Structural And Functional Studies On The Pyoverdine Mature Related Proteins In The Periplasm Of Pseudomonas Aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that can cause in10%-20%of infection in the hospital. Therefore, the prevention of P. aeruginosa pathogenicity is very important. An important virulence factor of Pseudomonas aeruginosa is its iron metabolic pathways, and its virulence factor is a small molecule which can capable of chelated iron material-iron carrier.All along, microbial siderophores as the main way to get iron, its synthesis and transport mechanism study has important theoretical and practical values. There are two more important iron carriers in Pseudomonas aeruginosa. One of siderophores is pyochelin, and the other one is pyoverdine (PVD) which is a water-soluble fluorescein siderophore. Since all types of PVD have the same highly conserved fluorophores, these proteins in a variety of fluorescent Pseudomonas are also highly conserved, so we can expect an efficient anti-Pseudomonas aeruginosa, specific drugs design for a new target.Pseudomonas aeruginosa PAO1of PVDI is the prototype of pyoverdine studies. There are dozens of genes involved in its synthesis. In this thesis, it is associated with the proteins of PVDI maturation for the study. The process of mature PVD is as follows:In the intracellular cytoplasmic, NRPS catalytic ferribactin precursors into ferribactin. Ferribactin, is a non-fluorescent PVDI precursor with an unformed chromophore. The molecule is transported across the inner membrane by the transporter PvdE. The chromophore is formed in the periplasm with catalyzing steps of PvdN, PvdO and PvdP in this process to form PVDI that has a fluorescent chromophore. PVDI is transported into by an unknown transporter.In order to clarify the role of PvdN, PvdO and PvdP in the pathway of PVD mature, we use molecular biology methods to cloning, expression and purification of the native protein PvdO, PvdN and selenomethionine protein, and these proteins have been crystallized and optimized. We got good single crystals, these data of these crystals are collected and processed with HKL2000, and with CCP4, COOT, Phenix, CNS and other structural biology software we solved the structures of PvdO and PvdN. By the same way, we also cloned, expressed and purified the protein PvdP, and optimized its crystal. Unfortunately, we can not get the data of the crystal. By structural analysis of the PvdN and PvdO, we hope to design experiments analysizing their functions, and finally, illuminating the catalytic mechanisms of PvdO and PvdN.