Abstract
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.
Highlights
Many fungi produce -1,3-glucans as the main components of the cell wall
In the carbohydrate-active enzymes (CAZy) data base (8 –13) enzymes having -1,3-glucanase activity are found in the glycoside hydrolase (GH)3 families 5, 16, 17, 55, 64, 72, and 81
The enzyme has no hydrolytic activity toward -1,6- or -1,3/1,4-glucan but shows high activity toward laminarin from Laminaria digitata [22], which is a -1,3/1,6-glucan with an average degree of polymerization (DP) of ϳ25
Summary
Materials—Laminarioligosaccharides (-1,3-linked oligomers of D-glucopyranose) with DPs of 2–7 (laminaribiose, L2; laminaritriose, L3; laminaritetraose, L4; laminaripentaose, L5; laminarihexaose, L6; laminariheptaose, L7) were purchased from Seikagaku Corp. (Tokyo, Japan). 6-O-Glucosyl-laminaritriose (LG4, -D-Glcp-(136)--D-Glcp-(133)--D-Glcp[133]-D-Glcp) was prepared using -1,3-glucanase Lam16A according to the method reported previously [23]. The column was equilibrated with 100 mM NaOH, and the reaction products were eluted with a linear gradient of 0 –500 mM sodium acetate in 100 mM NaOH at a flow rate of 1 ml1⁄7minϪ1 over 30 min. A solution of 1 mM L7 was incubated with 12 nM Lam55A for various times, and the reaction mixtures were boiled and applied to the column equilibrated with 100 mM NaOH. The reaction products were eluted with a linear gradient of 0 –500 mM sodium acetate in 100 mM NaOH at a flow rate of 1 ml1⁄7minϪ1 over 30 min as described previously [25]. Each substrate (20 mM) was incubated with 1.2 M Lam55A for 1 min, and the reaction mixture was applied to a TSK-GEL Amide-80 column (4.6 ϫ 250 mm; Tosoh Co., Tokyo, Japan).
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