Expression and Characterization of P. aeruginosa Cytochrome P450 CYP168A1

Abstract

BACKGROUND Pseudomonas aeruginosa (P. aeruginosa) is an encapsulated gram-negative bacterium regularly associated with the persistent opportunistic infections of the lungs of cystic fibrosis patients. Further, it is one of six pathogens that are the major causes of nosocomial infections in the United States and important worldwide due to increasing levels of multidrug resistance. Cytochrome P450 (CYP) from pathogenic bacteria such as M. tuberculosis have been defined as virulence factors and investigated for the ability to metabolize antibacterial drugs as well their roles in both cholesterol catabolism and fatty acid metabolism. However, the four putative P. aeruginosa CYPs have yet to be characterized. HYPOTHESIS P. aeruginosa CYPs regulate the bacterial metabolism of cholesterol, fatty acids and antibiotics resulting in selective advantages in both growth and the perpetuation of opportunistic bacterial colonization. METHODS The DNA sequence for the putative CYP168A1 was cloned and expressed in E. coli resulting in the expression of an approximately 49kDa protein. Total CYP168A1 was then assayed by the ferrous CO versus ferric difference spectrum resulting in 315 nmole/L of protein at a final concentration of 30µM. UV-visible spectroscopy was then utilized to characterize Type II binding to antifungal azoles, Type I binding to various fatty acids and reverse Type I binding to cholesterol. Further, CYP activity was investigated using LC-MS/MS to identify hydroxylation of fatty acid ligands. RESULTS UV-visible spectroscopy using revealed differential Type II binding affinities for ketoconazole, miconazole, and econazole defined by a Bmax of 0.045, 0.036 and 0.026 and a Kd of 0.697, 0.882 and 2.457µM, respectively. Interestingly, CYP168A1 showed no affinity for fluconazole. The Type I binding affinities of 1µM CYP168A1 have increasing Bmax and affinity corresponding to increasing carbon chain length for saturated fatty acids: lauric acid (12 carbons), myristic acid (14 carbons), and stearic acid (18 carbons). The binding of the monounsaturated oleic acid (18 carbons) results in Bmax = 0.0992 and Kd = 0.374µM and binding the polyunsaturated arachidonic acid (20 carbons) results in a Bmax = 0.058 and Kd = 0.9595µM. Further, CYP168A1 has been shown by LC-MS/MS to successfully hydroxylate lauric acid at the Ω and Ω-1 position utilizing either oxygen surrogates tert-butyl or cumene hydroperoxide. Initial work using spinach ferredoxin and ferredoxin reductase surrogates have also shown successful CYP168A1 metabolism of lauric acid. CONCLUSIONS As an initial step in defining the possible role that CYPs may play in P. aeruginosa growth and pathogenesis, a functional CYP168A1 was expressed in E. coli and characterized. The ability to differentially bind several fatty acids leads us to conclude that this CYP may be involved in energy metabolism. However, the ability of CYP168A1 to bind arachidonic acid leads us to postulate that CYP168A1 may also actively inhibit host lipid peroxidation shown to kill other opportunistic pathogens.