Structure–function analysis of Gynuella sunshinyii chitosanase uncovers the mechanism of substrate binding in GH family 46 members

Chitooligosaccharides (COS) is a kind of functional carbohydrates with great application potential as its various biological functions in food, cosmetics, and pharmaceutical fields. Exploring the relationship between structure and function of chitosanase is essential for the controllable preparation of chitooligosaccharides with the specific degree of polymerization (DP). GsCsn46A is a cold-adapted glycosyl hydrolase (GH) family 46 chitosanase with application potential for the controllable preparation of chitooligosaccharides. Here, we present two complex structures with substrate chitopentaose and chitotetraose of GsCsn46A, respectively. The overall structure of GsCsn46A contains nine α-helices and two β-strands that folds into two globular domains with the substrate between them. The unique binding positions of both chitopentaose and chitotetraose revealed two novel sugar residues in the negatively-numbered subsites of GH family 46 chitosanases. The structure–function analysis of GsCsn46A uncovers the substrate binding and catalysis mechanism of GH family 46 chitosanases. Structural basis mutagenesis in GsCsn46A indicated that altering interactions near +3 subsite would help produce hydrolysis products with higher DP. Specifically, the mutant N21W of GsCsn46A nearly eliminated the ability of hydrolyzing chitotetraose after long-time degradation.