The crystal structure of mature Polygonatum cyrtonema lectin (PCL) showed three similar carbohydrate-binding sites (CBS Ⅰ,CBS Ⅱ,and CBS Ⅲ).The Gin58 and Asp60 residues of CBS Ⅱ are substituted with His58 and Asn60.To establish the relationship between the key amino acid residues and structure or activity of PCL,we constructed four recombinant mutants in CBS Ⅰ,CBS Ⅱ,and CBS Ⅲ.The experimental results indicate that CBS Ⅰ,CBS Ⅲ and the disulfide bond play vital roles in the binding with mannose.Furthermore,molecular dynamics simulations and binding free energy calculation illustrate that CBS Ⅰ has a direct and strong relationship with the activity of PCL.CBS Ⅱ does not play a critical role in the model for mannose binding by PCL.Although CBS Ⅲ does not enhance the activity,it helps to maintain the activity and 3D structure.These results suggest that the carbohydrate-binding site of PCL may be in a hydrophilic environment,and Asn and Tyr are the key amino acids involved in its binding with sugar,but Gln and Asp are not necessary to maintain its activity.