Structure of a novel class II phospholipase D: Catalytic cleft is modified by a disulphide bridge

Abstract

Phospholipases D (PLDs) are principally responsible for the local and systemic effects of Loxosceles envenomation including dermonecrosis and hemolysis. Despite their clinical relevance in loxoscelism, to date, only the SMase I from Loxosceles laeta, a class I member, has been structurally characterized. The crystal structure of a class II member from Loxosceles intermedia venom has been determined at 1.7Å resolution. Structural comparison to the class I member showed that the presence of an additional disulphide bridge which links the catalytic loop to the flexible loop significantly changes the volume and shape of the catalytic cleft. An examination of the crystal structures of PLD homologues in the presence of low molecular weight compounds at their active sites suggests the existence of a ligand-dependent rotamer conformation of the highly conserved residue Trp230 (equivalent to Trp192 in the glycerophosphodiester phosphodiesterase from Thermus thermophofilus, PDB code: 1VD6) indicating its role in substrate binding in both enzymes. Sequence and structural analyses suggest that the reduced sphingomyelinase activity observed in some class IIb PLDs is probably due to point mutations which lead to a different substrate preference.

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders

“…As a class IIb enzyme, St_␤IB1i is most similar to the latter, as all class II enzymes contain two disulfide bonds. The largest structural variations were found in the flexible loop regions, as observed previously in comparisons of class I and class IIa structures (2,28). Docking experiments with LPC and LPE substrates and St_␤IB1i suggested a location for the headgroup recognition pocket deep in the active site, away from variable loops.…”

“…This disulfide bonding pattern, along with low SMase D activity, places St_␤IB1i in the class IIb group (27,28). The flexible, catalytic, and variable loop regions of St_␤IB1i all exhibit large temperature factors, as also seen in both LiRecDT1 and SMase I.…”

“…2 and 5B) (2,27,28,59). With the exception of Asp-52, which is in the highly flexible loop region of the molecule (Fig.…”

“…SicTox proteins with confirmed low SMase D activity have also been termed "class IIb" PLDs (27,28); all these are members of the SicTox ␤ clade.…”

Acute Kidney Injury Induced by Snake and Arthropod Venoms

“…As some Loxosceles sphingomyelinases D have broad substrate specificity, being able to hydrolyze not only sphingophospholipids but also lysoglycerophospholipids, they are now classified as phospholipases D (Lee and Lynch, 2005). Due to sequence, structural and biochemical differences these toxins are grouped in two classes and their structures and substrate specificities have been recently elucidated (de Giuseppe et al, 2011;Murakami et al, 2005). Other important components of Loxosceles venom are the metalloproteinases.…”