Carbohydrate binding domains facilitate efficient oligosaccharides synthesis by enhancing mutant catalytic domain transglycosylation activity

Abstract

Chemoenzymatic approaches using carbohydrate-active enzymes (CAZymes) offer a promising avenue for synthesis of glycans like oligosaccharides. Here, we report a novel chemoenzymatic route for cellodextrins synthesis employed by chimeric CAZymes, akin to native glycosyltransferases, involving the unprecedented participation of a 'noncatalytic' lectin-like or carbohydrate-binding domains (CBMs) in the catalytic step for glycosidic bond synthesis using cellobiosyl donor sugars as activated substrates. CBMs are often thought to play a passive substrate targeting role in enzymatic glycosylation reactions mostly via overcoming substrate diffusion limitations for tethered catalytic domains (CDs) but are not known to participate directly in any nucleophilic substitution mechanisms that impact the actual glycosyl transfer step. Our study provides evidence for the direct participation of CBMs in the catalytic reaction step for -glucan glycosidic bonds synthesis enhancing activity for CBM-based CAZyme chimeras by >140-fold over CDs alone. Dynamic intra-domain interactions that facilitate this poorly understood reaction mechanism were further revealed by small-angle X-ray scattering structural analysis along with detailed mutagenesis studies to shed light on our current limited understanding of similar transglycosylation-type reaction mechanisms. In summary, our study provides a novel strategy for engineering similar CBMbased CAZyme chimeras for synthesis of bespoke oligosaccharides using simple activated sugar monomers. Lombard et al., 2014), and still growing with newly sequenced genomes and metagenomes becoming readily available.CAZymes like GHs or GTs are further classified either as retaining or inverting type, depending on whether the stereochemistry at the anomeric carbon for the product (versus the reactant) is either retained or inverted (Davies and Henrissat, 1995), respectively. As first proposed by Koshland for glycosidases (Koshland, 1953), retaining enzymes require a classical two-step double displacement hydrolysis mechanism (i.e., SN2) with a glycosyl-enzyme intermediate (GEI) formed via a covalent bond between the cleaved substrate and the protein in the alternate orientation (e.g., GEI for retaining enzyme will have an -bond, as opposed to the -orientation of the reactant and product). While most native glycosidases catalyze the hydrolysis of glycosidic linkages, many GHs can also produce oligosaccharides following a competing transglycosylation mechanism if a suitably localized glycosyl acceptor group is present instead of a water molecule within the enzyme active site (Bissaro et al., 2015). Retaining GHs that show significant transglycosylation reactivity are also thought to utilize a similar SN2 type general mechanism, except that rather than nucleophilic attack by water, the attack is instigated by a hydroxyl oxygen on an acceptor sugar placed adjacent to the GEI complex (Figure 1).