Molecular Engineering of Phospholipase D for Enhanced Phosphatidyldisaccharide Synthesis and Elucidation of Its Transphosphatidylation Mechanism

Abstract Phosphatidylsaccharides with diverse glycosyl headgroups have distinct functional applications, yet efficient synthesis of phosphatidyldisaccharides remains challenging because phospholipase D (PLD) preferentially accepts monosaccharides, and its transphosphatidylation mechanism is poorly understood. Here, structure-guided engineering of residues near the glycosyl donor site identified R34(W217N), which increased phosphatidylmaltoside (PtdMal) conversion from 0.56 ± 0.03% to 75.45 ± 0.49%. The mutant also markedly enhanced production of phosphatidylsucroside, phosphatidyllactoside, and phosphatidyllactofructoside, exceeding previous benchmarks. Molecular dynamics simulations indicated that W217N relieved steric crowding at the active-site entrance, strengthened van der Waals and hydrogen-bond interactions near the HKD motifs, and favored donor hydroxyl positioning for nucleophilic attack, thereby stabilizing the enzyme–substrate complex and improving catalytic efficiency. These findings provide mechanistic insight into PLD transphosphatidylation and establish a basis for engineering PLDs to synthesize structurally diverse phosphatidyloligosaccharides.