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

Area of Science:

• Enzymology
• Structural Biology
• Biochemistry

Background:

• α-Agarases are crucial enzymes for producing bioactive agarooligosaccharides from agarose.
• Understanding their complex structures and catalytic mechanisms is vital for industrial applications.
• Previous studies faced challenges due to the intricate architecture of these enzymes.

Purpose of the Study:

• To elucidate the structure-based catalytic mechanism of α-agarase CmAga from *Catenovulum maritimum* STB14.
• To identify key catalytic residues and substrate-binding sites.
• To explore the cooperative roles of the catalytic domain and carbohydrate-binding modules.

Main Methods:

• Integrated Cryo-Electron Microscopy (Cryo-EM) and AlphaFold2 for structural determination.
• Molecular dynamics simulations to analyze enzyme dynamics.
• Neural relational inference modeling to understand interdomain communication.

Main Results:

• Identified D994 and E1129 as critical catalytic residues, with E1129 showing selectivity for α-1,3-glycosidic bonds.
• Mapped substrate-binding sites (-3 to +3 subsites) involving residues Y858, W1201, Y1164, and W1166.
• Revealed a cooperative mechanism involving the catalytic domain and four carbohydrate-binding modules (CBMs) in substrate capture, transfer, and active site stabilization.
• Identified D149 and L608 as key nodes in interdomain communication pathways.

Conclusions:

• The study provides a detailed structural and mechanistic understanding of α-agarase CmAga.
• These findings lay the groundwork for mechanistic studies and the rational engineering of multi-CBM containing carbohydrate-active enzymes (CAZymes).
• The identified cooperative mechanism highlights the importance of CBMs in enzyme function.