Abstract

It is known that the family of phospholipases D (PLD) from spiders of the genus Loxosceles, hydrolyze the substrates sphingomyelin and lisophosphatidylcholine, by their catalytic acid-base action which involves two histidines. However, little is known about the amino acids that participate on substrate recognition. In this study we identified highly conserved amino acids of the glycerophosphoiy1 diester phosphodiesterase (GDPD) domain of recombinant LIPLD1, which interact with the substrate sphingomyelin. The mutation of W256 to serine and D259 to glycine decreased significantly the sphingomyelinase and hemolytic activity when compared to wild type LIPLD1. The interaction of LIPLD1 with sphingomyelin was also strongly reduced in both mutants LIPLD1-W256S and LIPLD1D259G. The results show the importance of these residues in the interaction of the protein with its substrate sphingomyelin in cell membranes. (C)2014 Elsevier Ltd. All rights reserved.