogenetic positions of isolates of both species. Cellulase and xylanase activity analyses revealed similar activity profiles for all F. succinogenes strains. However, the F(E) strain, phylogenetically close to S85, presented a poor xylanolytic system and weak specific activities. Furthermore, the HM2 strain, genetically distant from the other F. succinogenes isolates, displayed a larger cellulolytic profile on zymograms and higher cellulolytic specific activity. F. intestinalis NR9 presented a higher cellulolytic specific activity and a stronger extracellular xylanolytic activity. Almost all glycoside hydrolase genes studied were found in the F. succinogenes isolates by PCR, except in the HM2 strain, and few of them were detected in F. intestinalis NR9. As expected, the fibrolytic genes of strains of the genus Fibrobacter as well as the cellulase and xylanase activities are better conserved in closely related phylogenetic isolates.

Animals↗

PubMed2001

Streptomyces matensis laminaripentaose hydrolase is an 'inverting' beta-1,3-glucanase.

The laminaripentaose-producing beta-1,3-glucanase of Streptomyces matensis is a member of the glycoside hydrolase family GH-64. We have constructed and purified a recombinant hexahistidine-tagged form of the enzyme for characterisation. The enzyme, which exists as a monomer in solution, hydrolyses beta-1,3-glucan by a mechanism leading to overall inversion of the anomeric configuration. This is the first determination of the mechanism prevailing in glycoside hydrolase family GH-64 and this is the first characterisation of an 'inverting' beta-1,3-glucanase.

Biopolymers↗

PubMed2006

Oligosaccharide binding in family 8 glycosidases: crystal structures of active-site mutants of the beta-1,4-xylanase pXyl from Pseudoaltermonas haloplanktis TAH3a in complex with substrate and product.

The structures of inactive mutants D144A and E78Q of the glycoside hydrolase family 8 (GH-8) endo-beta-1,4-d-xylanase (pXyl) from the Antarctic bacterium Pseudoalteromonas haloplanktis TAH3a in complex with its substrate xylopentaose (at 1.95 A resolution) and product xylotriose (at 1.9 A resolution) have been determined by X-ray crystallography. A detailed comparative analysis of these with the apo-enzyme and with other GH-8 structures indicates an induced fit mechanism upon ligand binding whereby a number of conformational changes and, in particular, a repositioning of the proton donor into a more catalytically competent position occurs. This has also allowed for the description of protein-ligand interactions in this enzyme and for the demarcation of subsites -3 to +3. An in-depth analysis of each of these subsites gives an insight into the structure-function relationship of this enzyme and the basis of xylose/glucose discrimination in family 8 glycoside hydrolases. Furthermore, the structure of the -1/+1 subsite spanning complex reveals that the substrate is distorted from its ground state conformation. Indeed, structural analysis and in silico docking studies indicate that substrate hydrolysis in GH-8 members is preceded by a conformational change, away from the substrate ground-state chair conformation, to a pretransition state local minimum (2)S(O) conformation.

Binding Sites↗

PubMed2005

Crystallization and preliminary X-ray analysis of alpha-xylosidase from Escherichia coli.

Glycoside hydrolases have been implicated in many biological processes. To date, they have been classified into 93 glycoside hydrolase (GH) families based on amino-acid sequence similarity. alpha-Xylosidase from Escherichia coli belongs to GH family 31 and catalyzes the release of alpha-xylose from the non-reducing terminal side of alpha-xyloside. Single crystals of alpha-xylosidase have been grown by vapour diffusion at 293 K from 10%(w/v) PEG 20K, 2%(v/v) 2-propanol, 2%(v/v) glycerol and 0.1 M 2-morpholinoethanesulfonic acid pH 5.5. These crystals belong to space group P2(1)2(1)2(1) and X-ray diffraction data were collected to a resolution of 2.75 A. Crystals of selenomethionyl-substituted alpha-xylosidase were also obtained, which diffracted to at least 3.0 A. Based on the value of VM, the asymmetric unit in these crystals was assumed to contain six molecules.

Escherichia coli↗

PubMed2004

Molecular characterization of a Rhodotorula-lytic enzyme from Paecilomyces lilacinus having beta-1,3-mannanase activity.

We cloned the gene and corresponding cDNA for an extracellular Rhodotorula-lytic enzyme which has beta-1,3-mannase activity, tentatively named MAN5C, from Paecilomyces lilacinus. MAN5C showed a high homology score with the members of glycoside hydrolase family 5 in a domain search with the Pfam database, indicating that MAN5C is a novel and unique member of glycoside hydrolase family 5.

Amino Acid Sequence↗

PubMed2006

Structural characterization of the Acetobacter xylinum endo-beta-1,4-glucanase CMCax required for cellulose biosynthesis.

Previous studies have demonstrated that endoglucanase is required for cellulose biosynthesis both in bacteria and plants. However, it has yet to be elucidated how the endoglucanases function in the mechanism of cellulose biosynthesis. Here we describe the crystal structure of the cellulose biosynthesis-related endo-beta-1,47-glucanase (CMCax; EC 3.2.1.4) from the cellulose-producing Gramnegative bacterium, Acetobacter xylinum (= Gluconacetobacter xylinus), determined at 1.65-A resolution. CMCax falls into the glycoside hydrolase family 8 (GH-8), and the structure showed that the overall fold of the CMCax is similar to those of other glycoside hydrolases belonging to GH-8. Structure comparison with Clostridium thermocellum CelA, the best characterized GH-8 endoglucanase, revealed that sugar recognition subsite +3 is completely missing in CMCax. The absence of the subsite +3 leads to significant broadness of the cleft at the cellooligosaccharide reducing-end side. CMCax is known to be a secreted enzyme and is present in the culture medium. However, electron microscopic analysis using immunostaining clearly demonstrated that a portion of CMCax is localized to the cell surface, suggesting a link with other known membrane-anchored endoglucanases that are required for cellulose biosynthesis.

Amino Acid Sequence↗

PubMed1988

Degradation of hemicellulose and pectin by horse caecum contents.

1. Polysaccharide depolymerases and glycoside hydrolases involved in the breakdown of plant structural polysaccharides (hemicellulose and pectins) were monitored in three fractions of the liquid phase of horse caecum digesta: acellular fluid (AF), bacteria (B) and protozoa plus bacteria (PB). 2. Both bacteria and protozoa were found to be involved in the decomposition of pectic substances, with two enzymic activities: depolymerase (polygalacturonase, EC 3.2.1.15; and pectin lyase, EC 4.2.2.10) and esterase (pectinesterase, EC 3.1.1.11). The activity of the PB fraction was higher than that of B. 3. With hemicellulosic substrates, all three fractions showed a significant xylan endo-1,3-beta-xylosidase (EC 3.2.1.32) activity. Mannan was hardly broken down. 4. Galactomannan and arabinogalactan were broken down more extensively by the PB fraction than by the B fraction. Glycosidase activities (xylan 1,4-beta-xylosidase, EC 3.2.1.37 and alpha-L-arabinofuranosidase, EC 3.2.1.55) were also observed.

Animals↗