Abstract
Here we report the cDNA cloning of a phospholipase A(2) (PLA(2)) from five Sparidae species. The deduced amino acid sequences show high similarity with pancreatic PLA(2). In addition, a phylogenetic tree derived from alignment of various available sequences revealed that Sparidae PLA(2) are closer to avian PLA(2) group 1B than to mammals' ones. In order to understand the structure-function relationships of these enzymes, we report here the recombinant expression in E.coli, the refolding and characterization of His-tagged annular seabream PLA(2) (AsPLA(2)). A single Ni-affinity chromatography step was used to obtain a highly purified recombinant AsPLA(2) with a molecular mass of 15 kDa as attested by gel electrophoresis and MALDI-TOF mass spectrometry data. The enzyme has a specific activity of 400 U.mg(-1) measured on phosphatidylcholine at pH 8.5 and 50 degrees C. The enzyme high thermo-activity and thermo-stability make it a potential candidate in various biological applications. The 3D structure models of these enzymes were compared with structures of phylogenetically related pancreatic PLA(2). By following these models and utilizing molecular dynamics simulations, the resistance of the AsPLA(2) at high temperatures was explained. Using the monomolecular film technique, AsPLA(2) was found to be active on various phospholipids spread at the air/water interface at a surface pressure between 12 and 25 dyn cm(-1). Interestingly, this enzyme was shown to be mostly active on dilauroyl-phosphatidylglycerol monolayers and this behavior was confirmed by molecular docking and dynamics simulations analysis. The discovery of a thermo-active new member of Sparidae PLA(2), provides new insights on structure-activity relationships of fish PLA(2). (C) 2017 Elsevier B.V. All rights reserved.