An Unusual His/Asp Dyad Operates Catalysis in Agar-Degrading Glycosidases

Agarose motifs, found in agars present in the cell walls of red algae, consist of alternating units of d-galactose (G) and α-3,6-anhydro-l-galactose (LA). Glycoside hydrolases from family 117 (GH117) cleave the terminal α-1,3-glycosidic bonds, releasing LA units. Structural studies have suggested that these enzymes use unconventional catalytic machinery, involving a histidine (His302) as a general acid rather than a carboxylic residue as in most glycosidases. By means of quantum mechanics/molecular mechanics metadynamics, we investigated the reaction mechanism of Phocaeicola plebeius GH117, confirming the catalytic role of His302. This residue shares a proton with a neighbor aspartate residue (Asp320), forming a His/Asp dyad. Our study also reveals that, even though the sugar unit at the -1 subsite (LA) can adopt two conformations, ⁴ C ₁ and ^1,4 B, only the latter is catalytically competent, defining a ^1,4 B → [⁴ E]^‡ → ^1,4 B (→ ⁴ C ₁) conformational itinerary. This mechanism may be applicable to similar enzymes with a His/Asp dyad in their active sites, such as GH3 β-N-acetylglucosaminidase and GH156 sialidase. These insights enhance our understanding of glycosidase catalytic strategies and could inform the engineering of enzymes for the more efficient processing of seaweed.