Dynamic loops and active site mediate substrate recognition of Streptomyces phospholipase D and its functional...

This study aimed to decipher the structure-function relationship of Streptomyces klenkii phospholipase D (SkPLD) and leverage its transphosphatidylation activity for synthesizing functional phospholipids in milk fat systems. Crystallography and mutagenesis analyses identified dynamic loops (c-f) as key mediators of substrate recognition. Targeted constraints, including proline substitution in loop (c) and disulfide bonds in loops (d/e), impaired transphosphatidylation, with loop (f) being essential for catalysis. Moreover, SkPLD employs dual HKD motifs: His167 initiates nucleophilic attack, while His440 activates acceptors, stabilized by Tyr459, Asp199, and Asp465. Hydrophobic residues and Ala122/Asn457 determine phospholipid alkyl side chain orientation. We achieved direct phosphatidylserine (8.1 mg/mL), phosphatidylethanolamine (42.8 mg/mL), and phosphatidylglycerol (13.2 mg/mL) synthesis in milk fat globule membranes using SkPLD, from initial levels of 1.5, 26.7, and 0 mg/mL, bypassing solvent-based systems. This work provides a mechanistic framework for Streptomyces phospholipase D catalysis and establishes an enzymatic strategy for functional phospholipid enrichment in dairy products.