Structural and Energetic Evidence Supports the Non-Covalent Phosphate Cyclization by the Class II Phospholipase D from .

Phospholipase D (PLD) enzymes from spider venom mediate envenomation pathology by cleaving phospholipid headgroups. We revisited the crystal structure of PLD (PDB: 3RLH) to evaluate two alternative mechanisms-covalent and non-covalent-for headgroup cleavage. The covalent mechanism involves a nucleophilic attack on the substrate's P atom by catalytic histidine, forming a phosphohistidine intermediate. It was originally suggested that this intermediate hydrolyzes, leading to linear phosphates. The non-covalent mechanism relies on the substrate's hydroxyl group performing an intramolecular attack on the P atom, thereby generating a cyclic phosphate. Structural refinement of the crystal structure revealed a cyclic phosphate bound at the active site, replacing previously assigned PEG molecules. This cyclic product, stabilized by His12, His47, and Mg, provides structural evidence that supports phosphate cyclization. The results of computational analyses, including molecular dynamics and quantum mechanics/molecular mechanics simulations, further support the non-covalent mechanism as the energetically preferred pathway, with a significantly lower activation barrier. Our findings highlight the role of substrate orientation and of the catalytic His residues in transphosphatidylation, advancing our understanding of PLD enzymology and providing insights for the design of inhibitors against envenomation.