Site-Directed Mutagenesis Confirms Structural and Functional Roles of Key Residues in D-Lac Activity

To further corroborate our prior conjectures regarding the configuration, binding cleft, and metal ion binding site, we conducted mutations of calcium coordination residues E80, N197, N252, and D311 to alanine. Additionally, we mutated tyrosine (Y215), which is more prevalent in the hydrolase pocket, to both alanine and phenylalanine. The outcome of these mutations was the complete annulment of D-Lac activity, thereby lending further support to our previous suppositions and predictions (Figure 1D).