Analysis of surface binding sites (SBSs) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77 — a mini-review

Abstract

Surface binding sites (SBSs) interact with carbohydrates outside of the enzyme active site. They are frequently situated on catalytic domains and are distinct from carbohydrate binding modules (CBMs). SBSs are found in a variety of enzymes and often seen in crystal structures. Notably about half of the > 45 enzymes (in 17 GH and two GT families) with an identified SBS are from GH13 and a few from GH77, both belonging to clan GH-H of carbohydrate active enzymes. The many enzymes of GH13 with SBSs provide an opportunity to analyse their distribution within this very large and diverse family. SBS containing enzymes in GH13 are spread among 15 subfamilies (two were not assigned a subfamily). Comparison of these SBSs reveals a complex evolutionary history with evidence of conservation of key residues and/or structural location between some SBSs, while others are found at entirely distinct structural locations, suggesting convergent evolution. An array of investigations of the two SBSs in barley α-amylase demonstrated they play different functional roles in binding and degradation of polysaccharides. MalQ from Escherichia coli is an α-1,4-glucanotransferase of GH77, a family that is known to have at least one member that contains an SBS. Whereas MalQ is a single domain enzyme lacking CBMs, its plant orthologue DPE2 contains two N-terminal CBM20s. Surface plasmon resonance binding studies showed that MalQ and DPE2 have a similar affinity for β-cyclodextrin and that MalQ binds malto-oligosaccharides of >DP4 at a second site in competition with β-cyclodextrin yielding a stoichiometry >1. This suggests that MalQ may have an SBS, though its structural location remains unknown.

GH62 arabinofuranosidases: Structure, function and applications

“…Carbohydrate surface binding sites (SBSs) are situated on the catalytic domain at a certain distance from the active site and have been identified in enzymes acting on different polysaccharides including xylan (Cockburn et al, 2016(Cockburn et al, , 2014Cuyvers et al, 2011). In AnAbf62A-m2,3 an SBS ~30 Å from the active site was identified by mutational analysis of Trp23 and Tyr44 (Fig.…”

The Starch Granule-Associated Protein EARLY STARVATION1 Is Required for the Control of Starch Degradation in Arabidopsis thaliana Leaves

“…Starch binding CBMs fall into distinct classes, for example, GWD and AMY3 both possess two CBM45 domains in tandem, and isoamylases, branching enzymes, and PTST possess CBM48 domains. SBSs are generally highly variable and require experimental definition (Cockburn et al, 2013;Meekins et al, 2014). Neither ESV nor LESV possesses a recognizable CBM, and further work will be required to discover whether they possess SBSs.…”

Raw starch degrading α-amylases: an unsolved riddle

“…Starch is usually bound to the enzymes via additional binding sites that can are located on starch binding domains (SBDs) situated far from the active site area, or they can be present in the form of surface binding sites (SBSs) on the surface of enzymes [38]. Illustrative examples of variety of GH13 α-amylases including those having SBD, SBS(s), both or none of them (or not yet determined) are presented in Figure 1.…”

“…SBSs have been described to carry out a suite of functions especially needed for enzymatic reactions with biological macromolecules and supramolecular structures as found in, e.g., plant cell walls, chitin, and starch granules [38]. The functions of SBSs are various, such as guiding the amylase to the substrate, directing starch , Hordeum vulgare (barley) α-amylase (PDB: 1P6W) isozyme 1 (AMY1) in complex with the substrate analogue, methyl 4I,4II,4III-trithiomaltotetraoside (thio-DP4).…”