Endo-beta-1,3-galactanase from winter mushroom Flammulina velutipes.
ABSTRACT: Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of ?-1,3-galactan main chains and ?-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-?-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed ?-1,3-galactan, but did not act on ?-1,3-glucan, ?-1,3:1,4-glucan, xyloglucan, and agarose. It released various ?-1,3-galactooligosaccharides together with Gal from ?-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on ?-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-?-1,3-glucanase and endo-?-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-?-1,3-galactanase.
Project description:Type II arabinogalactan (AG-II) is a suitable carbohydrate source for Bifidobacterium longum subsp. longum, but the degradative enzymes have never been characterized. In this study, we characterized an exo-?-1,3-galactanase, BLLJ_1840, belonging to glycoside hydrolase family 43 from B. longum subsp. longum JCM1217. The recombinant BLLJ_1840 expressed in Escherichia coli hydrolyzed ?-1,3-linked galactooligosaccharides but not ?-1,4- and ?-1,6-linked galactooligosaccharides. The enzyme also hydrolyzed larch wood arabinogalactan (LWAG), which comprises a ?-1,3-linked galactan backbone with ?-1,6-linked galactan side chains. The kcat/Km ratio of dearabinosylated LWAG was 24-fold higher than that of ?-1,3-galactan. BLLJ_1840 is a novel type of exo-?-1,3-galactanase with a higher affinity for the ?-1,6-substituted ?-1,3-galactan than for nonsubstituted ?-1,3-galactan. BLLJ_1840 has 27% to 28% identities with other characterized exo--1,3-galactanases from bacteria and fungi. The homologous genes are conserved in several strains of B. longum subsp. longum and B. longum subsp. infantis but not in other bifidobacteria. Transcriptional analysis revealed that BLLJ_1840 is intensively induced with BLLJ_1841, an endo-?-1,6-galactanase candidate, in the presence of LWAG. This is the first report of exo-?-1,3-galactanase in bifidobacteria, which is an enzyme used for the acquisition of AG-II in B. longum subsp. longum.
Project description: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.
Project description:A novel ?-(1,3)-glucanase gene designated lamC, cloned from Corallococcus sp. strain EGB, contains a fascin-like module and a glycoside hydrolase family 16 (GH16) catalytic module. LamC displays broad hydrolytic activity toward various polysaccharides. Analysis of the hydrolytic products revealed that LamC is an exo-acting enzyme on ?-(1,3)(1,3)- and ?-(1,6)-linked glucan substrates and an endo-acting enzyme on ?-(1,4)-linked glucan and xylan substrates. Site-directed mutagenesis of conserved catalytic Glu residues (E304A and E309A) demonstrated that these activities were derived from the same active site. Excision of the fascin-like module resulted in decreased activity toward ?-(1,3)(1,3)-linked glucans. The carbohydrate-binding assay showed that the fascin-like module was a novel ?-(1,3)-linked glucan-binding module. The functional characterization of the fascin-like module and catalytic module will help us better understand these enzymes and modules.IMPORTANCE In this report of a bacterial ?-(1,3)(1,3)-glucanase containing a fascin-like module, we reveal the ?-(1,3)(1,3)-glucan-binding function of the fascin-like module present in the N terminus of LamC. LamC displays exo-?-(1,3)/(1,6)-glucanase and endo-?-(1,4)-glucanase/xylanase activities with a single catalytic domain. Thus, LamC was identified as a novel member of the GH16 family.
Project description:Exo-beta-1,3-galactanase from Phanerochaete chrysosporium (Pc1,3Gal43A) consists of a glycoside hydrolase family 43 catalytic domain and a substrate-binding domain that belongs to carbohydrate-binding module family 35. It catalyzes the hydrolysis of beta-1,3-galactan, which is the backbone of the arabinogalactan proteins; the C-terminal carbohydrate-binding module family 35 domain increases the local concentration of the enzyme around beta-1,3-galactan by its high affinity for the substrate. To enable phase determination using the multiwavelength anomalous dispersion method, selenomethionyl Pc1,3Gal43A was crystallized at 298 K using the hanging-drop vapour-diffusion method. The presence of selenium in the crystals was confirmed from the X-ray absorption spectrum. The crystals belonged to space group P2(1) and diffracted to 1.8 A resolution.
Project description:The putative endo-beta-1,6-galactanase gene from Streptomyces avermitilis was cloned and expressed in Escherichia coli, and the enzymatic properties of the recombinant enzyme were characterized. The gene consisted of a 1,476-bp open reading frame and encoded a 491-amino-acid protein, comprising an N-terminal secretion signal sequence and glycoside hydrolase family 5 catalytic module. The recombinant enzyme, Sa1,6Gal5A, catalyzed the hydrolysis of beta-1,6-linked galactosyl linkages of oligosaccharides and polysaccharides. The enzyme produced galactose and a range of beta-1,6-linked galacto-oligosaccharides, predominantly beta-1,6-galactobiose, from beta-1,6-galactan chains. There was a synergistic effect between the enzyme and Sa1,3Gal43A in degrading tomato arabinogalactan proteins. These results suggest that Sa1,6Gal5A is the first identified endo-beta-1,6-galactanase from a prokaryote.
Project description:Glycans are major nutrients for the human gut microbiota (HGM). Arabinogalactan proteins (AGPs) comprise a heterogenous group of plant glycans in which a ?1,3-galactan backbone and ?1,6-galactan side chains are conserved. Diversity is provided by the variable nature of the sugars that decorate the galactans. The mechanisms by which nutritionally relevant AGPs are degraded in the HGM are poorly understood. Here we explore how the HGM organism Bacteroides thetaiotaomicron metabolizes AGPs. We propose a sequential degradative model in which exo-acting glycoside hydrolase (GH) family 43 ?1,3-galactanases release the side chains. These oligosaccharide side chains are depolymerized by the synergistic action of exo-acting enzymes in which catalytic interactions are dependent on whether degradation is initiated by a lyase or GH. We identified two GHs that establish two previously undiscovered GH families. The crystal structures of the exo-?1,3-galactanases identified a key specificity determinant and departure from the canonical catalytic apparatus of GH43 enzymes. Growth studies of Bacteroidetes spp. on complex AGP revealed 3 keystone organisms that facilitated utilization of the glycan by 17 recipient bacteria, which included B. thetaiotaomicron. A surface endo-?1,3-galactanase, when engineered into B. thetaiotaomicron, enabled the bacterium to utilize complex AGPs and act as a keystone organism.
Project description:?-1,3:1,4-Glucan is a major cell wall component accumulating in endosperm and young tissues in grasses. The mixed linkage glucan is a linear polysaccharide mainly consisting of cellotriosyl and cellotetraosyl units linked through single ?-1,3-glucosidic linkages, but it also contains minor structures such as cellobiosyl units. In this study, we examined the action of an endo-?-1,3(4)-glucanase from Trichoderma sp. on a minor structure in barley ?-1,3:1,4-glucan. To find the minor structure on which the endo-?-1,3(4)-glucanase acts, we prepared oligosaccharides from barley ?-1,3:1,4-glucan by endo-?-1,4-glucanase digestion followed by purification by gel permeation and paper chromatography. The endo-?-1,3(4)-glucanase appeared to hydrolyze an oligosaccharide with degree of polymerization 5, designated C5-b. Based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF)/ToF-mass spectrometry (MS)/MS analysis, C5-b was identified as ?-Glc-1,3-?-Glc-1,4-?-Glc-1,3-?-Glc-1,4-Glc including a cellobiosyl unit. The results indicate that a type of endo-?-1,3(4)-glucanase acts on the cellobiosyl units of barley ?-1,3:1,4-glucan in an endo-manner.
Project description:Background:The Bacillus subtilis endo-?-1,4-glucanase (BsCel5A) hydrolyzes ?-1,3-1,4-linked glucan, and the enzyme includes a family 3 carbohydrate-binding module (CBM3) that binds ?-1,4-linked glucan. Methods:Here we investigate the BsCel5A ?-1,3-1,4 glucanase activity after exchanging the CBM3 domain for the family 11 CBM from Ruminiclostridium thermocellum celH (RtCBM11) having ?-1,3-1,4 glucan affinity. Results:The BsCel5A-RtCBM11 presents a 50.4% increase in Vmax, a 10% reduction in K0.5, and a 2.1-fold increase in catalytic efficiency. Enzyme mobility and binding to barley ?-1,3-1,4 glucan and pre-treated sugarcane bagasse were investigated using Electron Paramagnetic Resonance (EPR) with Site-Directed Spin Labeling (SDSL) of the binding site regions of the CBM3 and RtCBM11 domains in the BsCel5A-CBM3 and BsCel5A-RtCBM11, respectively. Although higher mobility than the RtCBM11 was shown, no interaction of the spin-labeled CBM3 with ?-1,3-1,4 glucan was observed. In contrast, a Ka value of 0.22 mg/mL was estimated from titration of the BsCel5A-RtCBM11 with ?-1,3-1,4 glucan. Enzyme binding as inferred from altered EPR spectra of the BsCel5A-RtCBM11 was observed only after xylan or lignin extraction from sugarcane bagasse. Binding to xylan- or lignin-free lignocellulose was correlated with a 4.5- to 5-fold increase in total reducing sugar release as compared to the milled intact sugarcane bagasse, suggesting that xylan impedes enzyme access to the ?-1,3-1,4 glucan. Conclusions:These results show that the non-specific binding of the BsCel5A-RtCBM11 to the lignin component of the cell wall is minimal, and represent the first reported use of EPR to directly study the interaction of glycoside hydrolyse enzymes with natural insoluble substrates.
Project description:Streptococcus bovis JB1 was found to produce a 25-kDa extracellular enzyme active against beta-(1,3-1,4)-glucans. A gene was isolated encoding a specific beta-(1,3-1,4)-glucanase that corresponds to this size and belongs to glycoside hydrolase family 16. A 4- to 10-fold increase in supernatant beta-glucanase activity was obtained when the cloned beta-glucanase gene was reintroduced into S. bovis JB1 by use of constructs based on the plasmid vector pTRW10 or pIL253. The beta-(1,3-1,4)-glucanase gene was also expressed upon introduction of the pTRW10 construct pTRWL1R into Lactococcus lactis IL2661 and Enterococcus faecalis JH2-SS, although extracellular activity was 8- to 50-fold lower than that in S. bovis JB1. The beta-(1,3-1,4)-glucanase purified from the culture supernatant of S. bovis JB1 carrying pTRWL1R showed a K(m) of 2.8 mg per ml and a Vmax of 338 mumol of glucose equivalents per min per mg of protein with barley beta-glucan as the substrate. The S. bovis beta-(1,3-1,4)-glucanase may contribute to the ability of this bacterium to utilize starch by degrading structural polysaccharides present in endosperm cell walls.
Project description:Marine bacteria residing on local red, green, and brown seaweeds were screened for exo-1,3-?-glucanase (ExoP) activity. Of the 90 bacterial species isolated from 32 seaweeds, only one, a Pseudoalteromonas sp., was found to display such activity. It was isolated from a Durvillaea sp., a brown kelp known to contain significant amounts of the storage polysaccharide laminarin (1,3-?-D-glucan with some 1,6-? branching). Four chromatographic steps were utilized to purify the enzyme (ExoP). Chymotryptic digestion provided peptide sequences for primer design and subsequent gene cloning. The exoP gene coded for 840 amino acids and was located just 50 bp downstream from a putative lichenase (endo-1,3-1,4-?-glucanase) gene, suggesting possible cotranscription of these genes. Sequence comparisons revealed ExoP to be clustered within a group of bacterial glycosidases with high similarity to a group of glycoside hydrolase (GH3) plant enzymes, of which the barley exo-1,3/1,4-?-glucanase (ExoI) is the best characterized. The major difference between the bacterial and plant proteins is an extra 200- to 220-amino-acid extension at the C terminus of the former. This additional sequence does not correlate with any known functional domain, but ExoP was not active against laminarin when this region was removed. Production of recombinant ExoP allowed substrate specificity studies to be performed. The enzyme was found to possess similar levels of exoglucanase activity against both 1,4-? linkages and 1,3-? linkages, and so ExoP is designated an exo-1,3/1,4-?-exoglucanase, the first such bacterial enzyme to be characterized. This broader specificity could allow the enzyme to assist in digesting both cell wall cellulose and cytoplasmic laminarin.