8XVR

Crystal structure of inulosucrase from Lactobacillus reuteri 121 mutant R544W

Summary for 8XVR

• Entry DOI: 10.2210/pdb8xvr/pdb
• Descriptor: Glycoside hydrolase family 68 protein, CALCIUM ION, 1,2-ETHANEDIOL, ... (7 entities in total)
• Functional Keywords: glycoside hydrolase 68 enzyme, hydrolase
• Biological source: Limosilactobacillus reuteri
• Total number of polymer chains: 4
• Total formula weight: 263556.62

Authors

Ni, D.,Hou, X.,Cheng, M.,Xu, W.,Rao, Y.,Mu, W. (deposition date: 2024-01-15, release date: 2025-01-22, Last modification date: 2025-12-24)

Primary citation

Ni, D.,Huang, Z.,Zhang, S.,Hou, X.,Xu, W.,Zhang, W.,Rao, Y.,Mu, W.
Structure-Guided Tunnel Engineering to Reveal the Molecular Basis of Sugar Chain Extension of Inulosucrase.
J.Agric.Food Chem., 73:16454-16467, 2025

PubMed Abstract: 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 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.

PubMed: 40523840
DOI: 10.1021/acs.jafc.5c02217

Experimental method

X-RAY DIFFRACTION (2.44 Å)