Structure-Guided Tunnel Engineering to Reveal the Molecular Basis of Sugar Chain Extension of Inulosucrase

Inulosucrase (IS) is a key enzyme in the synthesis of inulin, a multifunctional polysaccharide with significant physiological benefits and wide-ranging applications. Lactobacillus IS has the unique capability to produce both high-molecular-weight polysaccharides and oligosaccharides with diverse degrees of polymerization. Understanding the mechanism of sugar chain extension by IS is essential for modulating chain length and engineering custom-designed inulin. In this study, we resolved the crystal structures of IS from <i>Lactobacillus reuteri</i> 121 and its mutant IS-R544W, revealing a unique C-terminal extension into the catalytic pocket. Notably, structure-guided rational design identified IS-Tyr695 in the C-terminal region, along with IS-Asn303, IS-Asn305, IS-Asn367, IS-Gln369, and IS-Asn419, as critical residues specifically required for polysaccharide synthesis without affecting oligosaccharide production. In contrast, IS-Arg544, IS-Tyr618, and IS-Arg622 were determined to be essential for oligosaccharide synthesis with no impact on polysaccharide production. Based on findings from rational design and molecular dynamics simulations, we propose a novel shunting mechanism for the synthesis of polysaccharides and oligosaccharides by IS. This study provides fundamental insights into the inulin chain extension mechanism of IS and lays a theoretical foundation for engineering GH68 enzymes for the production of tailor-made fructans.