The 1.3 Å crystal structure of a novel endo‐β‐1,3‐glucanase of glycoside hydrolase family 16 from alkaliphilic <i>Nocardiopsis</i> sp. strain F96

Fibriansah, Guntur; Masuda, Sumiko; Koizumi, Naoya; Nakamura, Satoshi; Kumasaka, Takashi

doi:10.1002/prot.21589

Cited by 60 publications

(51 citation statements)

References 43 publications

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“…strain F96 (9) and the hyperthermophile Pyrococcus furiosus (10). Although the latter report modeled the existence of laminarin trisaccharide in the protein catalytic cleft, the bacterial GH-16 laminarinase-sugar complex structure has not been reported so far.…”

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confidence: 98%

Crystal Structures of the Laminarinase Catalytic Domain from Thermotoga maritima MSB8 in Complex with Inhibitors

Jeng

Wang

Lin

et al. 2011

Journal of Biological Chemistry

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Laminarinases hydrolyzing the ␤-1,3-linkage of glucans play essential roles in microbial saccharide degradation. Here we report the crystal structures at 1.65-1.82 Å resolution of the catalytic domain of laminarinase from the thermophile Thermotoga maritima with various space groups in the ligand-free form or in the presence of inhibitors gluconolactone and cetyltrimethylammonium. Ligands were bound at the cleft of the active site near an enclosure formed by Trp-232 and a flexible GASIG loop. A closed configuration at the active site cleft was observed in some molecules. The loop flexibility in the enzyme may contribute to the regulation of endo-or exo-activity of the enzyme and a preference to release laminaritrioses in long chain carbohydrate hydrolysis. Glu-137 and Glu-132 are proposed to serve as the proton donor and nucleophile, respectively, in the retaining catalysis of hydrolyzation. Calcium ions in the crystallization media are found to accelerate crystal growth. Comparison of laminarinase and endoglucanase structures revealed the subtle difference of key residues in the active site for the selection of ␤-1,3-glucan and ␤-1,4-glucan substrates, respectively. Arg-85 may be pivotal to ␤-1,3-glucan substrate selection. The similarity of the structures between the laminarinase catalytic domain and its carbohydrate-binding modules may have evolutionary relevance because of the similarities in their folds.Thermotoga maritima is a hyperthermophilic, anaerobic, and fermentive saccharolytic bacterium, catabolizing sugars and its polymers to make energy. Laminarinase (3-␤-D-glucan glucanohydrolase; EC 3.2.1.39, Lam), 3 an endoglucosidase, hydrolyzes internal ␤-1,3-glucosyl linkages in ␤-D-glucans and is therefore crucial in carbohydrate degradation for nutrient uptake and energy production in bacteria. According to the sequenced genome of T. maritima MSB8 (1), the laminarinase gene encodes the enzyme composed of a catalytic domain and two carbohydrate-binding modules (CBMs) connected by a linker region on each terminus. The structure of TmLamCBM2, located on the C terminus, has previously been determined by Boraston et al. (2). The coexistence of CBM with catalytic domains is widespread in many modular bacterial polysaccharide hydrolases that contain separately folding modules (3). The prevalent role of CBMs is to facilitate the association of substrates with the catalytic module; moreover, they sometime boost the reaction efficiency of the catalytic domain (4, 5). The modularity in biological macromolecules draws scientists' attention in biocatalyst designs (6). Because of the substrate diversity among glycosyl hydrolases (GHs), it is not easy to classify these enzymes according to their substrate specificity. Henrissat and Bairoch (7) developed a sequence similarity-based classification to categorize GH enzymes as an alternative to the traditional enzyme classification system. Except for a laminaripentaose-producing ␤-1,3-glucanase from Streptomyces matensis, which belongs to GH-64 (8), most of the bacterial la...

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confidence: 98%

Crystal Structures of the Laminarinase Catalytic Domain from Thermotoga maritima MSB8 in Complex with Inhibitors

Jeng

Wang

Lin

et al. 2011

Journal of Biological Chemistry

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“…The Phyre program predicted a secondary structure of FvEn3GAL closely related to an endo-␤-1,3-glucanase from Nocardiopsis sp. F96, BglF (NspBglF), which is a member of GH family 16 (2.3 ϫ 10 Ϫ20 ) (28,38). The overall structure of FvEn3GAL proposed by the program was a ␤-jellyroll fold, which is typical for GH 16 enzymes (39,40).…”

Section: Purification Of the Enzyme From The Culture Medium Of F Velmentioning

confidence: 99%

Endo-β-1,3-galactanase from Winter Mushroom Flammulina velutipes

Kotake

Hirata

Degi

et al. 2011

Journal of Biological Chemistry

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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.

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“…3). The amino acid sequence analysis indicated that LamC contained a single catalytic domain with the two catalytic residues (Glu304 and Glu309) that are highly conserved among GH16 members (10,28).…”

Section: Resultsmentioning

confidence: 99%

“…6, both rLamC-ΔC and rLamC The protein sequence alignments were generated using the MUSCLE alignment in MEGA 7.0 (38). Secondary structures are labeled based on their appearance in 2HYK following a previous annotation (28). The stars identify the catalytic amino acids, including Glu-304, Asp-306, and Glu-309 (LamC numbering), and the conserved Trp residues are marked by the symbol OE above the column.…”

Section: Resultsmentioning

confidence: 99%

Functional Analysis of a Novel β-(1,3)-Glucanase from Corallococcus sp. Strain EGB Containing a Fascin-Like Module

Zhou

et al. 2017

Appl Environ Microbiol

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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 exoacting enzyme on ␤-(1,3)(1,3)-and ␤-(1,6)-linked glucan substrates and an endoacting 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 carbohydratebinding assay showed that the fascin-like module was a novel ␤-(1,3)-linked glucanbinding module. The functional characterization of the fascin-like module and catalytic module will help us better understand these enzymes and modules. -(1,3)-glucan, which is the main cell wall component in yeast and filamentous fungi and a structural polysaccharide (e.g., callose) in plants and is also found in exopolysaccharides produced by some bacteria (1). Based on their amino acid sequence similarity and secondary structure, ␤-(1,3)-glucanases are classified mainly into glycoside hydrolase family 16 (GH16) and GH17. However, these two families have the same hydrolytic mechanism with anomeric retention (2).Numerous genes encoding ␤-(1,3)-glucanase have been cloned and characterized from different sources, including varieties of plants (3-5), bacteria, and archaea, such as Bacillus circulans (6), Paenibacillus (7, 8), Thermotoga neapolitana (1), Rhodothermus marinus (9), and Pyrococcus furiosus (10). Few glucanases exhibit broad substrate linkage specificity, although Lafond et al. cloned a gene from Podospora anserina that encodes a broad-specificity ␤-glucanase acting on ␤-(1,3)-, ␤-(1,4)-, and ␤-(1,6)-glucans (11).Many polysaccharide-degrading enzymes display a modular structure, in which a catalytic module is attached to one or more noncatalytic modules (8, 12, 13). The impact of the noncatalytic modules on the enzymatic properties of ␤-(1,3)-glucanase

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The 1.3 Å crystal structure of a novel endo‐β‐1,3‐glucanase of glycoside hydrolase family 16 from alkaliphilic Nocardiopsis sp. strain F96

Cited by 60 publications

References 43 publications

Crystal Structures of the Laminarinase Catalytic Domain from Thermotoga maritima MSB8 in Complex with Inhibitors

Crystal Structures of the Laminarinase Catalytic Domain from Thermotoga maritima MSB8 in Complex with Inhibitors

Endo-β-1,3-galactanase from Winter Mushroom Flammulina velutipes

Functional Analysis of a Novel β-(1,3)-Glucanase from Corallococcus sp. Strain EGB Containing a Fascin-Like Module

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