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

文献类型： 外文期刊

作者： Ni, Dawei 1 ; Huang, Zhaolin 1 ; Zhang, Shuqi 1 ; Hou, Xiaodong 3 ; Xu, Wei 1 ; Zhang, Wenli 1 ; Rao, Yijian 4 ; Mu, Wanmeng 1 ;

作者机构： 1.Jiangnan Univ, State Key Lab Food Sci & Resources, Wuxi 214122, Jiangsu, Peoples R China

2.Jiangnan Univ, Sch Food Sci & Technol, Wuxi 214122, Jiangsu, Peoples R China

3.Jiangsu Acad Agr Sci, Inst Germplasm Resources & Biotechnol, Prov Key Lab Agrobiol, Nanjing 210014, Jiangsu, Peoples R China

4.Jiangnan Univ, Sch Biotechnol, Key Lab Carbohydrate Chem & Biotechnol, Minist Educ, Wuxi 214122, Jiangsu, Peoples R China

关键词： inulosucrase; levansucrase; tunnel; sugar chain extension; shunting mechanism

期刊名称：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY （ 影响因子：6.2； 五年影响因子：6.4 ）

ISSN： 0021-8561

年卷期： 2025 年 73 卷 26 期

页码：

收录情况： SCI

摘要： 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 Lactobacillus reuteri 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.

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