title: His-311 and Arg-559 are key residues involved in fatty acid oxygenation in pathogen-inducible oxygenase.
abstract: Pathogen-inducible oxygenase (PIOX) oxygenates fatty acids into 2R-hydroperoxides. PIOX belongs to the fatty acid alpha-dioxygenase family, which exhibits homology to cyclooxygenase enzymes (COX-1 and COX-2). Although these enzymes share common catalytic features, including the use of a tyrosine radical during catalysis, little is known about other residues involved in the dioxygenase reaction of PIOX. We generated a model of linoleic acid (LA) bound to PIOX based on computational sequence alignment and secondary structure predictions with COX-1 and experimental observations that governed the placement of carbon-2 of LA below the catalytic Tyr-379. Examination of the model identified His-311, Arg-558, and Arg-559 as potential molecular determinants of the dioxygenase reaction. Substitutions at His-311 and Arg-559 resulted in mutant constructs that retained virtually no oxygenase activity, whereas substitutions of Arg-558 caused only moderate decreases in activity. Arg-559 mutant constructs exhibited increases of greater than 140-fold in Km, whereas no substantial change in Km was observed for His-311 or Arg-558 mutant constructs. Thermal shift assays used to measure ligand binding affinity show that the binding of LA is significantly reduced in a Y379F/R559A mutant construct compared with that observed for Y379F/R558A construct. Although Oryza sativa PIOX exhibited oxygenase activity against a variety of 14-20-carbon fatty acids, the enzyme did not oxygenate substrates containing modifications at the carboxylate, carbon-1, or carbon-2. Taken together, these data suggest that Arg-559 is required for high affinity binding of substrates to PIOX, whereas His-311 is involved in optimally aligning carbon-2 below Tyr-379 for catalysis.
authors.affiliation: Hauptman-Woodward Medical Research Institute, State University of New York, Buffalo, New York 14203, USA.
authors.forename: Mary
authors.initials: M
authors.lastname: Koszelak-Rosenblum
authors.forename: Adam C
authors.initials: AC
authors.lastname: Krol
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Simmons
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Goulah
authors.forename: Liliana
authors.initials: L
authors.lastname: Wroblewski
authors.forename: Michael G
authors.initials: MG
authors.lastname: Malkowski
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites / Q000502:physiology; D020134:Catalytic Domain / Q000502:physiology; D051545:Cyclooxygenase 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D019787:Linoleic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D020125:Mutation, Missense; D012275:Oryza / Q000201:enzymology*; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D008024:Ligands; D010940:Plant Proteins; D019787:Linoleic Acid; D049308:Dioxygenases; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2