title: Biochemical and Cellular Characterization and Inhibitor Discovery of Pseudomonas aeruginosa 15-Lipoxygenase.
abstract: Pseudomonas aeruginosa is an opportunistic pathogen that can cause nosocomial and chronic infections in immunocompromised 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 those of s15-LOX-1 (at pH 6.5, Kcat = 181 ± 6 s(-1) and Kcat/KM = 16 ± 2 μM(-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 LoxA 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 biofilm 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California , Santa Cruz, California 95064, United States.
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Deschamps
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh , Pittsburgh, Pennsylvania 15219, United States.
authors.forename: Abiola F
authors.initials: AF
authors.lastname: Ogunsola
authors.affiliation: Department of Chemistry and Biochemistry, University of California , Santa Cruz, California 95064, United States.
authors.forename: J Brian
authors.initials: JB
authors.lastname: Jameson
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health , 9800 Medical Center Drive, MSC 3370, Bethesda, Maryland 20892, United States.
authors.forename: Adam
authors.initials: A
authors.lastname: Yasgar
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh , Pittsburgh, Pennsylvania 15219, United States.
authors.forename: Becca A
authors.initials: BA
authors.lastname: Flitter
authors.affiliation: Department of Chemistry and Biochemistry, University of California , Santa Cruz, California 95064, United States.
authors.forename: Cody J
authors.initials: CJ
authors.lastname: Freedman
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh , Pittsburgh, Pennsylvania 15219, United States.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Melvin
authors.affiliation: Department of Chemistry and Biochemistry, University of California , Santa Cruz, California 95064, United States.
authors.forename: Jason V M H
authors.initials: JV
authors.lastname: Nguyen
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health , 9800 Medical Center Drive, MSC 3370, Bethesda, Maryland 20892, United States.
authors.forename: David J
authors.initials: DJ
authors.lastname: Maloney
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health , 9800 Medical Center Drive, MSC 3370, Bethesda, Maryland 20892, United States.
authors.forename: Ajit
authors.initials: A
authors.lastname: Jadhav
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health , 9800 Medical Center Drive, MSC 3370, Bethesda, Maryland 20892, United States.
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh , Pittsburgh, Pennsylvania 15219, United States.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Bomberger
authors.affiliation: Department of Chemistry and Biochemistry, University of California , Santa Cruz, California 95064, United States.
authors.forename: Theodore R
authors.initials: TR
authors.lastname: Holman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D001093:Arachidonate 15-Lipoxygenase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism*; D007700:Kinetics; D016859:Lipoxygenase Inhibitors / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011817:Rabbits; D013379:Substrate Specificity
chemical_list: D006893:Hydroxyeicosatetraenoic Acids; D016859:Lipoxygenase Inhibitors; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D001093:Arachidonate 15-Lipoxygenase