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

Chandra Kanth Bandi, Antonio Goncalves, Sai Venkatesh Pingali, Shishir P.S. Chundawat

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)2944-2956
Number of pages13
JournalBiotechnology and Bioengineering
Volume117
Issue number10
DOIs
StatePublished - Oct 1 2020

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Keywords

  • carbohydrate-active enzymes
  • carbohydrate-binding modules
  • oligosaccharides
  • transglycosylation
  • β-glucan synthase

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