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Abstract 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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Oriented display of cello-oligosaccharides for pull-down binding assays to distinguish binding preferences of glycan binding proteins
The production of biofuels from lignocellulosic biomass using carbohydrate-active enzymes like cellulases is key to a sustainable energy production. Understanding the adsorption mechanism of cellulases and associated binding domain proteins down to the molecular level details will help in the rational design of improved cellulases. In nature, carbohydrate-binding modules (CBMs) from families 17 and 28 often appear in tandem appended to the C-terminus of several endocellulases. Both CBMs are known to bind to the amorphous regions of cellulose non-competitively and show similar binding affinity towards soluble cello-oligosaccharides. Based on the available crystal structures, these CBMs may display a uni-directional binding preference towards cello-oligosaccharides (based on how the oligosaccharide was bound within the CBM binding cleft). However, molecular dynamics (MD) simulations have indicated no such clear preference. Considering that most soluble oligosaccharides are not always an ideal substrate surrogate to study the binding of CBMs to the native cell wall or cell surface displayed glycans, it is critical to use alternative reagents or substrates. To better understand the binding of type B CBMs towards smaller cello-oligosaccharides, we have developed a simple solid-state depletion or pull-down binding assay. Here, we specifically orient azido-labeled carbohydrates from the reducing end to alkyne-labeled micron-sized bead surfaces, using click chemistry, to mimic insoluble cell wall surface-displayed glycans. Our results reveal that both family 17 and 28 CBMs displayed a similar binding affinity towards cellohexaose-modified beads, but not cellopentaose-modified beads, which helps rationalize previously reported crystal structure and MD data. This may indicate a preferred uni-directional binding of specific CBMs and could explain their co-evolution as tandem constructs appended to endocellulases to increase amorphous cellulose substrate targeting efficiency. Overall, our proposed workflow can be easily translated to measure the affinity of glycan-binding proteins to click-chemistry based immobilized surface-displayed carbohydrates or antigens.
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- PAR ID:
- 10516385
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Carbohydrate Research
- Volume:
- 534
- Issue:
- C
- ISSN:
- 0008-6215
- Page Range / eLocation ID:
- 108943
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/J.CARRES.2023.108943
