Structural Elucidation and Functional Insights into D-Lac: A Calcium-Dependent Enzyme

Previously, the calcium-dependent enzyme D-Lac was purified and identified. However, the absence of structural data and a homologous template with high similarity hampered efforts to design and engineer this enzyme. The advent of AlphaFold2 provided a breakthrough by predicting the 3D structure of D-Lac, serving as a foundation for subsequent structure-guided engineering. A point cloud segmentation method, PointSite, was employed to precisely identify the potential binding region of D-Lac to D-PL. Furthermore, conserved sequence analysis guided rational engineering, highlighting the high degree of conservation within the D-Lac binding pocket. To validate the predicted structure, binding pocket, and metal ion binding site, key residues involved in calcium coordination (E80, N197, N252, and D311) were mutated to alanine. Additionally, tyrosine (Y215), frequently observed within the hydrolase pocket, was mutated to alanine and phenylalanine, respectively. These mutations resulted in the complete loss of D-Lac activity, confirming the critical role of these residues in enzyme function. These findings provide valuable insights into the structural and functional characteristics of D-Lac, paving the way for further engineering and optimization of this enzyme.