Catalytic properties of rice alpha-oxygenase. A comparison with mammalian prostaglandin H synthases.
Long-chain fatty acids can be metabolized to C(n)(-1) aldehydes by alpha-oxidation in plants. The reaction mechanism of the enzyme has not been elucidated. In this study, a complete nucleotide sequence of fatty acid alpha-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 homogeneous 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 alpha-oxygenase formed no product having a lambda(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 alpha-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 alpha-oxygenase has a high similarity with that of PGHS. The rice alpha-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 alpha-oxygenase when palmitic acid 2-hydroperoxide was used as a substrate. From these findings, the catalytic resemblance between alpha-oxygenase and PGHS seems to be evident, although there still are differences in their substrate recognitions and peroxidation activities.
Koeduka
Takao
T
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan.
Matsui
Kenji
K
Akakabe
Yoshihiko
Y
Kajiwara
Tadahiko
T
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D020224:Expressed Sequence Tags; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000201:enzymology* / Q000235:genetics*; D010100:Oxygen / Q000378:metabolism; D010101:Oxygen Consumption; D010105:Oxygenases / Q000737:chemistry* / Q000378:metabolism; D010802:Phylogeny; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013997:Time Factors
D011993:Recombinant Fusion Proteins; D006861:Hydrogen Peroxide; D010105:Oxygenases; C426838:fatty acid alpha-hydroperoxylase; D011451:Prostaglandin-Endoperoxide Synthases; D010100:Oxygen