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Laminarinase from Flavobacterium sp. reveals the structural basis of thermostability and substrate specificity.

ABSTRACT: Laminarinase from Flavobacterium sp. strain UMI-01, a new member of the glycosyl hydrolase 16 family of a marine bacterium associated with seaweeds, mainly degrades ?-1,3-glucosyl linkages of ?-glucan (such as laminarin) through the hydrolysis of glycosidic bonds. We determined the crystal structure of ULam111 at 1.60-Å resolution to understand the structural basis for its thermostability and substrate specificity. A calcium-binding motif located on the opposite side of the ?-sheet from catalytic cleft increased its degrading activity and thermostability. The disulfide bridge Cys31-Cys34, located on the ?2-?3 loop near the substrate-binding site, is responsible for the thermostability of ULam111. The substrates of ?-1,3-linked laminarin and ?-1,3-1,4-linked glucan bound to the catalytic cleft in a completely different mode at subsite -3. Asn33 and Trp113, together with Phe212, formed hydrogen bonds with preferred substrates to degrade ?-1,3-linked laminarin based on the structural comparisons. Our structural information provides new insights concerning thermostability and substrate recognition that will enable the design of industrial biocatalysts.

SUBMITTER: Qin HM

PROVIDER: S-EPMC5595797 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Laminarinase from Flavobacterium sp. reveals the structural basis of thermostability and substrate specificity.

Qin Hui-Min HM Miyakawa Takuya T Inoue Akira A Nakamura Akira A Nishiyama Ryuji R Ojima Takao T Tanokura Masaru M

Scientific reports 20170912 1

Laminarinase from Flavobacterium sp. strain UMI-01, a new member of the glycosyl hydrolase 16 family of a marine bacterium associated with seaweeds, mainly degrades β-1,3-glucosyl linkages of β-glucan (such as laminarin) through the hydrolysis of glycosidic bonds. We determined the crystal structure of ULam111 at 1.60-Å resolution to understand the structural basis for its thermostability and substrate specificity. A calcium-binding motif located on the opposite side of the β-sheet from catalyti ...[more]

PMID: 28900273

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Project description:Laminarin is an abundant algal polysaccharide that serves as carbon storage and fuel to meet the nutrition demands of heterotrophic microbes. Laminarin depolymerization catalyzed by microbial extracellular enzymes initiates remineralization, a key process in ocean biogeochemical cycles. Here, we described a glycoside hydrolase 16 (GH16) family laminarinase from a marine alga-associated Flavobacterium at the biochemical and structural levels. We found that the endolytic enzyme cleaved laminarin with a preference for β-1,3-glycoside linkages and showed transglycosylation activity across a broad range of acceptors. We also solved and compared high-resolution crystal structures of laminarinase in the apo form and in complex with β-1,3-tetrasaccharides, revealing an expanded catalytic cleft formed following substrate binding. Moreover, structure and mutagenesis studies identified multiple specific contacts between the enzyme and glucosyl residues essential for the substrate specificity for β-1,3-glucan. These results provide novel insights into the structural requirements for substrate binding and catalysis of GH16 family laminarinase, enriching our understanding of bacterial utilization of algal laminarin.IMPORTANCE Heterotrophic bacterial communities are key players in marine biogeochemical cycling due to their ability to remineralize organic carbon. Processing of complex organic matter requires heterotrophic bacteria to produce extracellular enzymes with precise specificity to depolymerize substrates to sizes sufficiently small for uptake. Thus, extracellular enzymatic hydrolysis initiates microbe-driven heterotrophic carbon cycling. In this study, based on biochemical and structural analyses, we revealed the depolymerization mechanism of β-1,3-glucan, a carbon reserve in algae, by laminarinase from an alga-associated marine Flavobacterium The findings provide new insights into the substrate recognition and catalysis of bacterial laminarinase and promote a better understanding of how extracellular enzymes are involved in organic matter cycling.

Dataset Information

Laminarinase from Flavobacterium sp. reveals the structural basis of thermostability and substrate specificity.

Publications

Laminarinase from Flavobacterium sp. reveals the structural basis of thermostability and substrate specificity.

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