Efficient heterologous expression, functional characterization and molecular modeling of annular seabream digestive...

Here we report the cDNA cloning of a phospholipase A₂ (PLA₂) from five Sparidae species. The deduced amino acid sequences show high similarity with pancreatic PLA₂. In addition, a phylogenetic tree derived from alignment of various available sequences revealed that Sparidae PLA₂ are closer to avian PLA₂ group IB 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 PLA₂ (AsPLA₂). A single Ni-affinity chromatography step was used to obtain a highly purified recombinant AsPLA₂ with a molecular mass of 15kDa as attested by gel electrophoresis and MALDI-TOF mass spectrometry data. The enzyme has a specific activity of 400U.mg^-1 measured on phosphatidylcholine at pH 8.5 and 50°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 PLA₂. By following these models and utilizing molecular dynamics simulations, the resistance of the AsPLA₂ at high temperatures was explained. Using the monomolecular film technique, AsPLA₂ was found to be active on various phospholipids spread at the air/water interface at a surface pressure between 12 and 25dyncm^-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 PLA₂, provides new insights on structure-activity relationships of fish PLA₂.