Carbohydrate-binding module assisting glycosynthase-catalysed polymerizations

Codera, Victoria; Gilbert, Harry J.; Faijes, Magda; Planas, Antoni

doi:10.1042/bj20150420

Cited by 11 publications

(3 citation statements)

References 26 publications

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“…This could explain why gene annotation/classification of these enzymes based on sequence similarities and protein folds often results in the significant overlap between the two enzyme classes (Hidaka et al, 2004; Lombard et al, 2014; Roston, Wang, Kuhn, & Benning, 2014). However, few studies have explored the role of CBMs on transglycosylation activity of GHs and none have so far explored the possibility of the reaction mechanism directly employing CBMs (Codera, Gilbert, Faijes, & Planas, 2015; Mizutani et al, 2012; Stockinger et al, 2015). While CBMs or lectin‐like domains have been never reported previously to directly participate in the catalytic reaction step for glycosidic bonds synthesis by GHs, these domains have been shown to play important roles in the functioning of several GTs.…”

Section: Discussionmentioning

confidence: 99%

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

Bandi

Gonçalves

Pingali

et al. 2020

Biotech & Bioengineering

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Chemoenzymatic approaches using carbohydrate‐active enzymes (CAZymes) offer a promising avenue for the 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 “non‐catalytic” lectin‐like domain or carbohydrate‐binding modules (CBMs) in the catalytic step for glycosidic bond synthesis using β‐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. This study provides evidence for the direct participation of CBMs in the catalytic reaction step for β‐glucan glycosidic bonds synthesis enhancing activity for CBM‐based CAZyme chimeras by >140‐fold over CDs alone. Dynamic intradomain 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 CBM‐based CAZyme chimeras for the synthesis of bespoke oligosaccharides using simple activated sugar monomers.

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Section: Discussionmentioning

confidence: 99%

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

Bandi

Gonçalves

Pingali

et al. 2020

Biotech & Bioengineering

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show abstract

“…This could explain why gene annotation/classification of these enzymes based on sequence similarities and protein folds often results in significant overlap between the two enzyme classes (Hidaka et al, 2004;Lombard et al, 2014;Roston et al, 2014). However, few studies have explored the role of CBMs on transglycosylation activity of GHs and none have so far explored the possibility of the reaction mechanism directly employing CBMs (Codera et al, 2015;Mizutani et al, 2012;Stockinger et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

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

Bandi

Gonçalves

Pingali

et al. 2020

Preprint

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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).

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“…The weight-average molecular weight of the products ranged from 9.5 kDa (dp 29) for (4G3G)n to 14.5 kDa (dp 30) for (4G4G3G)n to 12.9 kDa for (4G4G4G3G)n. Recently, the Planas laboratory reported that, in some cases, polymers with a slightly higher DP are obtained when a carbohydrate-binding module (CBM), recognizing the respective polymer, is added to the polymerization reaction. 42 The presence of a CBM may reduce precipitation of the higher molecular weight polysaccharides, thus promoting the temporary generation of an oversaturated solution. Similar effects were observed when a CBM was covalently attached to a glycosynthase via a short peptide linker.…”

Section: ■ Introductionmentioning

confidence: 99%

Enzymatic Synthesis of Artificial Polysaccharides

Smith

Ortiz-Soto

Roth

et al. 2020

ACS Sustainable Chem. Eng.

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Polysaccharides are the most important renewable polymers on Earth and hold an enormous potential for the production of ecofriendly functional materials. In addition to being sustainable, they have superior properties to synthetic polymers, particularly in the biomedical field where biocompatibility and biodegradability are vital. Derivatization of polysaccharides obtained from plant biomass paves the path forward for the design and manufacturing of advanced materials with specific properties adapted to meet definitive needs. However, these advances have been severely limited due to issues with establishing structure− property relationships, which are hampered by the heterogeneity of target polysaccharides and the random distribution of functional groups obtained after their chemical modification. An accurate correlation of structure−property relationships at multiple length scales requires substrates with defined sizes, sequences, and substitution patterns. Such tailor-made polysaccharides may be obtained by implementing a bottom-up approach, starting from monosaccharide or oligosaccharide building blocks followed by their polymerization and substitution through catalysis by different carbohydrate-active enzymes such as glycosynthases, phosphorylases, sucrases, and glycosyltransferases. Recent progress in the enzymatic synthesis of artificial polysaccharides is reviewed, with an emphasis on the potential of the synthesized products, either as new materials or as tools to study structure−property relationships. The obtained information will guide future developments of rationally designed biobased materials for industrial and biomedical applications.

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Carbohydrate-binding module assisting glycosynthase-catalysed polymerizations

Cited by 11 publications

References 26 publications

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

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

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

Enzymatic Synthesis of Artificial Polysaccharides

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