Discovery and Characterisation of a Plant GH1 beta-Glucosidase Exhibiting Hydrolytic Activity on an N-Linked Glucopyranoside

{beta}-Glucosidases (Bgls) catalyse the hydrolysis of the glycosidic bond of {beta}-D-glycosides. Ubiquitous in nature, they play vital biological roles across diverse organisms, and their versatility has made them valuable for industrial applications. Bgls are commonly known to hydrolyse O- and S-linked glycopyranosides, but their activity on N-linked glycopyranosides has yet to be demonstrated. In a previous study, we discovered and biocatalytically produced a novel N-glucopyranoside, methyl anthranilate-N-{beta}-D-glucopyranoside (MANT-N-glucose). Building on this, we sought to develop a biocatalytic method for the degradation of MANT-N-glucose into its two main components, methyl anthranilate and glucose. Through screening of a eukaryotic Bgl library, we identified ZmGlu1 as an enzyme capable of hydrolysing MANT-N-glucose. This reaction displayed substantially reduced catalytic efficiency (0.31 min-1 mM-1) relative to ZmGlu1s activity with its native substrate and other O-glucopyranosides. Structural modelling of enzyme-substrate complexes revealed key interactions likely contributing to the reduced activity. These findings provide a foundation for future investigations into N-glycopyranoside Bgl reactivity and highlight the broader potential of Bgls in biocatalytic applications. ImportanceIn this study, we uncover a previously unknown function within the widely used enzyme, {beta}-glucosidases (Bgls). These enzymes catalyse the hydrolysis of O- and S-linked glycopyranosides, yet their ability to act on N-linked glycopyranosides has remained unrecognised until now. Using both human gut bacteria and a library of eukaryotic Bgls, we identified and characterised an enzyme capable of hydrolysing an N-linked substrate, methyl anthranilate-N-{beta}-D-glucopyranoside. This finding expands the recognised activity range of Bgls and highlights their broader industrial relevance. In addition, this finding opens avenues for further investigation into Bgls substrate selectivity, as well as enzyme engineering aimed at enhancing activity toward N-linked glycopyranosides and elucidating the underlying structure-function.