*2.2. Homology Modeling and Substrate Binding Specificity of ScCDA2*

The crystal structures of several CDAs have already been determined, while CDA/substrate complex structure determination is less well defined and the interaction between the enzyme and the substrate is poorly understood. To study the characteristics of *Sc*CDA2 and chitin molecule interactions, we performed molecular docking to understand the binding mechanism of *Sc*CDA2. Homology modelling of *Sc*CDA2 (Figure 3A) revealed that the secondary structure consists of a conserved (α/β)8 folded barrel structure and six loops. The model was further evaluated for protein geometry by SAVES. Evaluation report shows that 97.3% residues in additional allowed regions and 85.57% of the residues have averaged 3D-1D score ≥0.2, and the quality factor is 91.2214, indicating that the structure quality was acceptable (Figure S1). The docking results (Figure 3B) show that chitin lies in the substrate-binding pocket which is surrounded by six loops, His250, Asp102, Asp103, His149 and His153. Asp103, His149 and His153 form a coordinate bond with Zn2+, and the metal ion serves as a Lewis acid to assist the water affinity attack on the carbon atom on the amide bond. The adjacent His250 and Asp102 play a catalytic role through protonation, and the common action of these amino acids leads to the cleaving of the acetyl group. In addition, the structural superposition of *Ar*CE4A (PDB ID: 5LFZ), *SL*CE4 (PDB ID: 2CC0), *Sp*PgdA (PDB ID: 2C1G) and model of *Sc*CDA2 reveal that there are six conserved loop domains in *Sc*CDA2 (Figure 3C).

**Figure 3.** Catalytic binding mode resulting from homologous modelling and molecular docking. (**A**) The stereo view of the overall structure of *Sc*CDA2 (**B**) Highlights the binding pocket of *Sc*CDA2 docked with GlcNAc. The pink sticks represent a catalytic amino acid, and the blue sticks represent the amino acid that forms a coordinate bond with Zn2+. The substrate GlcNAc is represented by a yellow stick. (**C**) Conservative loops were found through multiple structure superposition. The model of *Sc*CDA2 was superposed with an ArCE4A structure from a marine *Arthrobacter* species (PDB ID: 5LFZ), a CE4 carbohydrate esterase structure from *Streptomyces lividans* (PDB ID: 2CC0) and an *Sp*PgdA structure from *Streptococcus pneumoniae* (PDB ID: 2C1G). The conservative loops also have been marked.
