Tryptophan and aspartic acid residues present in the glycerophosphoryl diester phosphodiesterase (GDPD) domain of the Loxosceles laeta phospholipase D are essential for substrate recognition.
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 glycerophosphoryl diester phosphodiesterase (GDPD) domain of recombinant LlPLD1, 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 LlPLD1. The interaction of LlPLD1 with sphingomyelin was also strongly reduced in both mutants LlPLD1-W256S and LlPLD1-D259G. The results show the importance of these residues in the interaction of the protein with its substrate sphingomyelin in cell membranes.
Laboratory of Molecular Parasitology, Department of Medical Technology, Faculty of Health Sciences, University of Antofagasta, P.O. Box 170, Antofagasta, Chile. Electronic address: acatalan@uantof.cl.
Alejandro
A
Catalán
Laboratory of Molecular Parasitology, Department of Medical Technology, Faculty of Health Sciences, University of Antofagasta, P.O. Box 170, Antofagasta, Chile.
William
W
Cortés
Laboratory of Molecular Parasitology, Department of Medical Technology, Faculty of Health Sciences, University of Antofagasta, P.O. Box 170, Antofagasta, Chile.
Christian
C
Muñoz
Laboratory of Molecular Parasitology, Department of Medical Technology, Faculty of Health Sciences, University of Antofagasta, P.O. Box 170, Antofagasta, Chile.
Jorge E
JE
Araya
D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D001224:Aspartic Acid / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010739:Phospholipase D / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013111:Spider Venoms / Q000737:chemistry*; D013112:Spiders / Q000201:enzymology; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry*
D060829:Arthropod Proteins; D013111:Spider Venoms; D001224:Aspartic Acid; D014364:Tryptophan; D010727:Phosphoric Diester Hydrolases; D010739:Phospholipase D; C038133:glycerophosphodiester phosphodiesterase
Glycerophosphoryl diester phosphodiesterases; Loxosceles laeta; Mutation site directed; Phospholipase D; Sphingomyelin