Structure, Mechanistic Action, and Essential Residues of a GH-64 Enzyme, Laminaripentaose-producing β-1,3-Glucanase

Wu, Hsin Mao; Liu, Sheng Wen; Hsu, Ming-Tsung; Hung, Chiu Lien; Lai, Chun-Chieh; Cheng, Wang-Yau; Wang, Hung Jung; Li, Yaw-Kuen; Wang, Weng Ching

doi:10.1074/jbc.m109.010983

Cited by 28 publications

(31 citation statements)

References 42 publications

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“…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 laminarinases have been classified in GH-16, which share a ␤-jelly roll fold and catalyze the glycosyl hydrolysis reaction in a retaining mechanism. At the active site, a glutamate residue acts as a nucleophile to attack the C1 atom in the absence of water molecules, and then another glutamate serves as a proton donor to complete the double displacement mechanism.…”

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confidence: 99%

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: 99%

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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“…2b). On the surface of the RmLam81A structure there is a prominent cleft which is about 10 Å deep, 10 Å Cartoon representation of -1,3-glucanases from other GH families: (a) GH family 16 (green; PDB entry 3azx; Jeng et al, 2011), (b) GH family 17 (cyan; PDB entry 3ur7; Wojtkowiak et al, 2012), (c) GH family 55 (purple; PDB entry 3eqn; Ishida et al, 2009) and (d) GH family 64 -1,3-glucanase (blue; PDB entry 3gd0; Wu et al, 2009). The bottom panel shows the superposition of RmLam81A (yellow) and other glucanases.…”

Section: Overall Structuresmentioning

confidence: 99%

The structure of a glycoside hydrolase family 81 endo-β-1,3-glucanase

Zhou

Chen

Yan

et al. 2013

Acta Crystallogr D Biol Crystallogr

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Endo-β-1,3-glucanases catalyze the hydrolysis of β-1,3-glycosidic linkages in glucans. They are also responsible for rather diverse physiological functions such as carbon utilization, cell-wall organization and pathogen defence. Glycoside hydrolase (GH) family 81 mainly consists of β-1,3-glucanases from fungi, higher plants and bacteria. A novel GH family 81 β-1,3-glucanase gene (RmLam81A) from Rhizomucor miehei was expressed in Escherichia coli. Purified RmLam81A was crystallized and the structure was determined in two crystal forms (form I-free and form II-Se) at 2.3 and 2.0 Å resolution, respectively. Here, the crystal structure of a member of GH family 81 is reported for the first time. The structure of RmLam81A is greatly different from all endo-β-1,3-glucanase structures available in the Protein Data Bank. The overall structure of the RmLam81A monomer consists of an N-terminal β-sandwich domain, a C-terminal (α/α)6 domain and an additional domain between them. Glu553 and Glu557 are proposed to serve as the proton donor and basic catalyst, respectively, in a single-displacement mechanism. In addition, Tyr386, Tyr482 and Ser554 possibly contribute to both the position or the ionization state of the basic catalyst Glu557. The first crystal structure of a GH family 81 member will be helpful in the study of the GH family 81 proteins and endo-β-1,3-glucanases.

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“…For example, the hexasaccharides found in carbohydrate-binding modules are described as 'U-shaped' (Boraston et al, 2002;van Bueren et al, 2005) and the two pentasaccharides present in the catalytic cleft of Gas2p transglycosylase (Hurtado-Guerrero et al, 2009) also adopt such a conformation. The tetrasaccharide found in complex with laminaripentaose-producing -1,3-glucanase (Wu et al, 2009) was also described as 'forming a distinct kink'.…”

Section: Figurementioning

confidence: 99%

“…Endo-1,3--glucanases have been well characterized in bacteria, fungi and plants. The bacterial enzymes represent two different glycoside hydrolase families, designated GH16 and GH64, which have distinct unrelated folds, namely a -sandwich jelly roll (Fibriansah et al, 2007) and a two-domain structure consisting of a -barrel and a mixed / domain (Wu et al, 2009). Fungal endo-1,3--glucanases belong to family GH55, with a ribcage-like overall architecture formed by two domains with a right-handed parallel -helix fold (Ishida et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Structures of an active-site mutant of a plant 1,3-β-glucanase in complex with oligosaccharide products of hydrolysis

Wojtkowiak

Witek

Hennig

et al. 2012

Acta Crystallogr D Biol Crystallogr

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Plant endo-1,3-β-glucanases are involved in important physiological processes such as defence mechanisms, cell division and flowering. They hydrolyze (1→3)-β-glucans, with very limited activity towards mixed (1→3,1→4)-β-glucans and branched (1→3,1→6)-β-glucans. Here, crystal structures of the potato (Solanum tuberosum) endo-1,3-β-glucanase GLUB20-2 with the nucleophilic Glu259 residue substituted by alanine (E259A) are reported. Despite this active-site mutation, the protein retained residual endoglucanase activity and when incubated in the crystallization buffer with a linear hexameric substrate derived from (1→3)-β-glucan (laminarahexose) cleaved it in two different ways, generating trisaccharides and tetrasaccharides, as confirmed by mass spectrometry. The trisaccharide (laminaratriose) shows higher binding affinity and was found to fully occupy the -1, -2 and -3 sites of the active-site cleft, even at a low molar excess of the substrate. At elevated substrate concentration the tetrasaccharide molecule (laminaratetrose) also occupies the active site, spanning the opposite sites +1, +2, +3 and +4 of the cleft. These are the first crystal structures of a plant glycoside hydrolase family 17 (GH17) member to reveal the protein-saccharide interactions and were determined at resolutions of 1.68 and 1.55 Å, respectively. The geometry of the active-site cleft clearly precludes any (1→4)-β-glucan topology at the subsites from -3 to +4 and could possibly accommodate β-1,6-branching only at subsites +1 and +2. The glucose units at subsites -1 and -2 interact with highly conserved protein residues. In contrast, subsites -3, +3 and +4 are variable, suggesting that the mode of glucose binding at these sites may vary between different plant endo-1,3-β-glucanases. Low substrate affinity is observed at subsites +1 and +2, as manifested by disorder of the glycosyl units there.

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Structure, Mechanistic Action, and Essential Residues of a GH-64 Enzyme, Laminaripentaose-producing β-1,3-Glucanase

Cited by 28 publications

References 42 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

The structure of a glycoside hydrolase family 81 endo-β-1,3-glucanase

Structures of an active-site mutant of a plant 1,3-β-glucanase in complex with oligosaccharide products of hydrolysis

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