Takao Koeduka

Kyoto University, Kioto, Kyōto, Japan

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Publications (22)79.18 Total impact

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    ABSTRACT: Prenyl residues confer divergent biological activities such as anti-pathogenic and anti-herbivorous activities on phenolic compounds, including flavonoids, coumarins, and xanthons. To date, about 1,000 prenylated phenolics have been isolated, with these compounds containing various prenyl residues. However, all currently described plant prenyltransferases (PTs) have been shown specific for dimethylallyl diphosphate (DMADP) as the prenyl donor, while most of the cDNAs encoding these genes have been isolated from the Leguminosae. In this study we describe the identification of a novel PT gene from Citrus limon, Cl-PT1, belonging to the homogentisate PT family. This gene encodes a PT that differs from other known PTs, including flavonoid-specific PTs, in polypeptide sequence. This membrane-bound enzyme was specific for geranyl diphosphate (GDP) as the prenyl donor and coumarin as the prenyl acceptor. Moreover, the gene product was targeted to plastid in plant cells. To our knowledge, this is the novel aromatic PT specific to GDP from citrus species.
    Plant physiology. 07/2014;
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    ABSTRACT: Furanocoumarins constitute a subfamily of coumarin compounds with important defense properties against pathogens and insects, as well as allelopathic functions in plants. Furanocoumarins are divided into two subgroups according to the alignment of the furan ring with the lactone structure; linear psoralen and angular angelicin derivatives. Determination of furanocoumarin type is based on the prenylation position of the common precursor to all furanocoumarins, umbelliferone, at C6 or C8, which gives rise to psoralen or angelicin derivatives, respectively. Here, we identified a membrane-bound prenyltransferase PcPT from parsley (Petroselinum crispum), and characterized the properties of the gene product. PcPT expression in different parsley tissues is increased by UV irradiation with a concomitant increase in furanocoumarin production. This enzyme has strict substrate specificity towards umbelliferone and dimethylallyl diphosphate, and a strong preference for the C6-position of the prenylated product (demethylsuberosin), leading to linear furanocoumarins. The C8-prenylated derivative (osthenol) is also formed, but to a much lesser extent. PcPT protein is targeted to the plastids in planta. Introduction of this PcPT in a coumarin-producing plant Ruta graveolens showed an increased consumption of endogenous umbelliferone. Expression of PcPT and a 4-coumaroyl-CoA 2'-hydroxylase gene in Nicotiana benthamiana, which does not produce furanocoumarins, resulted in demethylsuberosin formation, indicating that furanocoumarin production may be reconstructed in a metabolic engineering approach. These results demonstrate that a single prenyltransferase, such as PcPT, opens the path to linear furanocoumarins in parsley, but might also catalyse the synthesis of osthenol, the first intermediate committed to the angular furanocoumarin pathway, in other plants. This article is protected by copyright. All rights reserved.
    The Plant Journal 12/2013; · 6.58 Impact Factor
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    ABSTRACT: A variety of volatile phenylpropenes, C6 -C3 compounds are widely distributed in the plant kingdom, whereas prenylated phenylpropenes are limited to a few plant species. In this study, we analysed the volatile profiles from Illicium anisatum leaves and identified two O-prenylated phenylpropenes, 4-allyl-2-methoxy-1-[(3-methylbut-2-en-1-yl)oxy]benzene [O-dimethylallyleugenol (9)] and 5-allyl-1,3-dimethoxy-2-(3-methylbut-2-en-1-yl)oxy]benzene [O-dimethylallyl-6-methoxyeugenol (11)] as major constituents. The structure-activity relationship of a series of eugenol derivatives showed that specific phenylpropenes, including eugenol (1), isoeugenol (2) and 6-methoxyeugenol (6), with a phenolic hydroxy group had antifungal activity for a fungal pathogen, whereas guaiacol, a simple phenolic compound, and allylbenzene had no such activity. The eugenol derivatives that exhibited antifungal activity, in turn, had no significant toxicant property for mite oviposition. Interestingly, O-dimethylallyleugenol (9) in which the phenolic oxygen was masked with a dimethylallyl group exhibited a specific, potent oviposition deterrent activity for mites. The sharp contrast in structural requirements of phenylpropenes suggested distinct mechanisms underlying the two biological activities and the importance of a phenolic hydroxy group and its dimethylallylation for the structure-based design of new functional properties of phenylpropenes.
    Plant Biology 07/2013; · 2.32 Impact Factor
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    ABSTRACT: Eugenol, a volatile phenylpropene found in many plant species, exhibits antibacterial and acaricidal activities. This study attempted to modify the production of eugenol and its glycosides by introducing petunia coniferyl alcohol acetyltransferase (PhCFAT) and eugenol synthase (PhEGS) into hybrid aspen. Gas chromatography analyses revealed that wild-type hybrid aspen produced small amount of eugenol in leaves. The heterologous overexpression of PhCFAT alone resulted in up to 7-fold higher eugenol levels and up to 22-fold eugenol glycoside levels in leaves of transgenic aspen plants. The overexpression of PhEGS alone resulted in a subtle increase in either eugenol or eugenol glycosides, and the overexpression of both PhCFAT and PhEGS resulted in significant increases in the levels of both eugenol and eugenol glycosides which were nonetheless lower than the increases seen with overexpression of PhCFAT alone. On the other hand, overexpression of PhCFAT in transgenic Arabidopsis and tobacco did not cause any synthesis of eugenol. These results indicate that aspen leaves, but not Arabidopsis and tobacco leaves, have a partially active pathway to eugenol that is limited by the level of CFAT activity and thus the flux of this pathway can be increased by the introduction of a single heterologous gene.
    Biochemical and Biophysical Research Communications 06/2013; 436(1):73-78. · 2.41 Impact Factor
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    ABSTRACT: Coumarins, a large group of polyphenols, play important roles in the defense mechanisms of plants, and they also exhibit various biological activities beneficial to human health, often enhanced by prenylation. Despite the high abundance of prenylated coumarins in citrus fruits, there has been no report on coumarin-specific prenyltransferase activity in citrus. In this study, we detected both O- and C-prenyltransferase activities of coumarin substrates in a microsome fraction prepared from lemon (Citrus limon) peel, where large amounts of prenylated coumarins accumulate. Bergaptol was the most preferred substrate out of various coumarin derivatives tested, and geranyl diphosphate (GPP) was accepted exclusively as prenyl donor substrate. Further enzymatic characterization of bergaptol 5-O-geranyltransferase activity revealed its unique properties: apparent K(m) values for GPP (9 µM) and bergaptol (140 µM) and a broad divalent cation requirement. These findings provide information towards the discovery of a yet unidentified coumarin-specific prenyltransferase gene.
    Bioscience Biotechnology and Biochemistry 07/2012; 76(7):1389-93. · 1.27 Impact Factor
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    ABSTRACT: Phenylbutanone raspberry ketone, accumulating in the mature fruits of raspberry (Rubus idaeus), imparts the characteristic aroma to the fruits. Here we describe the isolation and characterization of raspberry ketone/zingerone synthase 1 (RZS1), which catalyzed the NADPH-dependent reduction of 4-hydroxybenzalacetone and 3-methoxy-4-hydroxybenzalacetone to raspberry ketone and zingerone (the latter not found in raspberry), respectively. Its apparent K(m) values for 4-hydroxybenzalacetone and NADPH were 88 μM and 202 μM, respectively. RZS1 preferred 4-hydroxybenzalacetone to 3-methoxy-4-hydroxybenzalacetone as a substrate by a factor of 1.7, and showed a 6-fold preference for 4-hydroxybenzalacetone over p-coumaraldehyde, and no activity for coniferaldehyde. Expression analysis of the RZS1 gene throughout the plant revealed that its transcript level was highest in mature fruits. We conclude that RZS1 is responsible for hydrogenation of the α,β-unsaturated double bond of phenylbutenones, the final step of the raspberry ketone biosynthesis, in the raspberry fruits.
    Biochemical and Biophysical Research Communications 08/2011; 412(1):104-8. · 2.41 Impact Factor
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    ABSTRACT: Flavonoids are natural compounds found in many plants, including the important fruit crop, tomato. Prenylated flavonoids consist of a large group of compounds, which often exhibit antitumour, antibacterial and/or anti-androgen activities. In this study, we engineered the biosynthesis of prenylated flavonoids using a Streptomyces prenyltransferase HypSc (SCO7190) possessing broad-range substrate specificity, in tomato as a host plant. LC/MS/MS analysis demonstrated the generation of 3'-dimethylallyl naringenin in tomato fruits when recombinant HypSc protein was targeted to the plastids, whereas the recombinant protein hardly produced this compound in vitro. This is the first report confirming the accumulation of a prenylated flavonoid using a bacterial prenyltransferase in transgenic plants, and our results suggest that the product specificities of prenyltransferases can be significantly influenced by the host plant.
    Plant Biology 03/2011; 13(2):411-5. · 2.32 Impact Factor
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    ABSTRACT: Floral scent has been extensively investigated in plants of the South American genus Petunia. Flowers of Petunia integrifolia emit mostly benzaldehyde, while flowers of Petunia axillaris subsp. axillaris emit a mixture of volatile benzenoid and phenylpropanoid compounds that include isoeugenol and eugenol. Flowers of the artificial hybrid Petunia hybrida, a cross between P. integrifolia and P. axillaris, emit a similar spectrum of volatiles as P. axillaris subsp. axillaris. However, the flowers of P. axillaris subsp. parodii emit neither isoeugenol nor eugenol but contain high levels of dihydroconiferyl acetate in the petals, the main scent-synthesizing and scent-emitting organs. We recently showed that both isoeugenol and eugenol in P. hybrida are biosynthesized from coniferyl acetate in reactions catalyzed by isoeugenol synthase (PhIGS1) and eugenol synthase (PhEGS1), respectively, via a quinone methide-like intermediate. Here we show that P. axillaris subsp. parodii has a functional EGS gene that is expressed in flowers, but its IGS gene contains a frame-shift mutation that renders it inactive. Despite the presence of active EGS enzyme in P. axillaris subsp. parodii, in the absence of IGS activity the coniferyl acetate substrate is converted by an as yet unknown enzyme to dihydroconiferyl acetate. By contrast, suppressing the expression of PhIGS1 in P. hybrida by RNA interference also leads to a decrease in isoeugenol biosynthesis, but instead of the accumulation of dihydroconiferyl acetate, the flowers synthesize higher levels of eugenol.
    The Plant Journal 03/2009; 58(6):961-9. · 6.58 Impact Factor
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    ABSTRACT: The phenylpropene t-anethole imparts the characteristic sweet aroma of anise (Pimpinella anisum, family Apiaceae) seeds and leaves. Here we report that the aerial parts of the anise plant accumulate t-anethole as the plant matures, with the highest levels of t-anethole found in fruits. Although the anise plant is covered with trichomes, t-anethole accumulates inside the leaves and not in the trichomes or the epidermal cell layer. We have obtained anise cDNA encoding t-anol/isoeugenol synthase 1 (AIS1), an NADPH-dependent enzyme that can biosynthesize t-anol and isoeugenol (the latter not found in anise) from coumaryl acetate and coniferyl acetate, respectively. In addition, we have obtained a cDNA encoding S-[methyl-14C]adenosyl-l-methionine:t-anol/isoeugenol O-methyltransferase 1 (AIMT1), an enzyme that can convert t-anol or isoeugenol to t-anethole or methylisoeugenol, respectively, via methylation of the para-OH group. The genes encoding AIS1 and AIMT1 were expressed throughout the plant and their transcript levels were highest in developing fruits. The AIS1 protein is 59% identical to petunia (Petunia hybrida) isoeugenol synthase 1 and displays apparent Km values of 145 microm for coumaryl acetate and 230 microm for coniferyl acetate. AIMT1 prefers isoeugenol to t-anol by a factor of 2, with Km values of 19.3 microm for isoeugenol and 54.5 microm for S-[methyl-14C]adenosyl-l-methionine. The AIMT1 protein sequence is approximately 40% identical to basil (Ocimum basilicum) and Clarkia breweri phenylpropene O-methyltransferases, but unlike these enzymes, which do not show large discrimination between substrates with isomeric propenyl side chains, AIMT1 shows a 10-fold preference for t-anol over chavicol and for isoeugenol over eugenol.
    Plant physiology 12/2008; 149(1):384-94. · 6.56 Impact Factor
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    ABSTRACT: Many plants synthesize the volatile phenylpropene compounds eugenol and isoeugenol to serve in defense against herbivores and pathogens and to attract pollinators. Clarkia breweri flowers emit a mixture of eugenol and isoeugenol, while Petunia hybrida flowers emit mostly isoeugenol with small amounts of eugenol. We recently reported the identification of a petunia enzyme, isoeugenol synthase 1 (PhIGS1) that catalyzes the formation of isoeugenol, and an Ocimum basilicum (basil) enzyme, eugenol synthase 1 (ObEGS1), that produces eugenol. ObEGS1 and PhIGS1 both utilize coniferyl acetate, are 52% sequence identical, and belong to a family of NADPH-dependent reductases involved in secondary metabolism. Here we show that C. breweri flowers have two closely related proteins (96% identity), CbIGS1 and CbEGS1, that are similar to ObEGS1 (58% and 59% identity, respectively) and catalyze the formation of isoeugenol and eugenol, respectively. In vitro mutagenesis experiments demonstrate that substitution of only a single residue can substantially affect the product specificity of these enzymes. A third C. breweri enzyme identified, CbEGS2, also catalyzes the formation of eugenol from coniferyl acetate and is only 46% identical to CbIGS1 and CbEGS1 but more similar (>70%) to other types of reductases. We also found that petunia flowers contain an enzyme, PhEGS1, that is highly similar to CbEGS2 (82% identity) and that converts coniferyl acetate to eugenol. Our results indicate that plant enzymes with EGS and IGS activities have arisen multiple times and in different protein lineages.
    The Plant Journal 06/2008; 54(3):362-74. · 6.58 Impact Factor
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    ABSTRACT: Oxylipin metabolism represents one of the important hormonal and defensive mechanisms employed by plants, algae, or animals. It begins mostly with the reaction of lipoxygenases (LOXs), which catalyze the oxygenation of polyunsaturated fatty acids to form the corresponding hydroperoxides. At present, little information about LOXs in cyanobacteria has been reported. Herein, we report the first isolation of two LOX genes (NpLOX1 and NpLOX2) from a cyanobacterium, Nostoc punctiforme ATCC29133. Incubations of recombinant NpLOX1 and NpLOX2 proteins expressed in Eschelichia coli with linoleic acid resulted in the predominant formation of linoleic acid 13-S-hydroperoxide. Other C18 and C20 fatty acids could also be substrates for NpLOX enzymes. Phylogenetic analysis of NpLOX sequences showed that the NpLOX enzymes shared a high homology with LOX sequence of a bacterial pathogen, Pseudomonas aeruginosa, and these bacterial LOXs formed a subfamily distinct from those of plants, algae, and mammals.
    Current Microbiology 05/2007; 54(4):315-9. · 1.52 Impact Factor
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    ABSTRACT: Petunia flower petals emit large amounts of isoeugenol, which has been shown to be synthesized by isoeugenol synthase (PhIGS1) from an ester of coniferyl alcohol, hypothesized to be coniferyl acetate. This paper describes the identification and characterization of a novel petunia gene encoding an enzyme belonging to the BAHD acyltransferase family whose expression correlates with isoeugenol biosynthesis. RNAi suppression of this gene results in inhibition of isoeugenol biosynthesis. Biochemical characterization of the protein encoded by this gene showed that it has acetyltransferase activity and is most efficient with coniferyl alcohol among the alcohol substrates tested. Overall, these data support the conclusion that coniferyl acetate is the substrate of isoeugenol synthase.
    The Plant Journal 02/2007; 49(2):265-75. · 6.58 Impact Factor
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    ABSTRACT: Phenylpropenes, a large group of plant volatile compounds that serve in multiple roles in defense and pollinator attraction, contain a propenyl side chain. Eugenol synthase (EGS) catalyzes the reductive displacement of acetate from the propenyl side chain of the substrate coniferyl acetate to produce the allyl-phenylpropene eugenol. We report here the structure determination of EGS from basil (Ocimum basilicum) by protein x-ray crystallography. EGS is structurally related to the short-chain dehydrogenase/reductases (SDRs), and in particular, enzymes in the isoflavone-reductase-like subfamily. The structure of a ternary complex of EGS bound to the cofactor NADP(H) and a mixed competitive inhibitor EMDF ((7S,8S)-ethyl (7,8-methylene)-dihydroferulate) provides a detailed view of the binding interactions within the EGS active site and a starting point for mutagenic examination of the unusual reductive mechanism of EGS. The key interactions between EMDF and the EGS-holoenzyme include stacking of the phenyl ring of EMDF against the cofactor's nicotinamide ring and a water-mediated hydrogen-bonding interaction between the EMDF 4-hydroxy group and the side-chain amino moiety of a conserved lysine residue, Lys132. The C4 carbon of nicotinamide resides immediately adjacent to the site of hydride addition, the C7 carbon of cinnamyl acetate substrates. The inhibitor-bound EGS structure suggests a two-step reaction mechanism involving the formation of a quinone-methide prior to reduction. The formation of this intermediate is promoted by a hydrogen-bonding network that favors deprotonation of the substrate's 4-hydroxyl group and disfavors binding of the acetate moiety, akin to a push-pull catalytic mechanism. Notably, the catalytic involvement in EGS of the conserved Lys132 in preparing the phenolic substrate for quinone methide formation through the proton-relay network appears to be an adaptation of the analogous role in hydrogen bonding played by the equivalent lysine residue in other enzymes of the SDR family.
    PLoS ONE 02/2007; 2(10):e993. · 3.53 Impact Factor
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    ABSTRACT: Propenyl- and allyl-phenols, such as methylchavicol, p-anol and eugenol, have gained importance as flavoring agents and also as putative precursors in the biosynthesis of 9,9'-deoxygenated lignans, many of which have potential medicinal applications. In spite of several decades of investigation, however, the complete biosynthetic pathway to a propenyl/allylphenol had not yet been reported. We have subjected a Thai basil variety accumulating relatively large amounts of the simplest volatile allylphenol, methylchavicol, to in vivo administration of radiolabeled precursors and assays of protein preparations in vitro. Through these experiments, the biosynthesis of chavicol was shown to occur via the phenylpropanoid pathway to p-coumaryl alcohol. Various possibilities leading to deoxygenation of the latter were examined, including reduction of the side-chain double bond to form p-dihydrocoumaryl alcohol, followed by dehydration to afford chavicol, as well as formation of p-methoxycinnamyl alcohol, with further side-chain modification to afford methylchavicol. A third possibility studied was activation of the side-chain alcohol of p-coumaryl alcohol, e.g.via esterification, to form a more facile leaving group via reductive elimination. The latter was shown to be the case using p-coumaryl esters as potential substrates for a NAD(P)H-dependent reductase to afford chavicol, which is then O-methylated to afford methylchavicol.
    Organic & Biomolecular Chemistry 08/2006; 4(14):2733-44. · 3.57 Impact Factor
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    ABSTRACT: Phenylpropenes such as chavicol, t-anol, eugenol, and isoeugenol are produced by plants as defense compounds against animals and microorganisms and as floral attractants of pollinators. Moreover, humans have used phenylpropenes since antiquity for food preservation and flavoring and as medicinal agents. Previous research suggested that the phenylpropenes are synthesized in plants from substituted phenylpropenols, although the identity of the enzymes and the nature of the reaction mechanism involved in this transformation have remained obscure. We show here that glandular trichomes of sweet basil (Ocimum basilicum), which synthesize and accumulate phenylpropenes, possess an enzyme that can use coniferyl acetate and NADPH to form eugenol. Petunia (Petunia hybrida cv. Mitchell) flowers, which emit large amounts of isoeugenol, possess an enzyme homologous to the basil eugenol-forming enzyme that also uses coniferyl acetate and NADPH as substrates but catalyzes the formation of isoeugenol. The basil and petunia phenylpropene-forming enzymes belong to a structural family of NADPH-dependent reductases that also includes pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase.
    Proceedings of the National Academy of Sciences 07/2006; 103(26):10128-33. · 9.81 Impact Factor
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    ABSTRACT: Plant fatty acid alpha-dioxygenases (DOXs) catalyze the stereospecific conversion of fatty acids into the corresponding (R)-2-hydroperoxy fatty acids. In several plant species the corresponding gene was shown to be induced by pathogen infection, herbivore attack and environmental stresses. The precise signaling pathway accountable for the induction remains unidentified. In the present study, the effects of bacterial infection, oxidative- and heavy metal-stresses, and plant signaling molecules such as jasmonate, salicylic acid (SA), and ethylene (ET) on expression of a fatty acid alpha-DOX (OsDOX) gene in rice seedlings were investigated. The rice blight bacteria, Xanthomonas oryzae, elicited the accumulation of OsDOX transcripts in the leaves in both the incompatible and compatible interactions. Treating the seedling with CuSO4 also significantly enhanced the OsDOX expression. The degree of induction was shown to be mostly parallel to the level of endogenous jasmonic acid (JA) in the leaves. In contrast, SA was little effective and ET down-regulated not only the OsDOX expression but also the endogenous level of JA in rice seedlings. These results suggested that the OsDOX gene expression by a variety of stress-related stimuli was activated through jasmonate signaling and was negatively regulated by ET.
    Journal of Plant Physiology 09/2005; 162(8):912-20. · 2.70 Impact Factor
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    ABSTRACT: The cDNA from barley coding FA hydroperoxide lyase (HPL) was cloned. A recombinant protein derived from the cDNA was expressed in Escherichia coli as an active enzyme. Thus far, there have been no reports on HPL in monocotyledonous plants. The recombinant protein was shown to be most active to linolenic acid 13-hydroperoxide, followed by linoleic acid 13-hydroperoxide. 9-Hydroperoxides of the FA could not be substrates for the recombinant HPL. The activity was dramatically enhanced in the presence of a detergent and/or a salt in the reaction mixture. At the same time, the kinetics of the reaction, including inactivation and the Vmax value of the HPL, were also greatly modulated, depending on the concentration of a monovalent cation and/or a detergent in the reaction mixture. These results suggest that these effectors induced a conformational change in barley HPL, resulting in an improvement in substrate binding and in enzyme activity.
    Lipids 12/2003; 38(11):1167-72. · 2.56 Impact Factor
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    ABSTRACT: 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.
    Journal of Biological Chemistry 07/2002; 277(25):22648-55. · 4.65 Impact Factor
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    ABSTRACT: Long-chain fatty acids can be metabolized to Cn −1 aldehydes by α-oxidation in plants. The reaction mechanism of the enzyme has not been elucidated. In this study, a complete nucleotide sequence of fatty acid α-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 (Cn), which is then spontaneously decarboxylated to form corresponding Cn −1 aldehyde. With linoleic or linoleic acids as a substrate, rice α-oxygenase formed no product having aλ 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 α-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 α-oxygenase has a high similarity with that of PGHS. The rice α-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 α-oxygenase when palmitic acid 2-hydroperoxide was used as a substrate. From these findings, the catalytic resemblance between α-oxygenase and PGHS seems to be evident, although there still are differences in their substrate recognitions and peroxidation activities.
    Journal of Biological Chemistry 06/2002; 277(25):22648-22655. · 4.65 Impact Factor
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    ABSTRACT: Genes encoding an alpha-oxygenase, in Nicotiana tabacum and Arabidopsis thaliana, have been recently isolated. However, the reaction mechanism of the enzyme has not so far been elucidated. In this study, a cDNA encoding the fatty acid alpha-oxygenase gene in rice plants was isolated. The deduced amino acid sequence showed high similarity (63.6%) to that of N. tabacum. The gene was cloned into an expression vector system, pQE-30, and expressed in Escherichia coli as a host cell. Palmitic acid as a substrate was incubated with the lysate of the cells, and the products were analysed by HPLC. A compound formed predominantly by the recombinant enzyme was shown to be n-pentadecanal. By incubating the mixture at 0 degrees C, 2-hydroperoxypalmitic acid was detected as a primary product and little formation of n-pentadecanal was detected. Furthermore, uptake of molecular oxygen was observed with an oxygen electrode. This indicated that the gene in rice plants encodes the alpha-oxygenase.
    Biochemical Society Transactions 01/2001; 28(6):765-8. · 2.59 Impact Factor

Publication Stats

283 Citations
79.18 Total Impact Points

Institutions

  • 2011–2014
    • Kyoto University
      • Research Institute for Sustainable Humanosphere
      Kioto, Kyōto, Japan
  • 2006–2009
    • University of Michigan
      • Department of Molecular, Cellular and Developmental Biology
      Ann Arbor, MI, United States
  • 2001–2007
    • Yamaguchi University
      • Department of Biological Chemistry
      Yamaguchi-shi, Yamaguchi-ken, Japan