Mechanistic Insights into β-1,6-Branch Recognition and Orientation-Selective Hydrolysis by Endo -β-1,3-Glucanase Using Synthetic β-Glucan Probes

High Resolution Image Download MS PowerPoint Slide Understanding how glycoside hydrolases structurally heterogeneous polysaccharides remains a central challenge due to the limited availability of well-defined substrates. Here, we establish a chemically defined, probe-based platform to systematically dissect the hydrolytic specificity of an endo -β-1,3-glucanase using a systematically designed library of branched β-1,3-glucan oligosaccharides with precisely controlled branch position and length. Synthetic access to these glycans enabled their conversion into fluorescent probes for sensitive, site-specific analysis of enzymatic cleavage. Our results revealed that β-1,6-linked branching acts as a key structural determinant governing both cleavage efficiency and positional selectivity in a branched length-dependent manner. While monosaccharide branches are broadly accommodated, disaccharide branches introduce steric constraints that significantly suppress hydrolysis. Notably, analysis using an extended octasaccharide substrate uncovered a dominant cleavage at the central β-1,3-glycosidic bond, allowing refinement and expansion of the enzyme subsite model beyond that accessible with shorter substrates. Collectively, these findings define branch architecture as a critical regulator of substrate orientation and cleavage-site selection, and establish chemically defined glycan libraries as a generalizable platform for mechanistic interrogation of glycoside hydrolases.