Siderophores are low molecular weight iron chelators produced by bacteria to scavenge iron from the environment, and are frequently required for bacterial virulence. In iron limiting environments such as the human host, if the production of the siderophore is disrupted, the bacteria cannot acquire the iron required for growth and survival. Pseudomonas aeruginosa are notoriously antibiotic-resistant and are able to colonize immuno-compromised patients and the lungs of patients with cystic fibrosis. The long-term goal of the research in this laboratory is to provide a detailed mechanistic and structural understanding of enzymes in the siderophore biosynthetic pathways that are targets for drug discovery. Toward that end, we propose to investigate the nonribosomal peptide synthetase accessory enzymes that produce salicylate, a precursor incorporated into the siderophore pyochelin from the bacteria P. aeruginosa. The two enzymes to be studied, the isochorismate synthase (PchA) and isochorismate-pyruvate lyase (PchB), are ideal model systems for making fundamental advances in understanding the reaction pathways of catalysis. We will use a multifaceted approach that includes enzyme kinetic analysis, structural biology and computational enzymology, and identify inhibitors of the enzymes through high-throughput screening. Our first specific aim will be to determine the relative catalytic contributions of a reactive substrate conformation and electrostatic transition state stabilization in the pericyclic reaction mechanisms performed by the isochorismate- pyruvate lyase that has adventitious chorismate mutase activity. We will define the structure- function relationships of the Mg+2 dependent isochorismate synthase, a member of the MST family of proteins in our second aim. Finally, our third aim is to identify small molecule inhibitors of salicylate production, and determine their mode of action.
Pseudomonas aeruginosa is a dangerous opportunistic pathogen that is a common cause of infections in susceptible hosts, including cancer patients undergoing chemotherapy, AIDS patients, those with immune deficiencies, cystic fibrosis patients, burn patients and other at risk individuals including infants. The goal of this work is to provide a fundamental understanding of two enzymes that promote virulence in P. aeruginosa so that this information can be exploited to generate new antimicrobial drugs.
|Meneely, Kathleen M; Luo, Qianyi; Riley, Andrew P et al. (2014) Expanding the results of a high throughput screen against an isochorismate-pyruvate lyase to enzymes of a similar scaffold or mechanism. Bioorg Med Chem 22:5961-9|
|Meneely, Kathleen M; Luo, Qianyi; Lamb, Audrey L (2013) Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities. Arch Biochem Biophys 539:70-80|
|Meneely, Kathleen M; Luo, Qianyi; Dhar, Prajnaparamita et al. (2013) Lysine221 is the general base residue of the isochorismate synthase from Pseudomonas aeruginosa (PchA) in a reaction that is diffusion limited. Arch Biochem Biophys 538:49-56|
|Olucha, Jose; Ouellette, Andrew N; Luo, Qianyi et al. (2011) pH Dependence of catalysis by Pseudomonas aeruginosa isochorismate-pyruvate lyase: implications for transition state stabilization and the role of lysine 42. Biochemistry 50:7198-207|
|Lamb, Audrey L (2011) Pericyclic reactions catalyzed by chorismate-utilizing enzymes. Biochemistry 50:7476-83|
|Luo, Qianyi; Meneely, Kathleen M; Lamb, Audrey L (2011) Entropic and enthalpic components of catalysis in the mutase and lyase activities of Pseudomonas aeruginosa PchB. J Am Chem Soc 133:7229-33|