Structural And Functional Studies Of Legionella Pneumophila Acid Phosphatase

Legionella pneumophila, the causative agent of Legionnaires’disease, is a Gram-negative facultative intracellular pathogen that is capable of multiplying in a wide spectrum of eukaryotic cell. Legionella pneumophila normally thrives in fresh water environments as free-living forms, in biofilms and as the intracellular parasite in protozoa. Human infection occurs when aerosolized droplets of contaminated water are inhaled and reach the alveolar mucosa. In the lungs Legionella pneumophila invades and replicates in macrophages, which are considered the primary target of Legionella pneumophila although, evidence indicates that Legionella pneumophila can also invade and replicate in epithelial cells.When the cells are infected by Legionella pneumophila, the virulence factors are released into the target cells by the type II secretion system and the type IV secretion system, which can achieve the effect of infection, and make Legionella pneumophila be able to proliferate in the host cell.MapA(The major acid phosphatase from Legionella pneumophila) is one of the virulence proteins which is secreted by the type II secretion system.The major acid phosphatase from Legionella pneumophila (MapA) belongs to the histidine acid phosphatase superfamily which contains the characteristic histidine acid phosphatase (HAP) sequence motif RHGXRXP. It catalyzes the hydroxylation of a phosphoryl group from phosphomonoesters by an active-site histidine under acidic conditon. Approximately 90% of acid phosphatase activity of Legionella pneumophila is contributed by MapA. This phosphatase activity is likely to play a role in the infection of the host cell or in some signal transduction processes.We have obtained a series of high resolution protein crystal structure of MapA. The overall structure of MapA is similar to other HAPs. The residues Glu201, Arg33, Arg37, Arg101, His34 and Asp281 are involved in phosphate binding. Further biochemical experiments show that the Glu201 and Asp281 is important for enzyme activity. To get insight into the mechanism of substrate recognition, the crystal structure of MapA mutant D281A in complex with 3’-AMP was solved at 2.9 A resolution. The structure of D281 A/3’-AMP complex reveals that the 3’-AMP completely fits the substrate binding tunnel, with the 2’-hydroxyl group of ribose moiety stabilized by Glu201, and the adenine moiety clamped between His205 and Phe237. In order to verify whether the amino acids in these key sites play a key role in the catalytic process, we performed a series of site directed mutations:E201 A, D281 A, H205A, F237A, H205A/F237A (All of them turned to Ala), respectively. At present, pNPP,5’-AMP,3’-AMP as the substrate of the enzyme data have been measured. We also performed ITC (isothermal titration calorimetry) experiments, using pNPP and 5 ’-AMP and 3’-AMP as ligands, what in order to determine the difference of binding capacity between these substrates with native protein and these substrates with the mutants. As we expected, the enzyme activity test and ITC data do show that MapA is indeed different from its mutants, and it also verifies the key residues which are analyzed by the structural biology.To the best of our knowledge, this is the second substrate bound structure of HAP, with a new conformation of the adenine and ribose groups of 3’-AMP in the active site. Not only makes us further understand the structure and biochemical properties of the protein, our studies but also indicate a new substrate recognition mechanism of HAP.