Catalytic Properties of Rice α-Oxygenase

Abstract

Long-chain fatty acids can be metabolized to C n؊1 aldehydes by ␣-oxidation in plants. The reaction mechanism of the enzyme has not been elucidated. In this study, a complete nucleotide sequence of fatty acid ␣-oxygenase gene in rice plants (Oryza sativa) was isolated. The deduced amino acid sequence showed some similarity with those of mammalian prostaglandin H synthases (PGHSs). The gene was expressed in Escherichia coli and purified to apparently homogenous state. It showed the highest activity with linoleic acid and predominantly formed 2-hydroperoxide of the fatty acid (C n ), which is then spontaneously decarboxylated to form corresponding C n؊1 aldehyde. With linoleic or linoleic acids as a substrate, rice ␣-oxygenase formed no product having a max at approximately 234 nm, which indicated that the enzyme could not oxygenize the pentadiene system in the substrate. The spectroscopic feature of the purified enzyme in its ferrous state is similar to that of mammalian PGHS, whereas that of dithionite-reduced state showed significant difference. Site-directed mutagenesis revealed that His-158, Tyr-380, and Ser-558 were essential for the ␣-oxygenase activity. These residues are conserved in PGHS and known as a heme ligand, a source of a radical species to initiate oxygenation reaction and a residue involved in substrate binding, respectively. This finding suggested that the initial step of the oxygenation reaction in ␣-oxygenase has a high similarity with that of PGHS. The rice ␣-oxygenase activity was inhibited by imidazole but hardly inhibited by nonsteroidal anti-inflammatory drugs, such as aspirin, ibuprofen, and flurbiprofen, which are known as typical PGHS inhibitors. In addition, peroxidase activity could not be detected with ␣-oxygenase when palmitic acid 2-hydroperoxide was used as a substrate. From these findings, the catalytic resemblance between ␣-oxygenase and PGHS seems to be evident, although there still are differences in their substrate recognitions and peroxidation activities.

Multifrequency Electron Paramagnetic Resonance Characterization of PpoA, a CYP450 Fusion Protein that Catalyzes Fatty Acid Dioxygenation

“…14 Tyrosyl radicals have also been implicated in other heme-containing peroxidases 21 and dioxygenases. 22,23 EPR spectroscopy and particularly high-field EPR 24 have emerged as an essential tool to directly detect the formation of protein radicals and to identify the nature of the amino acid site. Furthermore, EPR spectroscopy provides a detailed characterization of the electronic structures of the radical intermediates, which correlates with its redox properties and hydrogen bond interactions.…”

His-311 and Arg-559 Are Key Residues Involved in Fatty Acid Oxygenation in Pathogen-inducible Oxygenase

“…The resulting 2R-hydroperoxides undergo spontaneous decarboxylation to shorter aldehydes and fatty acids (11,14). Using stereospecifically deuterated LA, Hamberg and colleagues (16) showed that the dioxygenation catalyzed by PIOX resulted in the stereoselective removal of the 2-pro-R hydrogen from carbon-2 of the fatty acid substrate, followed by the stereospecific addition of molecular oxygen.…”

“…PIOX belongs to a larger family of heme-containing proteins that oxygenate fatty acids (4), which include the mammalian cyclooxygenases (COX-1 and COX-2; (5)), linoleate diol synthase (LDS) from the fungus Gaeumannomyces graminis (6,7), and a Pseudomonas alcalignes protein of unknown function encoded by OrfX (8). PIOX has also been identified in many plant species, including Nicotiana attenuata (9), Nicotiana tabacum (3), Arabidopsis thaliana (3,10), O. sativa (11), Capsicum annuum (12), and Lycopersicon esculentum (13).…”

“…PIOX belongs to a larger family of heme-containing proteins that oxygenate fatty acids (4), which include the mammalian cyclooxygenases (COX-1 and COX-2; (5)), linoleate diol synthase (LDS) from the fungus Gaeumannomyces graminis (6, 7), and a Pseudomonas alcalignes protein of unknown function encoded by OrfX (8). PIOX has also been identified in many plant species, including Nicotiana attenuata (9), Nicotiana tabacum (3), Arabidopsis thaliana (3, 10), O. sativa (11), Capsicum annuum (12), and Lycopersicon esculentum (13).PIOX utilizes stereoselective oxygenation to convert linoleic acid (LA) (18:2, n-6) and other fatty acid substrates to their corresponding 2R-hydroperoxides, generating a novel class of oxylipins (3,11,14,15). The resulting 2R-hydroperoxides undergo spontaneous decarboxylation to shorter aldehydes and fatty acids (11,14).…”

“…PIOX utilizes stereoselective oxygenation to convert linoleic acid (LA) (18:2, n-6) and other fatty acid substrates to their corresponding 2R-hydroperoxides, generating a novel class of oxylipins (3,11,14,15). The resulting 2R-hydroperoxides undergo spontaneous decarboxylation to shorter aldehydes and fatty acids (11,14).…”