Structural and functional insights into metal coordination and substrate recognition of Akkermansia muciniphila sialidase Amuc_1547.

Abstract

Sialidases in Akkermansia muciniphila are pivotal for mucin degradation, enabling energy acquisition, modulating gut microbiota balance, and influencing host health. However, their structural and functional mechanisms remain poorly characterized. This study resolved the magnesium-bound crystal structure of Amuc_1547, revealing a six-bladed β-propeller fold linked to a carbohydrate-binding module (CBM)-like β-sandwich domain. Structural characterization ​identified a conserved S-x-D-x-G-x-x-W motif, a unique metal-binding pocket coordinated by residues Glu289, Glu299, and Asp300, and a putative carbohydrate substrate-binding pocket within the CBM-like domain. Enzymatic assays confirmed the functional relevance of these structural elements and demonstrated that both metal ions and glycans significantly enhance enzymatic activity. Molecular docking, dynamics simulations, and enzyme kinetics analysis identified critical residue substitutions involved in sialic acid substrate binding and catalysis: Gln367 replaces an arginine in the classical Arg-triplet, while Gln350 and His349 ​replace the nucleophilic tyrosine. These substitutions collectively mediate substrate binding, nucleophilic attack, and transition state stabilization, distinguishing the catalytic mechanism of Amuc_1547 from other six-bladed β-propeller sialidases. Additionally, comparative analysis of the four A. muciniphila sialidases highlights sequence divergence and domain architecture variations, suggesting niche-specific roles in gut microenvironments. Our work not only deciphers the structural basis of metal-dependent substrate recognition in Amuc_1547 but also advances our understanding of the adaptation of A. muciniphila to gut niches, offering a blueprint for leveraging sialidase-driven mucin metabolism in microbiota-targeted therapies.