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  • Carbohydrate‐binding domains facilitate efficient oligosaccharides synthesis by enhancing mutant catalytic domain transglycosylation activity
Title: 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 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.  more » « less
Award ID(s):
1704679
PAR ID:
10456212
Author(s) / Creator(s):
 ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Biotechnology and Bioengineering
Volume:
117
Issue:
10
ISSN:
0006-3592
Page Range / eLocation ID:
p. 2944-2956
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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