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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.

SUBMITTER: Koszelak-Rosenblum M

PROVIDER: S-EPMC2529013 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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His-311 and Arg-559 are key residues involved in fatty acid oxygenation in pathogen-inducible oxygenase.

Koszelak-Rosenblum Mary M Krol Adam C AC Simmons Danielle M DM Goulah Christopher C CC Wroblewski Liliana L Malkowski Michael G MG

The Journal of biological chemistry 20080702 36

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 align ...[more]

PMID: 18596034

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Dataset Information

His-311 and Arg-559 are key residues involved in fatty acid oxygenation in pathogen-inducible oxygenase.

Publications

His-311 and Arg-559 are key residues involved in fatty acid oxygenation in pathogen-inducible oxygenase.

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