Abstact

Arcanobacterium haemolyticum, an emerging human pathogen, expresses phospholipase D (PLD(AH)), a multifunctional virulence factor capable of cleaving sphingomyelin and lysophospholipids from plasma membranes, in addition to promoting host cell adhesion and necrosis. Here, we report the first crystal structure of PLD(AH), determined at 2.45 A resolution, which reveals a canonical (alpha/beta)(8) TIM-barrel fold typical of glycerophosphodiester phosphodiesterase (GDPD)-like PLD enzymes, but with distinct structural features. PLD(AH) contains two disulfide bonds arranged in a unique pattern not observed in homologous brown spider PLDs, defining a new structural variant within the GDPD-like PLD family, designated class IIa-alpha. Comparative structural analysis with PLD from Loxosceles intermedia (PLD(LI)) revealed differences in loop architecture and local amino acid composition in the vicinity of the active site, including point substitutions that modulate cavity volume and flexibility. Despite exhibiting a melting temperature (Tm) between 45 and 51 degrees C, PLD(AH) retained residual enzymatic activity up to 95 degrees C, indicating exceptional thermostability among GDPD-like PLDs. Molecular dynamics simulations showed that increasing temperature selectively enhanced the flexibility of specific loops (C and G) without perturbing the catalytic core, suggesting that localized structural adaptability contributes to thermal resilience. These findings establish PLD(AH) as a structurally distinct GDPD-like sphingomyelinase D enzyme and provide insights into the molecular features underlying its multifunctional activity and thermostability.