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The structure of a family 110 glycoside hydrolase provides insight into the hydrolysis of α-1,3-galactosidic linkages in λ-carrageenan and blood group antigens.

ABSTRACT: α-Linked galactose is a common carbohydrate motif in nature that is processed by a variety of glycoside hydrolases from different families. Terminal Galα1-3Gal motifs are found as a defining feature of different blood group and tissue antigens, as well as the building block of the marine algal galactan λ-carrageenan. The blood group B antigen and linear α-Gal epitope can be processed by glycoside hydrolases in family GH110, whereas the presence of genes encoding GH110 enzymes in polysaccharide utilization loci from marine bacteria suggests a role in processing λ-carrageenan. However, the structure-function relationships underpinning the α-1,3-galactosidase activity within family GH110 remain unknown. Here we focus on a GH110 enzyme (PdGH110B) from the carrageenolytic marine bacterium Pseudoalteromonas distincta U2A. We showed that the enzyme was active on Galα1-3Gal but not the blood group B antigen. X-ray crystal structures in complex with galactose and unhydrolyzed Galα1-3Gal revealed the parallel β-helix fold of the enzyme and the structural basis of its inverting catalytic mechanism. Moreover, an examination of the active site reveals likely adaptations that allow accommodation of fucose in blood group B active GH110 enzymes or, in the case of PdGH110, accommodation of the sulfate groups found on λ-carrageenan. Overall, this work provides insight into the first member of a predominantly marine clade of GH110 enzymes while also illuminating the structural basis of α-1,3-galactoside processing by the family as a whole.

SUBMITTER: McGuire BE

PROVIDER: S-EPMC7939477 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Degradation of lambda-carrageenan by Pseudoalteromonas carrageenovora lambda-carrageenase: a new family of glycoside hydrolases unrelated to kappa- and iota-carrageenases.

Project description:Carrageenans are sulfated galactans found in the cell walls of red seaweeds. They are classified according to the number and the position of sulfate ester groups. lambda-Carrageenan is the most sulfated carrageenan and carries at least three sulfates per disaccharide unit. The sole known depolymerizing enzyme of lambda-carrageenan, the lambda-carrageenase from Pseudoalteromonas carrageenovora, has been purified, cloned and sequenced. Sequence analyses have revealed that the lambda-carrageenase, referred to as CglA, is the first member of a new family of GHs (glycoside hydrolases), which is unrelated to families GH16, that contains kappa-carrageenases, and GH82, that contains iota-carrageenases. This large enzyme (105 kDa) features a low-complexity region, suggesting the presence of a linker connecting at least two independent modules. The N-terminal region is predicted to fold as a beta-propeller. The main degradation products have been purified and characterized as neo-lambda-carratetraose [DP (degree of polymerization) 4] and neo-lambda-carrahexaose (DP6), indicating that CglA hydrolyses the beta-(1-->4) linkage of lambda-carrageenan. LC-MALLS (liquid chromatography-multi-angle laser light scattering) and (1)H-NMR monitoring of the enzymatic degradation of lambda-carrageenan indicate that CglA proceeds according to an endolytic mode of action and a mechanism of inversion of the anomeric configuration. Using 2-aminoacridone-labelled neo-lambda-carrabiose oligosaccharides, in the present study we demonstrate that the active site of CglA comprises at least 8 subsites (-4 to +4) and that a DP6 oligosaccharide binds in the subsites -4 to +2 and can be hydrolysed into DP4 and DP2.

| S-EPMC1868830 | biostudies-literature

Crystalline cellulose hydrolysis in microbial communities via actinobacterial glycoside hydrolase family 12

Project description:Proteomic data from supernatants from thermophilic cellulolytic microbial communities, for the identification of specific hydrolases from Thermobispora bispora related actinobacterium.

2016-08-25 | PXD004204 | Pride

Modulating Glycoside Hydrolase Activity between Hydrolysis and Transfer Reactions Using an Evolutionary Approach.

Project description:The proteins within the CAZy glycoside hydrolase family GH13 catalyze the hydrolysis of polysaccharides such as glycogen and starch. Many of these enzymes also perform transglycosylation in various degrees, ranging from secondary to predominant reactions. Identifying structural determinants associated with GH13 family reaction specificity is key to modifying and designing enzymes with increased specificity towards individual reactions for further applications in industrial, chemical, or biomedical fields. This work proposes a computational approach for decoding the determinant structural composition defining the reaction specificity. This method is based on the conservation of coevolving residues in spatial contacts associated with reaction specificity. To evaluate the algorithm, mutants of α-amylase (TmAmyA) and glucanotransferase (TmGTase) from Thermotoga maritima were constructed to modify the reaction specificity. The K98P/D99A/H222Q variant from TmAmyA doubled the transglycosydation/hydrolysis (T/H) ratio while the M279N variant from TmGTase increased the hydrolysis/transglycosidation ratio five-fold. Molecular dynamic simulations of the variants indicated changes in flexibility that can account for the modified T/H ratio. An essential contribution of the presented computational approach is its capacity to identify residues outside of the active center that affect the reaction specificity.

| S-EPMC8587830 | biostudies-literature

The Structure of an Archaeal ?-Glucosaminidase Provides Insight into Glycoside Hydrolase Evolution.

Project description:The archaeal exo-?-d-glucosaminidase (GlmA) is a dimeric enzyme that hydrolyzes chitosan oligosaccharides into monomer glucosamines. GlmA is a member of the glycosidase hydrolase (GH)-A superfamily-subfamily 35 and is a novel enzyme in terms of its primary structure. Here, we present the crystal structure of GlmA in complex with glucosamine at 1.27 Å resolution. The structure reveals that a monomeric form of GlmA shares structural homology with GH42 ?-galactosidases, whereas most of the spatial positions of the active site residues are identical to those of GH35 ?-galactosidases. We found that upon dimerization, the active site of GlmA changes shape, enhancing its ability to hydrolyze the smaller substrate in a manner similar to that of homotrimeric GH42 ?-galactosidase. However, GlmA can differentiate glucosamine from galactose based on one charged residue while using the "evolutionary heritage residue" it shares with GH35 ?-galactosidase. Our study suggests that GH35 and GH42 ?-galactosidases evolved by exploiting the structural features of GlmA.

| S-EPMC5377812 | biostudies-literature

Crystal Structure of Bifidobacterium bifidum Glycoside Hydrolase Family 110 α-Galactosidase Specific for Blood Group B Antigen.

Project description:To overcome incompatibility issues and increase the possibility of blood transfusion, technologies that enable efficient conversion of A- and B-type red blood cells to the universal donor O-type is desirable. Although several blood type-converting enzymes have been identified, detailed understanding about their molecular functions is limited. α-Galactosidase from Bifidobacterium bifidum JCM 1254 (AgaBb), belonging to glycoside hydrolase (GH) 110 subfamily A, specifically acts on blood group B antigen. Here we present the crystal structure of AgaBb, including the catalytic GH110 domain and part of the C-terminal uncharacterized regions. Based on this structure, we deduced a possible binding mechanism of blood group B antigen to the active site. Site-directed mutagenesis confirmed that R270 and E380 recognize the fucose moiety in the B antigen. Thermal shift assay revealed that the C-terminal uncharacterized region significantly contributes to protein stability. This region is shared only among GH110 enzymes from B. bifidum and some Ruminococcus species. The elucidation of the molecular basis for the specific recognition of blood group B antigen is expected to lead to the practical application of blood group conversion enzymes in the future.

| S-EPMC11368712 | biostudies-literature

Differential recognition and hydrolysis of host carbohydrate antigens by Streptococcus pneumoniae family 98 glycoside hydrolases.

Project description:The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-beta-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-beta-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

| S-EPMC2758015 | biostudies-literature

Insight into Dominant Cellulolytic Bacteria from Two Biogas Digesters and Their Glycoside Hydrolase Genes.

Project description:Diverse cellulolytic bacteria are essential for maintaining high lignocellulose degradation ability in biogas digesters. However, little was known about functional genes and gene clusters of dominant cellulolytic bacteria in biogas digesters. This is the foundation to understand lignocellulose degradation mechanisms of biogas digesters and apply these gene resource for optimizing biofuel production. A combination of metagenomic and 16S rRNA gene clone library methods was used to investigate the dominant cellulolytic bacteria and their glycoside hydrolase (GH) genes in two biogas digesters. The 16S rRNA gene analysis revealed that the dominant cellulolytic bacteria were strains closely related to Clostridium straminisolvens and an uncultured cellulolytic bacterium designated BG-1. To recover GH genes from cellulolytic bacteria in general, and BG-1 in particular, a refined assembly approach developed in this study was used to assemble GH genes from metagenomic reads; 163 GH-containing contigs ≥ 1 kb in length were obtained. Six recovered GH5 genes that were expressed in E. coli demonstrated multiple lignocellulase activities and one had high mannanase activity (1255 U/mg). Eleven fosmid clones harboring the recovered GH-containing contigs were sequenced and assembled into 10 fosmid contigs. The composition of GH genes in the 163 assembled metagenomic contigs and 10 fosmid contigs indicated that diverse GHs and lignocellulose degradation mechanisms were present in the biogas digesters. In particular, a small portion of BG-1 genome information was recovered by PhyloPythiaS analysis. The lignocellulase gene clusters in BG-1 suggested that it might use a possible novel lignocellulose degradation mechanism to efficiently degrade lignocellulose. Dominant cellulolytic bacteria of biogas digester possess diverse GH genes, not only in sequences but also in their functions, which may be applied for production of biofuel in the future.

| S-EPMC4466528 | biostudies-literature

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

Project description: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.

| S-EPMC3406117 | biostudies-literature

Crystal structure of glycoside hydrolase family 55 {beta}-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium.

Project description:Glycoside hydrolase family 55 consists of beta-1,3-glucanases mainly from filamentous fungi. A beta-1,3-glucanase (Lam55A) from the Basidiomycete Phanerochaete chrysosporium hydrolyzes beta-1,3-glucans in the exo-mode with inversion of anomeric configuration and produces gentiobiose in addition to glucose from beta-1,3/1,6-glucans. Here we report the crystal structure of Lam55A, establishing the three-dimensional structure of a member of glycoside hydrolase 55 for the first time. Lam55A has two beta-helical domains in a single polypeptide chain. These two domains are separated by a long linker region but are positioned side by side, and the overall structure resembles a rib cage. In the complex, a gluconolactone molecule is bound at the bottom of a pocket between the two beta-helical domains. Based on the position of the gluconolactone molecule, Glu-633 appears to be the catalytic acid, whereas the catalytic base residue could not be identified. The substrate binding pocket appears to be able to accept a gentiobiose unit near the cleavage site, and a long cleft runs from the pocket, in accordance with the activity of this enzyme toward various beta-1,3-glucan oligosaccharides. In conclusion, we provide important features of the substrate-binding site at the interface of the two beta-helical domains, demonstrating an unexpected variety of carbohydrate binding modes.

| S-EPMC2665064 | biostudies-literature

Engineering of family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.

Project description:Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.

| S-EPMC3681849 | biostudies-literature

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