Structural Basis for Metal Ion Coordination and the Catalytic Mechanism of Sphingomyelinases D

Abstract

Sphingomyelinases D (SMases D) fromLoxosceles spider venom are the principal toxins responsible for the manifestation of dermonecrosis, intravascular hemolysis, and acute renal failure, which can result in death. These enzymes catalyze the hydrolysis of sphingomyelin, resulting in the formation of ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidyl choline, generating the lipid mediator lysophosphatidic acid. This report represents the first crystal structure of a member of the sphingomyelinase D family from Loxosceles laeta (SMase I), which has been determined at 1.75-Å resolution using the "quick cryo-soaking" technique and phases obtained from a single iodine derivative and data collected from a conventional rotating anode x-ray source. SMase I folds as an (␣/␤) 8 barrel, the interfacial and catalytic sites encompass hydrophobic loops and a negatively charged surface. Substrate binding and/or the transition state are stabilized by a Mg 2؉

Molecular Cloning, Expression, Function, Structure and Immunoreactivities of a Sphingomyelinase D from Loxosceles adelaida, a Brazilian Brown Spider from Karstic Areas

“…This feature, not present in class I SMases D, diminishes significantly the active-site volume and also alters the inherent flexibility exhibited by the flexible loop. Beyond that, all the structural features observed in class I SMases D are fully conserved and details concerning the action mechanism are well described in Murakami et al, 2005 andMurakami et al, 2006. 3.6 Sphingomyelin-binding mode to SMases D Despite the structure of a SMase D member has been solved, there is neither structural nor biophysical data relating the binding mode of the sphingomyelinase (SM) into the active-site cleft of SMases D. Thus, in order to shed light into the structural determinants for recognition and binding of SM by SMases D, a MD simulation was performed using the crystal structure of the SMase D I and the SM docked into enzyme taking into account the crystallographic position of the sulphate ion, which provides a good notion how the phosphate group from SM is oriented in the active-site cavity.…”

“…L. adelaida SMase D A1 is a class II member and conserves all structural features for catalytic activity upon sphingomyelin. MD simulations indicated the binding mode of SM in the SMase I, a class I member that already has its crystallographic structure solved (Murakami et al, 2005) and they demonstrated the role exerted by Trp230 in the orientation of choline head, the magnesium ion in the coordination of the phosphate group and other aliphatic residues in the stabilization of substrate at the active site. In conclusion, we have cloned, expressed and biochemically and structurally characterized a new sphingomyelinase D from Loxosceles adelaida spider and shown that it displays all the functional characteristics of whole venom.…”

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

“…4) (55). This type of SMase D lacks the HKD motif seen in phospholipase Ds (PLDs), explaining why they do not act on phosphatidylcholine (PC) and other glycerophospholipids, although sometimes they are mistakenly referred to as PLDs (56)(57)(58).…”

“…These bacterial proteins are homologous to enzymes from several fungi and from spiders of the Sicariidae family (54), such as the SMase I from Loxosceles laeta (PDB ID 1XX1) (55).…”

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.…”