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The Quaternary Structure of a Glycoside Hydrolase Dictates Specificity toward ?-Glucans.

ABSTRACT: In the Carbohydrate-Active Enzyme (CAZy) database, glycoside hydrolase family 5 (GH5) is a large family with more than 6,000 sequences. Among the 51 described GH5 subfamilies, subfamily GH5_26 contains members that display either endo-?(1,4)-glucanase or ?(1,3;1,4)-glucanase activities. In this study, we focused on the GH5_26 enzyme fromSaccharophagus degradans(SdGluc5_26A), a marine bacterium known for its capacity to degrade a wide diversity of complex polysaccharides.SdGluc5_26A displays lichenase activity toward ?(1,3;1,4)-glucans with a side cellobiohydrolase activity toward ?(1,4)-glucans. The three-dimensional structure ofSdGluc5_26A adopts a stable trimeric quaternary structure also observable in solution. The N-terminal region ofSdGluc5_26A protrudes into the active site of an adjacent monomer. To understand whether this occupation of the active site could influence its activity, we conducted a comprehensive enzymatic characterization ofSdGluc5_26A and of a mutant truncated at the N terminus. Ligand complex structures and kinetic analyses reveal that the N terminus governs the substrate specificity ofSdGluc5_26A. Its deletion opens the enzyme cleft at the -3 subsite and turns the enzyme into an endo-?(1,4)-glucanase. This study demonstrates that experimental approaches can reveal structure-function relationships out of reach of current bioinformatic predictions.

SUBMITTER: Lafond M

PROVIDER: S-EPMC4807298 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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The Quaternary Structure of a Glycoside Hydrolase Dictates Specificity toward β-Glucans.

Lafond Mickael M Sulzenbacher Gerlind G Freyd Thibaud T Henrissat Bernard B Berrin Jean-Guy JG Garron Marie-Line ML

The Journal of biological chemistry 20160111 13

In the Carbohydrate-Active Enzyme (CAZy) database, glycoside hydrolase family 5 (GH5) is a large family with more than 6,000 sequences. Among the 51 described GH5 subfamilies, subfamily GH5_26 contains members that display either endo-β(1,4)-glucanase or β(1,3;1,4)-glucanase activities. In this study, we focused on the GH5_26 enzyme fromSaccharophagus degradans(SdGluc5_26A), a marine bacterium known for its capacity to degrade a wide diversity of complex polysaccharides.SdGluc5_26A displays lich ...[more]

PMID: 26755730

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Project description:BackgroundPlant pathogens secrete enzymes that degrade plant cell walls to enhance infection and nutrient acquisition.ResultsA novel endotransglucosylase catalyzes cleavage and transfer of β-glucans and decreases the physical strength of plant cell walls.ConclusionEndotransglucosylation causes depolymerization and polymerization of β-glucans, depending on substrate molecular size.SignificanceEnzymatic degradation of plant cell walls is required for wall loosening, which enhances pathogen invasion. A Magnaporthe oryzae enzyme, which was encoded by the Mocel7B gene, was predicted to act on 1,3-1,4-β-glucan degradation and transglycosylation reaction of cellotriose after partial purification from a culture filtrate of M. oryzae cells, followed by liquid chromatography-tandem mass spectrometry. A recombinant MoCel7B prepared by overexpression in M. oryzae exhibited endo-typical depolymerization of polysaccharides containing β-1,4-linkages, in which 1,3-1,4-β-glucan was the best substrate. When cellooligosaccharides were used as the substrate, the recombinant enzyme generated reaction products with both shorter and longer chain lengths than the substrate. In addition, incorporation of glucose and various oligosaccharides including sulforhodamine-conjugated cellobiose, laminarioligosaccharides, gentiobiose, xylobiose, mannobiose, and xyloglucan nonasaccharide into β-1,4-linked glucans were observed after incubation with the enzyme. These results indicate that the recombinant enzyme acts as an endotransglucosylase (ETG) that cleaves the glycosidic bond of β-1,4-glucan as a donor substrate and transfers the cleaved glucan chain to another molecule functioning as an acceptor substrate. Furthermore, ETG treatment caused greater extension of heat-treated wheat coleoptiles. The result suggests that ETG functions to induce wall loosening by cleaving the 1,3-1,4-β-glucan tethers of plant cell walls. On the other hand, use of cellohexaose as a substrate for ETG resulted in the production of cellulose II with a maximum length (degree of polymerization) of 26 glucose units. Thus, ETG functions to depolymerize and polymerize β-glucans, depending on the size of the acceptor substrate.

Dataset Information

The Quaternary Structure of a Glycoside Hydrolase Dictates Specificity toward ?-Glucans.

Publications

The Quaternary Structure of a Glycoside Hydrolase Dictates Specificity toward β-Glucans.

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