Biochemical and Cellular Characterization and Inhibitor Discovery of Pseudomonas aeruginosa 15-Lipoxygenase

Pseudomonas aeruginosa is an opportunistic pathogen that can cause nosocomial and chronic infections in immunotompromised patients. P. aeruginosa secretes a lipoxygenase, LoxA, but the biological role of this enzyme is-currently unknown. LoxA is poorly similar in sequence to both soybean LOX-1 (s15-LOX-1) and human 15-LOX-1 (37 and, 39%, respectively) yet has kinetics comparably fast versus these of s15-LOX-1 (at pH 6.5, K-cat, = 181 +/- 6 s(-1) and K-cat/K-m.716 +/- 2 mu M-1 s(-1)). LoxA is capable of efficiently catalyzing the peroxidation of a broad range of free fatty acid (FA) substrates.(e.g., AA and LA) with high positional specificity, indicating a 15-LOX. Its mechanism includes hydrogen atom abstraction [a kinetic isotope effect (KIE) of >30], yet LoxA. is a poor catalyst against phosphoester FAs, suggesting that LoxA is not involved in membrane decomposition. LoxA also does not react with 5- or 15-HETEs, indicating poor involvement in lipoxin production. A LOX high-throughput screen of the LOPAC library yielded a variety of low-micromolar inhibitors; however, none selectively targeted Loth over the human LOX isozymes. With respect to cellular activity, the level of LoxA expression is increased when P. aeruginosa undergoes the transition to a biofilin mode of growth, but LoxA is not required for biofilm growth on abiotic surfaces. However, LoxA does appear to be required for biofilm growth in association with the-host airway epithelium, suggesting, a role for LoxA in mediating bacterium-host interactions during, colonization.