Abstract
Legionella pneumophila encodes an extracellular secreted phospholipase A named PlaA that is translocated by the type II secretion system. It plays an essential role in maintaining the integrity of Legionella-containing vacuoles in L. pneumophila pathogenesis. Here, it is shown that PlaA has a main lysophospholipase activity to hydrolyze fatty-acyl groups in lysophospholipids. Although it has a very low phospholipase A activity to catalyze the hydrolysis of fatty-acyl groups in phospholipids, PlaA can bind phospholipids such as 1,2-dipalmitoylphosphatidylcholine with a dissociation constant of 11.1 µM. Sequence-alignment analysis combined with activity assays revealed that PlaA contains a distinct substrate-binding site among the known structures of the phospholipase A family, implying that PlaA may present a novel mechanism for substrate recognition. Native PlaA and its selenomethionine (SeMet)-substituted form were purified and crystallized by vapour diffusion in hanging drops at 296 K. Diffraction data were collected to a resolution of 2.0 Å for native PlaA protein and to a resolution of 2.7 Å for SeMet-substituted PlaA protein. The crystals of native PlaA belonged to the monoclinic space group P2(1), while the crystals of SeMet-substituted PlaA belonged to the primitive orthorhombic space group P2(1)2(1)2(1). Initial phases for PlaA were obtained from SeMet SAD data sets.