Structural insights into glycoside hydrolase family 1 β-glucosidase: Selective oligosaccharide hydrolysis, synthesis, and product profiling

TL;DR

This study reveals how a specific β-glucosidase enzyme selectively breaks down and synthesizes sugars, offering new opportunities for enzyme engineering and biotechnology.

Contribution

The paper identifies a unique subsite in a GH1 β-glucosidase that governs substrate specificity and transglycosylation selectivity.

Findings

The enzyme Td2F2 has a subsite +1′ that stabilizes sophorose in a nonproductive binding mode.

Mutants T225N and E296D show enhanced hydrolytic activity toward sophorose.

Td2F2 preferentially forms β-1→2 and β-1→3 linkages during transglycosylation.

Abstract

β-Glucosidases are essential enzymes in plant cell wall metabolism and have diverse biotechnological applications, including cellulose degradation and prebiotic oligosaccharide synthesis. Td2F2, a glycoside hydrolase family 1 (GH1) β-glucosidase derived from a compost metagenome, exhibits a unique preference for sophorose. However, the molecular basis of this specificity remains unclear. In this study, we determined high-resolution crystal structures of Td2F2 in complex with sophorose (1.64 Å) and laminaribiose (1.16 Å) using sodium malonate as a cryoprotectant. Structural analysis, complemented by molecular dynamics simulations, revealed a distinct subsite +1′, where Asn223, Thr225, Glu296, and Arg325 form hydrogen bonds with the reducing-end glucose of sophorose, stabilizing an alternative, nonproductive binding mode adjacent to the catalytic subsites. Site-directed mutagenesis confirmed that residues in subsite +1′ are critical for substrate specificity. Guided by structural insights, we designed T225N and E296D mutants, which exhibited enhanced hydrolytic activity toward sophorose. To further investigate the transglycosylation potential of Td2F2, we characterized its dynamic product profile, ranging from disaccharides to tetrasaccharides, using porous graphitic carbon liquid chromatography–orbitrap tandem mass spectrometry. When p-nitrophenyl β-D-glucopyranoside and glucose were used as substrates, Td2F2 preferentially formed β-1→2 and β-1→3 linkages. These findings provide structural evidence that the subsite +1′ is a “waiting position” in the GH1 β-glucosidase, offering novel insights into its role in hydrolysis and transglycosylation selectivity. This structural and functional framework paves the way for future GH1 enzyme engineering and expanded biotechnological applications.

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