Enzymes in 3D: Synthesis, remodelling, and hydrolysis of cell wall (1,3;1,4)-β-glucans

Abstract

Recent breakthroughs in structural biology have provided valuable new insights into enzymes involved in plant cell wall metabolism. More specifically, the molecular mechanism of synthesis of (1,3;1,4)-β-glucans, which are widespread in cell walls of commercially important cereals and grasses, has been the topic of debate and intense research activity for decades. However, an inability to purify these integral membrane enzymes or apply transgenic approaches without interpretative problems associated with pleiotropic effects has presented barriers to attempts to define their synthetic mechanisms. Following the demonstration that some members of the CslF sub-family of GT2 family enzymes mediate (1,3;1,4)-β-glucan synthesis, the expression of the corresponding genes in a heterologous system that is free of background complications has now been achieved. Biochemical analyses of the (1,3;1,4)-β-glucan synthesized in vitro, combined with 3-dimensional (3D) cryogenic-electron microscopy and AlphaFold protein structure predictions, have demonstrated how a single CslF6 enzyme, without exogenous primers, can incorporate both (1,3)- and (1,4)-β-linkages into the nascent polysaccharide chain. Similarly, 3D structures of xyloglucan endo-transglycosylases and (1,3;1,4)-β-glucan endo- and exohydrolases have allowed the mechanisms of (1,3;1,4)-β-glucan modification and degradation to be defined. X-ray crystallography and multi-scale modeling of a broad specificity GH3 β-glucan exohydrolase recently revealed a previously unknown and remarkable molecular mechanism with reactant trajectories through which a polysaccharide exohydrolase can act with a processive action pattern. The availability of high-quality protein 3D structural predictions should prove invaluable for defining structures, dynamics, and functions of other enzymes involved in plant cell wall metabolism in the immediate future.

Glycosyl transferase GT2 genes mediate the biosynthesis of an unusual (1,3;1,4)‐β‐glucan exopolysaccharide in the bacterium Sarcina ventriculi

“…Depending on the register of the translocating glucan, interactions of the Tyr the C6(OH) groups lead to repositioning of the acceptor glucosyl residue at the active site, thereby controlling the incorporation of (1,3)-or (1,4)β-glucosidic linkages (Hrmova et al, 2023;Purushotham et al, 2022).…”

“…It was proposed that a Tyr residue of the switch motif (which is a His residue in plant cellulose synthases) interacts with the nascent glucan as it transitions through the channel. Depending on the register of the translocating glucan, interactions of the Tyr with the glucosyls' C6(OH) groups lead to repositioning of the acceptor glucosyl residue at the active site, thereby controlling the incorporation of (1,3)‐ or (1,4)‐β‐glucosidic linkages (Hrmova et al., 2023; Purushotham et al., 2022).…”

“…where the arrows indicate the points of hydrolysis, G represents a glucosyl residue, 3 and 4 represent linkage configurations, red is the reducing end of the polysaccharide chain and the released oligosaccharides are underlined (Woodward et al, 1983). MLGs from grasses and cereals have DP3:DP4 ratios ranging from 2:1 to 6:1 (Collins et al, 2010;Guillon et al, 2011;Hrmova et al, 2023;Wood et al, 1991) and are usually at least partly soluble in water. In the monilophyte genus Equisetum (horsetails), the DP3:DP4 ratio of the MLGs can be as low as 1:10 (Fry et al, 2008;Sørensen et al, 2008).…”

“…When these MLGs are incubated with a specific (1,3;1,4)‐β‐glucan endohydrolase (lichenase; EC 3.2.1.73), which hydrolyses (1,4)‐β‐glucosidic linkages that are adjacent to and towards the reducing end of single (1,3)‐β‐glucosyl residues, trisaccharides (G4G3G red ; DP3) and tetrasaccharides (G4G4G3G red ; DP4) are the major products released, as follows:

where the arrows indicate the points of hydrolysis, G represents a glucosyl residue, 3 and 4 represent linkage configurations, red is the reducing end of the polysaccharide chain and the released oligosaccharides are underlined (Woodward et al., 1983). MLGs from grasses and cereals have DP3:DP4 ratios ranging from 2:1 to 6:1 (Collins et al., 2010; Guillon et al., 2011; Hrmova et al., 2023; Wood et al., 1991) and are usually at least partly soluble in water. In the monilophyte genus Equisetum (horsetails), the DP3:DP4 ratio of the MLGs can be as low as 1:10 (Fry et al., 2008; Sørensen et al., 2008).…”