Charoenwongpaiboon T.Benini S.Field R.A.Klaewkla M.Lorthongpanich C.Pongsawasdi P.Pichyangkura R.Wangpaiboon K.Mahidol University2026-02-062026-02-062026-02-01Carbohydrate Polymers Vol.373 (2026)01448617https://repository.li.mahidol.ac.th/handle/123456789/114571Levansucrase catalyzes the polymerization of fructose units from sucrose into a β-2,6-linked fructan called “Levan”, which are versatile in many applications. While levansucrases from Gram-positive bacteria have well-characterized levan-binding track, the molecular basis for levan elongation by Gram-negative bacteria enzymes remains unclear. Here, we integrated rational mutagenesis, biochemical assays, and molecular dynamics (MD) simulations to elucidate the levan-binding architecture of levansucrease from the Gram-negative bacteria Erwinia tasmaniensis ( Et Lsc). Using surface topology and residue-frequency mapping, we identified three potential carbohydrate-binding tracks. Alanine scanning of 19 residues revealed that mutations in a distinct surface loop (track II) significantly decreased levan production, particularly F376A, which also abolished gel retention in levan-affinity PAGE. MD simulations proposed strong interactions between levan oligosaccharide and residues F376 and F349, and highlighted a conformational change within the 368–378 loop upon substrate binding. Interestingly, mutations in a second region (track III) selectively altered the product spectrum of β-2,1 fructooligosaccharides and promote levan biosynthesis, suggesting a dual-track model for fructan synthesis. Taken together, the results reveal a unique elongation mechanism in Gram-negative bacteria levansucrases that diverges from that of enzymes from Gram-positive bacteria. These findings provide a structural framework for engineering levansucrases with tunable product profiles for carbohydrate biotechnology applications.Materials ScienceChemistryArticleSCOPUS10.1016/j.carbpol.2025.1246162-s2.0-1050209315281879134441320395