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

Abstract

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.

A phylogenetic approach to study the origin and evolution of plasmodesmata-localized glycosyl hydrolases family 17

“…Research on GHL17 protein structure revealed two strictly conserved glutamate residues that act as the proton donor and the nucleophile in all reactions catalyzed by glycosyl hydrolases (Jenkins et al, 1995; Wojtkowiak et al, 2013). A number of aromatic and hydrophilic residues located near the catalytic cleft, presumably involved in substrate specificity and enzyme activity, are also conserved among all plant GHL17 proteins (Wojtkowiak et al, 2013).…”

“…A number of aromatic and hydrophilic residues located near the catalytic cleft, presumably involved in substrate specificity and enzyme activity, are also conserved among all plant GHL17 proteins (Wojtkowiak et al, 2013). …”

“…Similarly, the residues surrounding the catalytic site are mostly conserved in all selected representatives (Supplementary data 1, Figure 2). Moreover a region contained the aromatic residues Tyr200 and Phe203 (location refer to At2g05790 sequence), which is involved in substrate interaction (Wojtkowiak et al, 2013), is also conserved in all streptophytes (Figure 2). …”

Synergy between Cell Surface Glycosidases and Glycan-Binding Proteins Dictates the Utilization of Specific Beta(1,3)-Glucans by Human Gut Bacteroides

“…In this system, laminarin must first be debranched by a GH30 exo-␤(1,6)-glucosidase before being hydrolyzed by a GH17 endo-␤(1,3)-glucanase, which has very low activity on ␤(1,6)-glucose-branched laminarin (55). In general, GH17 endoglucanases have a very narrow active-site cleft that does not effectively accom- (47,55). This is unlike BuGH16 and BuGH158 enzymes, which allow the 1,3GUL system to bypass the initial debranching requirement.…”

One-Step Process for Environment-Friendly Preparation of Agar Oligosaccharides From Gracilaria lemaneiformis by the Action of Flammeovirga sp. OC4

“…Some oligosaccharides are biologically active substances that possess antimicrobial activity, immunoenhancing ability, antitumor activity (Fang et al, 2012; Zou et al, 2016; Chen et al, 2017), and antioxidant activity (Chen et al, 2015; Malunga and Beta, 2015; Hamley-Bennett et al, 2016) and have great potential in nutrition (Kashtanova et al, 2016; Marcotuli et al, 2016), health care (Mensink et al, 2015; Zhang et al, 2016; Di et al, 2017), disease diagnosis, and control (Kazłowski et al, 2012; Zhang et al, 2016). To date, oligosaccharides derived from plants and chitins have been used widely (Wojtkowiak et al, 2013; Hamley-Bennett et al, 2016; Yin et al, 2016). On the other hand, algae, an inexpensive and abundant biomass, is attracting increasing attention because it can be used for the production of algae oligosaccharides that have better physiological activity and a higher value than algae polysaccharides (such as agar, carrageenan, and alginate).…”