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PubMed2000

Cloning and sequencing of cel5Z gene from Erwinia chrysanthemi PY35.

The phytopathogenic bacterium Erwinia chrysanthemi (Ech) secretes multiple isozymes of plant cell wall disrupting enzymes such as pectate lyase and endoglucanases. We cloned genomic DNA from Ech PY35 digested with Sau3AI and ligated into pBluescript II SK+. One of the E. coli XL1-blue clones had the ability to hydrolyze carboxymethyl cellulose and polygalacturonic acid. By subsequent subcloning from this 2.9 kb fragment, we obtained a 2.0 kb (pPY401), designated cel5Z, which had the activity of hydrolyzation of carboxymethyl cellulose. The cel5Z gene had an open reading frame (ORF) of 1,281 bp starting with an ATG start codon and followed by a TAA stop codon, encoding 426 amino acids with a signal peptide of 41 amino acids. Since the deduced amino acid sequence of this protein was very similar to that of CelE of Pseudomonas fluorescens, and had the conserved region, VIYEIYNEPL, it belonged to the glycoside hydrolase family 5 of EC 3.2.1.4. The molecular mass of Cel5Z protein from E. coli XL1-blue, as analyzed by CMC-SDS-PAGE, appeared to be 42 kDa. The optimum pH was 6, and the optimum temperature was about 40 degrees C for its enzymatic activity.

Amino Acid Sequence↗

PubMed2005

Molecular cloning of the gene encoding beta-1,3(4)-glucanase A from a marine bacterium, Pseudomonas sp. PE2, an essential enzyme for the degradation of Pythium porphyrae cell walls.

The beta-1,3(4)-glucanase A (GluA)-encoding gene named gluA was cloned from the genomic library of a marine bacterium Pseudomonas sp. PE2 by expression in Escherichia coli, and the complete DNA sequence was determined. The recombinant enzyme from Pseudomonas sp. PE2 was examined to determine the essential enzymes for degrading Pythium porphyrae cell walls, comparatively using other two recombinant enzymes, chitinase A and beta-1,3-glucanase B from the same bacterial strain. GluA most degraded the cell walls among these three enzymes, suggesting that GluA seems to be most important to P. porphyrae cell-wall-degrading activity. The deduced GluA is a modular enzyme composed of an N-terminal signal peptide, the tandem-duplicated carbohydrate-binding module family 6 (CBM(GluA)-1 and CBM(GluA)-2), and a glycoside hydrolase family 16 catalytic domain. Deletion analysis clearly indicated that GluA lacking CBM(GluA)-1 and CBM(GluA)-2 does not bind to Avicel and xylan. These results suggest that the tandem-repeated CBM of GluA may play a key role in the binding of Avicel and xylan as well as beta-1,3- and beta-1,3;1,4-glucans and is very important to bind to insoluble polysaccharides.

Amino Acid Sequence↗

PubMed2005

Glycan foraging in vivo by an intestine-adapted bacterial symbiont.

Germ-free mice were maintained on polysaccharide-rich or simple-sugar diets and colonized for 10 days with an organism also found in human guts, Bacteroides thetaiotaomicron, followed by whole-genome transcriptional profiling of bacteria and mass spectrometry of cecal glycans. We found that these bacteria assembled on food particles and mucus, selectively induced outer-membrane polysaccharide-binding proteins and glycoside hydrolases, prioritized the consumption of liberated hexose sugars, and revealed a capacity to turn to host mucus glycans when polysaccharides were absent from the diet. This flexible foraging behavior should contribute to ecosystem stability and functional diversity.

Adaptation, Physiological↗

PubMed2003

The three-dimensional structures of two beta-agarases.

Agars are important gelifying agents for biochemical use and the food industry. To cleave the beta-1,4-linkages between beta-d-galactose and alpha-l-3,6-anhydro-galactose residues in the red algal galactans known as agars, marine bacteria produce polysaccharide hydrolases called beta-agarases. Beta-agarases A and B from Zobellia galactanivorans Dsij have recently been biochemically characterized. Here we report the first crystal structure of these two beta-agarases. The two proteins were overproduced in Escherichia coli and crystallized, and the crystal structures were determined at 1.48 and 2.3 A for beta-agarases A and B, respectively. The structure of beta-agarase A was solved by the multiple anomalous diffraction method, whereas beta-agarase B was solved with molecular replacement using beta-agarase A as model. Their structures adopt a jelly roll fold with a deep active site channel harboring the catalytic machinery, namely the nucleophilic residues Glu-147 and Glu-184 and the acid/base residues Glu-152 and Glu-189 for beta-agarases A and B, respectively. The structures of the agarases were compared with those of two lichenases and of a kappa-carrageenase, which all belong to family 16 of the glycoside hydrolases in order to pinpoint the residues responsible for their widely differing substrate specificity. The relationship between structure and enzymatic activity of the two beta-agarases from Z. galactanivorans Dsij was studied by analysis of the degradation products starting with different oligosaccharides. The combination of the structural and biochemical results allowed the determination of the number of subsites present in the catalytic cleft of the beta-agarases.

Bacterial Proteins↗

PubMed1976

Inhibition of glucan and levan synthesis and neuraminidase activity of oral streptococci by monkey antiserums.

A demonstration that antiserums from monkeys immunized with fructosyltransferase and glucosyltransferase reduced the activities of the enzymes in producing glucans and levans was done. The nature of the inhibiting factor has not yet been identified. The local immunization of monkeys with glycosidic hydrolases produced a sufficient amount of inhibiting principle, presumably antibody, to inhibit neuraminidase one month after the first immunization and this degree of immunization, which reached degrees of 50%, remained constant throughout 11 months of immunization of these monkeys. The immunizing principle has not yet been found in saliva. A criterion for the feasibility for immunization has been met. A significant reduction in streptoccal glucosyltransferase, fructosyltransferase, and neuraminidase activity was noted after immunization with enzymes.

Animals↗

PubMed2004

Crystallization and preliminary X-ray analyses of the active and the inactive forms of family GH-8 chitosanase with subclass II specificity from Bacillus sp. strain K17.

Chitosanase from Bacillus sp. strain K17 (ChoK) belongs to glycoside hydrolase family 8 and exhibits subclass II specificity. The purified protein is structurally stable over a wide pH range (3-10), but is active in a much narrower pH range (4.5-7.5), with optimal activity around pH 6.0. The protein has been successfully crystallized at two different pH values corresponding to the active and inactive states. The crystals diffract to 1.5 and 2.0 A resolution, respectively.

Bacillus↗

PubMed2004

A novel family of carbohydrate-binding modules identified with Ruminococcus albus proteins.

We recently showed that some of the enzymes underpinning cellulose solubilization by Ruminococcus albus 8 lack the conventional type of dockerin module characteristic of cellulosomal proteins and instead, bear an "X" domain of unknown function at their C-termini. We have now subcloned and expressed six X domains and showed that five of them bind to xylan, chitin, microcrystalline and phosphoric-acid swollen cellulose, as well as more heterogenous substrates such as alfalfa cell walls, banana stem and wheat straw. The X domain that did not bind to these substrates was derived from a family-5 glycoside hydrolase (Cel5G), which possesses two X domains in tandem. Whereas the internal X domain failed to bind to the substrates, the recombinant dyad exhibited markedly enhanced binding relative to that observed for the C-terminal X domain alone. The evidence supports a distinctive carbohydrate-binding role of broad specificity for this type of domain, and we propose a novel family (designated family 37) of carbohydrate-binding modules that appear to be peculiar to R. albus.

Bacterial Proteins↗

PubMed2003

Relation between domain evolution, specificity, and taxonomy of the alpha-amylase family members containing a C-terminal starch-binding domain.

The alpha-amylase family (glycoside hydrolase family 13; GH 13) contains enzymes with approximately 30 specificities. Six types of enzyme from the family can possess a C-terminal starch-binding domain (SBD): alpha-amylase, maltotetraohydrolase, maltopentaohydrolase, maltogenic alpha-amylase, acarviose transferase, and cyclodextrin glucanotransferase (CGTase). Such enzymes are multidomain proteins and those that contain an SBD consist of four or five domains, the former enzymes being mainly hydrolases and the latter mainly transglycosidases. The individual domains are labelled A [the catalytic (beta/alpha)8-barrel], B, C, D and E (SBD), but D is lacking from the four-domain enzymes. Evolutionary trees were constructed for domains A, B, C and E and compared with the 'complete-sequence tree'. The trees for domains A and B and the complete-sequence tree were very similar and contain two main groups of enzymes, an amylase group and a CGTase group. The tree for domain C changed substantially, the separation between the amylase and CGTase groups being shortened, and a new border line being suggested to include the Klebsiella and Nostoc CGTases (both four-domain proteins) with the four-domain amylases. In the 'SBD tree' the border between hydrolases (mainly alpha-amylases) and transglycosidases (principally CGTases) was not readily defined, because maltogenic alpha-amylase, acarviose transferase, and the archaeal CGTase clustered together at a distance from the main CGTase cluster. Moreover the four-domain CGTases were rooted in the amylase group, reflecting sequence relationships for the SBD. It appears that with respect to the SBD, evolution in GH 13 shows a transition in the segment of the proteins C-terminal to the catalytic (beta/alpha)8-barrel(domain A).

Binding Sites↗

PubMed2007

Molecular cloning, expression, and characterization of a beta-agarase gene, agaD, from a marine bacterium, Vibrio sp. strain PO-303.

The beta-agarase-d gene (agaD) from a marine bacterium, Vibrio sp. strain PO-303, was cloned and expressed in Escherichia coli. The gene consists of 1,362 bp and encodes a protein of 453 amino acids with a predicted molecular weight of 50,824. The full length of agarase-d consists of a signal peptide, a glycoside hydrolase family 16 catalytic module (CM), and a carbohydrate binding module (CBM). The full length of agarase-d without the signal peptide (rAgaDDeltafull), the catalytic module (rAgaDCM), or the CBM (rAgaDCBM) was expressed in E. coli as recombinant proteins. rAgaDCM exhibited higher enzyme activity (63.6 units/mg) than rAgaDDeltafull (1.20 units/mg) against agarose. rAgaDCM hydrolyzed agar and porphyran to several oligosaccharides and acted on neoagarohexaose to produce neoagarotetraose and neoagarobiose, but did not act on neoagarotetraose. rAgaDCBM bound to agarose.

Amino Acid Sequence↗

PubMed1987

Delaying carbohydrate absorption in noninsulin-dependent diabetes mellitus: useful therapy?

Epidemiological and psychological studies have revealed major difficulties in motivating diabetic patients to observe a long-term dietary regimen. Therefore, manipulation of intestinal digestion or absorption appears to be a feasible therapeutic approach in the management of diabetes. The addition of natural or chemically processed fiber has been shown to decrease both the postprandial and fasting blood glucose in type-2 diabetics by delaying carbohydrate absorption. Recently, selective enzyme inhibitors of glycoside hydrolases in the upper intestine have been found which create a moderate degree of malabsorption of carbohydrates. The postprandial blood sugar response can be reduced by 50%. However, both these forms of treatment may not be accepted by patients because of impalatability or gastrointestinal side effects. At present only short-term studies with each group of substances are available. Whether the reduction of hyperglycemia is sufficient for the prevention of complications must be clarified in long-term trials.

Acarbose↗

PubMed2026

Synthesis and Application of a Suite of 2,5-Aryl Tetrazole Photoaffinity-Based Probes for Profiling Microbial Carbohydrate and Mucin Metabolism in Gut Microbiota.

Photoaffinity-based chemoproteomics provides a strategy for interrogating protein engagement and networks within complex biological systems. In the context of carbohydrate metabolism, however, linking the probe structure to glycan-processing networks remains challenging due to the diversity and redundancy of carbohydrate-active enzymes (CAZymes). Here, we employ 2,5-tetrazoles as photoreactive groups to develop a suite of monosaccharide-bearing probes designed to capture carbohydrate-associated protein environments in gut microorganisms. Across defined bacterial cultures and human fecal lysates, tetrazole probes enriched glycoside hydrolases (GHs) and additional carbohydrate-associated proteins, including transporters and regulatory elements. Notably, enrichment profiles were functionally biased toward glycan-processing modules, despite minimal shifts in global protein abundance under different growth conditions. These findings demonstrate that tetrazole chemoproteomics complements abundance-based proteomics by reporting on glycan-associated protein engagement and organization. Together, this probe suite provides a substrate-centric approach to studying carbohydrate-processing networks in defined microbes and complex microbiomes.

Tetrazoles↗

PubMed1981

Influence of growth medium on adsorption of Streptococcus mutans, Actinomyces viscosus, and Actinomyces naeslundii to saliva-treated hydroxyapatite surfaces.

The influence of the growth medium on the ability of strains of Streptococcus mutans, Actinomyces viscosus and A. naeslundii to attach to saliva-treated hydroxyapatite (S-HA) surfaces was studied. Preliminary experiments indicated that cells of each species harvested in lag, log, and early stationary phases of growth adsorbed comparably to S-HA; thus, early stationary phase cells were used in all subsequent assays. Strains were grown in chemically defined medium (CDM), in CDM supplemented with gastric mucin or with filter-sterilized or (60)Co-irradiated saliva from human donors of blood types A, B, or O, and in Trypticase soy broth (BBL Microbiology Systems) and Todd-Hewitt broth. Adherence of S. mutans H12 to S-HA tended to vary when the streptococci were grown in saliva-supplemented CDM, but the number of cells which attached was generally within twofold of that of CDM-grown cells. Attachment of A. viscosus S2 and LY7 and of A. naeslundii S4 and L13 was generally similar when grown in CDM or in CDM supplemented with saliva, but it tended to increase for organisms grown in CDM supplemented with gastric mucin. None of the strains studied appeared to destroy the blood group reactivity of the added salivary components, and they attached equally well to HA treated with homologous or heterogous saliva from that present in the medium in which they were grown. The A. viscosus strains adsorbed in 25 to 40% higher numbers to HA treated with blood type B saliva than with type A saliva, irrespective of the medium used for growth. S. mutans H12 cells displayed alpha- and beta-glucosidase and alpha-galactosidase activity; the Actinomyces strains exhibited these activities plus beta-galactosidase when grown in all media. However, the levels of these glycoside hydrolases did not correlate with cell adsorption to S-HA. The apparent weak influence of the growth medium on attachment of S. mutans was studied further. Strains of S. mutans isolated from the saliva of five human donors were made resistant to streptomycin, grown in CDM, and then added to new saliva samples from the respective donors from which they were obtained. The in vitro-grown cells were found to attach to S-HA comparably to S. mutans cells present naturally in the saliva.

ABO Blood-Group System↗

PubMed2002

The membrane-bound alpha-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylan.

The microbial degradation of xylan is a key biological process. Hardwood 4-O-methyl-D-glucuronoxylans are extensively decorated with 4-O-methyl-D-glucuronic acid, which is cleaved from the polysaccharides by alpha-glucuronidases. In this report we describe the primary structures of the alpha-glucuronidase from Cellvibrio mixtus (C. mixtus GlcA67A) and the alpha-glucuronidase from Pseudomonas cellulosa (P. cellulosa GlcA67A) and characterize P. cellulosa GlcA67A. The primary structures of C. mixtus GlcA67A and P. cellulosa GlcA67A, which are 76% identical, exhibit similarities with alpha-glucuronidases in glycoside hydrolase family 67. The membrane-associated pseudomonad alpha-glucuronidase released 4-O-methyl-D-glucuronic acid from 4-O-methyl-D-glucuronoxylooligosaccharides but not from 4-O-methyl-D-glucuronoxylan. We propose that the role of the glucuronidase, in combination with cell-associated xylanases, is to hydrolyze decorated xylooligosaccharides, generated by extracellular hemicellulases, to xylose and 4-O-methyl-D-glucuronic acid, enabling the pseudomonad to preferentially utilize the sugars derived from these polymers.

Base Sequence↗

PubMed2001

Identification of novel beta-mannan- and beta-glucan-binding modules: evidence for a superfamily of carbohydrate-binding modules.

Many glycoside hydrolases, which degrade long-chain carbohydrate polymers, possess distinct catalytic modules and non-catalytic carbohydrate-binding modules (CBMs). On the basis of conserved protein secondary structure, we describe here the identification and experimental characterization of novel type of mannanase-associated mannan-binding module and also characterization of two CBM family 4 laminarinase-associated beta-glucan-binding modules. These modules are predicted to belong to a superfamily of CBMs which include families 4, 16, 17, 22 and a proposed new family, family 27.

Amino Acid Sequence↗

PubMed2000

Expression, purification, crystallization and preliminary X-ray analysis of the iota-carrageenase from Alteromonas fortis.

This is the first crystallization report of a glycoside hydrolase which belongs to family 82. A recombinant form of His-tagged iota-carrageenase from Alteromonas fortis was expressed, purified and crystallized. Crystals were obtained by the vapour-diffusion method using polyethylene glycol (M(W) = 6000) as a precipitant. They belong to space group P2(1), with unit-cell parameters a = 56. 75, b = 91.04, c = 125.01 A, beta = 93.41 degrees. The unit cell contains two molecules in the asymmetric unit related by a non-crystallographic twofold axis. Crystals diffracted to 2.0 A resolution on a synchrotron beamline.

Alteromonas↗

PubMed2004

Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from the novel marine isolate, JAMB-A94.

A gene, agaA, for a novel beta-agarase from the marine bacterium JAMB-A94 was cloned and sequenced. The 16S rDNA of the isolate had the closest match, of only 94.8% homology, with that from Microbulbifer salipaludis JCM11542(T). The agaA gene encoded a protein with a calculated molecular mass of 48,203 Da. The deduced amino acid sequence showed 37-66% identity to those of known agarases in glycoside hydrolase family 16. A carbohydrate-binding module-like amino acid sequence was found in the C-terminal region. The recombinant enzyme was hyper-produced extracellularly when Bacillus subtilis was used as a host. The purified enzyme was an endo-type beta-agarase, yielding neoagarotetraose as the main final product. It was very thermostable up to 60 degrees C. The optimal pH and temperature for activity were around 7.0 and 55 degrees C respectively. The activity was not inhibited by EDTA (up to 100 mM) and sodium dodecyl sulfate (up to 30 mM).

Alteromonadaceae↗

PubMed2004

A new thermostable alpha-L-arabinofuranosidase from a novel thermophilic bacterium.

An alpha-L-arabinofuranosidase gene was identified in a sequenced genome of a novel thermophilic bacterium, which belongs to the recently described phylum of Thermomicrobia. Amino acid sequence comparison of the enzyme (designated AraF) revealed similarity to glycoside hydrolases of family 51. The gene was cloned into Escherichia coli and its recombinant product expressed and purified. The enzyme appeared to be a hexamer. AraF was optimally active at 70 degrees C (over 10 min) and pH 6 having 92% residual activity after 1 h at 70 degrees C. AraF had a Km) value of 0.6 mM and V(max) value of 122 U mg(-1) on p-nitrophenyl-alpha-L-arabinofuranoside. AraF was almost equally active on branched arabinan and debranched arabinan, properties not previously found in alpha-L-arabinofuranosidases in GH family 51.

Bacterial Proteins↗

PubMed1977

Plant glycoprotein biosynthesis. Uridine diphosphate N-acetyl-glucosaminyltransferase from horseradish root.

A particulate enzyme preparation from horseradish root tissue was shown to catalyze the transfer of 2-acetamido-2-deoxy-D-[14C1]glucose from uridine diphosphate 2-acetamido-2-deoxy-D-[14C1]glucose to an exogenous acceptor molecule derived from horseradish peroxidase. The acceptor was produced from purified peroxidase by the action of a mixture of glycoside hydrolases covalently bound to Sepharose. The membrane preparation containing the transferase was purified approximately 12-fold by aqueous two phase distribution and by discontinuous sucrose density gradient centrifugation. Hydrolysis of the reaction product yielded glucosamine as the only radio-labeled substance. Precipitation of the reaction product by antiserum against peroxidase showed that the label was incorporated into peroxidase. The transferase utilized the acceptor most efficiently when only 12% of the 2-acetamido-2-deoxy-D-glucose was removed from the acceptor. The acceptor lost no accepting capabilities when heated to 100 degrees C for 3 min prior to assay. Trypsin treatment caused a 14% decrease in label incorporated while pronase treatment caused a 93% decrease,

Glycoproteins↗