Catalytic Mechanism and Secondary-Structure-Type-Dependent Allosteric Regulation of β-Agarase Amaga

It remains unclear whether enzymic allosteric sites on distinct secondary structure types mediate differential dynamic conformational transitions. Herein, we investigated the catalytic mechanism and secondary-structure-type-dependent allosteric regulation of β-agarase Amaga, specifically producing neoagaro-tetraose (NA4) and neoagaro-hexaose (NA6). Using homology modeling, molecular docking, point mutations, structural quantification, and kinetic analysis, we identified six key catalytic residues (Glu147, Asp149, Glu152, His169, His173, and Glu281) forming a retaining mechanism: Glu152, His169, Glu281, and Glu147 facilitate glycosylation, while Glu152 and His173 coordinate deglycosylation, with Asp149 maintaining the charge equilibrium. Notably, five β-sheet-localized residues of these six highlight their structural dominance. Allosteric site mutations revealed that random coil-localized sites enhance activity by converting random coils into α-helices, whereas β-sheet-localized sites drive transitions from β-sheets to α-helices, β-turns, and random coils. Despite differing pathways, higher-activity mutants exhibited faster structural transitions and increased NA4 production. These findings highlight secondary-structure-type-dependent design in engineering enzymes with tailored product specificity.