Structure-Informed Insights into Catalytic Mechanism and Multidomain Collaboration in α-Agarase CmAga

α-Agarases are glycoside hydrolases that cleave α-1,3-glycosidic bonds in agarose to produce bioactive agarooligosaccharides. Despite their great industrial potential, the structures and functional mechanisms of α-agarases remain unclear due to their complex and flexible architecture. Here, we investigated the structure-based catalytic mechanism of α-agarase CmAga from Catenovulum maritimum STB14 by integrated Cryo-EM and AlphaFold2. D994 and E1129 were identified as catalytic residues, with E1129 selectively recognizing α-1,3-glycosidic bonds. Y858, W1201, Y1164, and W1166 facilitate preferential substrate binding at the −3 ∼ +3 subsites. Molecular dynamics simulations and neural relational inference modeling revealed a cooperative mechanism involving the catalytic domain (CD) and four carbohydrate-binding modules (CBMs), with CBM6–1 and CBM6–2 capturing substrates, CBM_like transferring them to the CD, and CBM6–3 stabilizing the active site. D149 and L608 served as pivotal nodes within the interdomain communication pathways. These insights provide a foundation for mechanistic investigations and rational engineering of carbohydrate-active enzymes (CAZymes) with multiple CBMs.