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J. Biol. Chem., Vol. 277, Issue 25, 22648-22655, June 21, 2002

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Catalytic Properties of Rice -Oxygenase

A COMPARISON WITH MAMMALIAN PROSTAGLANDIN H SYNTHASES^*

Takao Koeduka, Kenji Matsui, Yoshihiko Akakabe, and Tadahiko Kajiwara

From the Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan

Received for publication, October 30, 2001, and in revised form, March 14, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Long-chain fatty acids can be metabolized to C_n1 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_n1 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Long-chain fatty acids can be metabolized by -, -, and -oxidation in both plant and animal tissues (1). The enzyme systems responsible for -oxidation have been isolated and extensively studied. It was confirmed that the enzymes localize on the mitochondria and peroxisomes. Cytochrome P450s in the endoplasmic reticulum catalyze the fatty acid -hydroxylation (2). There are at least three types of -oxidation systems. In mammals, -oxidation is essential for degradation of branched-chain fatty acids with a methyl group at the -position, which prevents the initial step of -oxidation. This reaction is carried out by phytanoyl-CoA hydroxylase, which needs 2-oxoglutarate as an essential cofactor (3). The mutation of a gene encoding this enzyme causes Refsum's disease in human. Phytanoyl-CoA hydroxylase can also act on straight-chain fatty acids; however, this activity seems to be important only in brain and nerve tissues. In Sphingomonas paucimobilis, a sphingolipid-rich and 2-hydroxymyristic acid-rich bacterium, the initial reaction of -oxidation is catalyzed by a novel type of cytochrome P450, fatty acid -hydroxylase (4). Fatty acid -hydroxylase requires hydrogen peroxide for the hydroxylation of myristic acid to produce 2-hydroxymyristic acid. In contrast, plants have a distinct type of -oxidation system. The addition of palmitic acid to the crude homogenate prepared from Ulva pertusa led to an increased yield of the corresponding (R)-2-hydroperoxy acid (5), which is further decarboxylated to form n-pentadecanal (Scheme 1). The fatty acid hydroperoxides as the reaction intermediates in plant -oxidation system were also proposed in higher plants, such as peas (Pisum sativum) leaf (6), germinating peanuts (Arachis hypogaea) (7), cucumbers (Cucumis sativus) (8), and potatoes (Solanum tuberosum) (9) as well as in marine green algae (U. pertusa). The enzyme system requires neither hydrogen peroxide nor 2-oxoglutarate, but it needs molecular oxygen to facilitate its catalytic reaction, which suggested that the enzyme involved in plant -oxidation system is distinct from those in mammals or bacteria.

Recently, a gene encoding pathogen-inducible oxygenase (PIOX)¹ and its homolog have been isolated in Nicotiana tabacum and Arabidopsis thaliana (10). The protein derived from the gene was expressed in insect cells and found to cause uptake of molecular oxygen in the presence of polyunsaturated fatty acids such as linoleic, linolenic, and arachidonic acids. Later, the protein was identified as fatty acid -oxygenase, which catalyzes the conversion of linoleic acid and the other fatty acids into the corresponding (R)-2-hydroperoxy fatty acids (11). Interestingly, the primary structures of plant -oxygenases show similarity with those of mammalian PGHSs, although the substrates and products of these two oxygenases are quite different from each other. So far, there has been no report on the catalytic properties of plant -oxygenases, and no one knows whether there exists some catalytic resemblance between plant -oxygenases and mammalian PGHSs. By a BLAST search on the rice EST data base, we noticed that one of the EST clones has high sequence similarity with tobacco PIOX. Fatty acid -oxygenase gene of the monocotyledonous plants has never been characterized. Thus, to elucidate the reaction mechanism of -oxidation and the function of a given amino acid residue for the reaction, the fatty acid -oxygenase gene of rice plants (Oryza sativa) was cloned and expressed in Escherichia coli, and the properties of the recombinant enzyme were analyzed.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plant Materials and Growth Condition-- Seeds of rice plants (O. sativa L. cv. Kinmaze) were sterilized with 10% sodium hypochlorite solution for 1 day and then grown hydroponically in a chamber at 28 °C under 14-h light/10-h dark photoperiod.

Expression and Purification of -Oxygenase-- EST clone S10043 from green shoots of rice (O. sativa L. cv. Nipponbare) was supplied by The Ministry of Agriculture, Forestry, and Fisheries DNA Bank (bank.dna.affrc.go.jp/). For the expression in E. coli, SacI site was introduced just before the initiation codon of the cDNA by PCR, and then the full-length of the open reading frame digested with SacI and XhoI was inserted into SacI-SalI site of an E. coli expression vector pQE-30 (Qiagen). The primers used for the PCR were 5'-ACGAGCTCATGGGTTCAGGACTCTTCAAGCC-3' (the initiation codon is underlined) as the sense primer and 5'-CCATATACTGCACTCCCATCCCACC-3' as the antisense primer. E. coli cells (strain M15) were transformed with the construct. An overnight culture (50 ml) of the transformant was inoculated into 500 ml of 2× YT medium supplemented with 100 µg/ml ampicillin and 30 µg/ml kanamycin. The cells were grown at 37 °C until the A₆₀₀ reached 0.6-0.8. Cultures were chilled to 25 °C, isopropyl-1-thio--D-galactopyranoside was added to a final concentration of 1 mM, and then the cells were further cultured for 12 h at 25 °C. Bacterial cells expressing the recombinant protein were harvested and suspended in 10 ml of 20 mM sodium phosphate (pH 7.0) containing 300 mM NaCl. The cells were lysed by using five 15-s pulses of sonication with a tip-type sonicator (UR-150P, Tomy Co., Tokyo, Japan). The lysate was centrifuged at 4000 × g for 10 min, and the resulting supernatant was recentrifuged at 100,000 × g for 60 min. The recombinant -oxygenase was solubilized with 8 ml/g wet weight of the cells of 20 mM sodium phosphate (pH 7.0) containing 300 mM NaCl and 0.1% Nonidet P-40 (buffer A) on ice for 45 min with stirring. The suspension was centrifuged at 100,000 × g for 60 min, and the supernatant was applied to a 1.6 × 1.5 cm TALON metal affinity column (CLONTECH Co.) preequilibrated with buffer A (pH 7.0). The column was washed with 100 ml of the buffer A at pH 7.0. Proteins were eluted from the column in buffer A at pH 5.0. The eluted fractions having O₂ uptake activity were pooled and immediately adjusted at pH 7.0.

Enzyme Assay and Protein Content-- Oxygen consumption was measured with a Clark-type oxygen electrode (YSI 5331, Yellow Springs Instrument) at 25 °C in 100 mM Hepes buffer (pH 7.2) with 1.8 ml of the total volume. The reaction was started by the addition of the enzyme to the reaction mixture containing 0.55 mM linoleic acid and 0.002% Nonidet P-40. The activity (1 katal) was defined as the quantity of enzyme catalyzing the consumption of 1 mol of O₂/sec at 25 °C. With some experiments, the aldehyde formed from the substrate was quantified with HPLC. Palmitic acid (500 nmol in total volume, 5 ml of the reaction mixture) was incubated with the enzyme for 40 min at 25 °C. The aldehyde (n-pentadecanal) formed was quantified as its hydrazone derivative as described below. In this case, the amount of aldehyde formed in 1 min was shown as the enzyme activity. Protein content was determined by the method of Bradford (12) with bovine serum albumin as a standard.

Product Analysis

Long-chain Aldehyde-- Reaction mixture consisting of 2 ml of linoleic acid solution (50 mM, suspended in 0.2% Nonidet P-40; final concentration, 100 µM), the enzyme solution corresponding to 0.95 µg of protein, and 100 mM Hepes buffer (pH 7.2) with 1 ml of the total volume were incubated at 25 °C for 10 min. Aldehydes formed during the incubation were analyzed by HPLC as their 2,4-dinitrophenylhydrazone derivatives. Reversed-phase HPLC analysis of the products was performed on LC-9A (Shimadzu) equipped with a Wakosil 5C8 column (4.6 mm × 250 mm) and with an SPD-6AV (Shimadzu) UV detector (detection at 350 nm). 2,4-dinitrophenylhydrazone derivatives were eluted by using a solvent system of acetonitrile:water:tetrahydrofuran (94:5:1, v/v) with a flow rate of 1 ml/min. The column temperature was 25 °C. Each hydrazone was assigned by comparing its retention time with the authentic one.

Fatty-acid Hydroperoxide-- Reaction mixture of 10 µl of palmitic acid solution (50 mM, suspended in 0.2% EtOH; final concentration, 500 µM), the enzyme solution corresponding to 0.95 µg of protein, and 100 mM Hepes buffer (pH 7.2) with 5 ml of the total volume were incubated on ice for 15 min. The hydroperoxides formed were converted into esters with 9-anthryldiazomethane (ADAM) reagent. The detection of the resultant ADAM esters of hydroperoxides was performed with reversed-phase HPLC as described previously (5). To reveal stereochemistry of 2-hydroperoxy acids, the hydroperoxy group of their ADAM esters was treated with (+)-Noe's reagent (13-15) in the presence of catalytic amount of p-toluenesulfonic acid in tetrahydrofuran, which resulted in the formation of peracetal derivatives of 9-anthrylmethyl,2-hydroperoxy carboxylate. HPLC of the derivatives was done as described previously (5). Racemic 2-hydroperoxypalmitic acid and (R)-2-hydroperoxypalmitic acid were synthesized as described previously (16).

Mutagenesis

In vitro mutagenesis was performed by using the QuikChange^TM site-directed mutagenesis kit (Stratagene) with the following primers (the bases changed are underlined): His¹⁵⁸-Gln sense primer (5'-ACAGTTCATGGTTCAGGACTGGATGGATCA-3') and antisense primer (5'-TGATCCATCCAGTCCTGAACCATGAACTGT-3'); His²⁷⁷-Gln sense primer (5'-TTTGTTAAGGAACAGAATGCAGTTTGTGAT-3') and antisense primer (5'-ATCACAAACTGCATTCTGTTCCTTAACAAA-3'); Tyr³⁸⁰-Phe sense primer (5'-TTTACCAGTGTTTTCAGAATGCACTCCCTA-3') and antisense primer (5'-TAGGGAGTGCATTCTGAAAACACTGGTAAA-3'); His³⁸³-Gln sense primer (5'-GTTTACAGAATGCAGTCCCTAATACCAAGC-3') and antisense primer (5'-GCTTGGTATTAGGGACTGCATTCTGTAAAC-3'); Ser⁵⁵⁸-Ala sense primer (5'-TTCATTTTAATGGCAGCAAGGAGGCTCGAA-3') and antisense primer (5'-TCGAGCCTCCTCCTTGCTGCCATTAAAATG-3'). Mutation was confirmed by DNA sequencing.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

-Oxidation System in Rice Seedlings-- In rice seedlings, it has been postulated that selective inactivation of alcohol dehydrogenase is accountable to fatty acid -oxidation activity (17), although it has not been known whether the activity is the same as those found in the other plants such as cucumber and bean (8, 18). When crude homogenate prepared from rice leaves was incubated with palmitic acid, n-pentadecanal was detected as a major product. The activity needed molecular oxygen (data not shown). Thus, it would be considered that rice leaves have the fatty-acid -oxidation system. No activity could be found in dry rice seeds and seedlings grown until day 7; thereafter, it increased gradually and became highest at 30-35 days after germination (~45 nmol of n-pentadecanal formed/min/g fresh weight from palmitic acid) (Fig. 1). Most of the activity was found in shoots, whereas roots had low activity. Sequential centrifugation revealed that the activity could be mainly recovered in a membrane fraction; however, no distinct localization in a certain organelle could be observed.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Developmental time course of -oxygenase activity in germinating rice plants. -Oxygenase activities in shoots () and roots () were determined by quantifying the aldehyde formed. Each data point represents the mean ± S.D. of two measurements.

Sequence Analysis-- In tobacco, the oxygenation on the -position of fatty acids was found to be carried out by a novel oxygenase named PIOX, which has a structural resemblance with mammalian PGHS (10). We found that an EST clone (S10043, the rice EST data base in MAFF DNA Bank) derived from green shoots of rice has high sequence similarity with tobacco PIOX. The clone has an insert of 2208 bp comprising 111 bp of 5'-untranslated leader sequence and 301 bp of 3'-untranslated sequence. Because a stop codon upstream of the putative initiation codon could be found in-frame, the cDNA seemed to be full-length. Kozack consensus sequence, 5'-GCCATGGG-3' (the initiation codon is underlined) was also found in an appropriate position. The open reading frame encodes a deduced protein of 619 amino acids with a calculated M_r of 70710 and a pI of 8.87 (Fig. 2A). The deduced amino acid sequence has a high similarity with those of tobacco and Arabidopsis PIOXs (63.6 and 61.2%, respectively). The rice sequence also shares sequence similarity with mammalian PGHSs, namely 25.7 and 31.6% amino acid identity with PGHS-1 of sheep (19) and PGHS-2 of mouse (20), respectively. Interestingly, the rice sequence also has high sequence homology with tomato Feebly gene, which is identified by transposon tagging (21). The insertional mutagenesis of the Feebly gene in tomato produced high anthocyanin levels, developed into small fragile plants, and were insensitive to the herbicide phosphinothiricin. Sequence analysis with PSORT algorithm (psort.nibb.ac.jp/form.html) suggested that the rice sequence has no signal to direct the protein to a distinct organelle. As shown in Fig. 2B, in a phylogenic tree of PGHS and PIOX homologs, the rice protein locates in a family of plant PIOXs but not in the family of Feebly proteins. Both the plant proteins are apparently distinct from the mammalian PGHSs. Linoleate diol synthase of the fungus Gaeumannomyces graminis, which also has the sequence similarity with a mammalian PGHS (22), locates separately in the tree.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Deduced amino acid sequence of rice -oxygenase (A) and a phylogenetic tree made with plant PIOXs, PGHSs, and Feeblys (B). Multialignment of the sequence was performed with ClustalW (hypernig.nig.ac.jp/homology/clustalw.shtml). The phylogenetic tree was made with Tree View PPC in a Phylip format. The scale bar (0.1) means 0.1 nucleotide substitutions per site. The sequences used making the tree are tobacco PIOX, rice PIOX (this study, GenBankTM accession number AAF64042), Arabidopsis PIOX, tomato Feebly, Arabidopsis Feebly, Neurospora Feebly, mouse PGHS-2, sheep PGHS-1, coral PGHS, and Gaeumannomyces linoleate diol synthase.

In sheep PGHS-1, His-309 is noted as one of the heme ligands. Tyr-385 is noted to form a tyrosyl radical, which initiates the PGHS reaction by abstracting the 13-pro-S hydrogen from arachidonate. They are conserved completely within the PGHS family. They were also found to be present in the rice sequence at the appropriate positions of His-277 and Tyr-380 (Fig. 3). The amino acid sequences surrounding them are also conserved, particularly near the Tyr-380. Another histidine residue (His-383), which is also reported to be essential to PGHS activity (His-388 in sheep PGHS-1), can be found at the same array. In addition, His-207 (in sheep PGHS-1), which is also essential for the PGHS activities (23), was also conserved in the rice sequence at the position His-158. However, the amino acid residue equivalent to Ser-530 (PGHS-1), which is known as the binding site for the substrate and the target residue for many NSAIDs (24), was not found at the appropriate position in the rice sequence. Although a serine residue could be found near the one conserved in mammalian PGHSs, the amino acid sequence around the residue showed low sequence similarity; therefore, this relevance seemed to have little significance. Epidermal growth factor-like domain found in PGHSs was absent in the rice -oxygenase sequence. In the rice sequence as well as in the other plant -oxygenase sequences, N-glycosylation site could not be found.

View larger version (66K): [in this window] [in a new window]   Fig. 3.   Comparison of rice -oxygenase with PGHSs, PIOXs, and Feeblys. The highlighted letters in black denote identical amino acid residues, and those highlighted in gray denote similar ones. Dashes indicate gaps introduced to optimize the alignment. Sequences compared were sheep PGHS-1, mouse PGHS-2, rice -oxygenase (RICE), tobacco PIOX (TOBACCO), Arabidopsis PIOX (ARAB), tomato Feebly (TOMATO), and Neurospora Feebly (NEUROSPORA). The amino acid residues mutated and discussed in this study are shown in red.

Purification of -Oxygenase-- To reveal enzymatic properties of rice -oxygenase, the corresponding cDNA was incorporated into an E. coli expression vector, and the recombinant protein was expressed and purified to apparent homogeneity on SDS-polyacrylamide gel electrophoresis (cf. Fig. 7). The recombinant protein was recovered with a membrane fraction and needed to be solubilized with a detergent. The purified -oxygenase had a specific activity of 0.162 microkatals/mg when oxygen uptake was followed with linoleic acid as a substrate. The specific activity was slightly lower than those reported for sheep PGHSs (ranging from 0.37 to 1.3 microkatals/mg) (25, 26). The molecular mass of the recombinant protein was estimated to be 70,000 kDa on SDS-PAGE, which is in good accordance with that calculated from the sequence.

Spectral Properties of -Oxygenase-- Absorption spectrum of the purified recombinant rice -oxygenase is shown in Fig. 4. It had a Soret absorption band with a maximum at 410 nm and shoulders at 529, 563, and 628 nm. The last one is characteristic of high spin heme species. This spectrophotometric feature was comparable with PGHS, which has the Soret / and charge-transfer transitions at 411, 501, and 633 nm (25, 27, 28). The molecular coefficient was 11.6 mM¹ cm¹ (410 nm). This value was somewhat lower than those reported for mammalian PGHSs (ranging from 123 to 165 mM¹ cm¹). Upon reduction of the enzyme with dithionite, only a slight change could be found at the Soret absorption band, namely the _max blue-shifted by only 1-2 nm and the molar extinct coefficient decreased slightly. On the contrary, the / and charge-transfer transitions changed significantly. With the reduced form, a broad band at ~507 nm was evident in the / band, and clear peaks at 636 and 661 nm appeared in the charge-transfer transitions. The spectrophotometric properties of reduced form of rice -oxygenase was totally different from those of mammalian PGHSs having peaks of 428, 530, and 558 nm for the Soret  and  bands, respectively (28). In short, the heme coordination state of ferrous form of the rice enzyme has a close resemblance with those of mammalian PGHSs, although that of ferric state is significantly different.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Absorption spectra of native and reduced form of recombinant -oxygenase. A, the spectrum of recombinant -oxygenase (1.29 mg/ml) in 20 mM Tris-HCl buffer (pH 7.0) containing 0.10% Nonidet P-40 was measured in the absence or after the addition of dithionite. In panel B, the region between 450 and 700 nm was enlarged. A representative spectra among those obtained with different preparations are shown.

Product and Substrate Specificities-- When the purified recombinant enzyme was reacted with linoleic acid at 25 °C, the formation of (8Z,11Z)-heptadecadienal, which is a fatty aldehyde with one methylene length shorter than the substrate, could be detected as shown in Fig. 5A. No formation of this compound could be detected when the E. coli cells harboring the expression plasmid without the insert were used instead. In algae as well as in higher plants (5), it has been reported that a long-chain fatty aldehyde would be formed through decarboxylation of the corresponding 2-hydroperoxy fatty acid (Scheme 1). 2-Hydroperoxy fatty acids are chemically unstable and have a half-life time of ~30 min in an aqueous buffer at 23 °C (11). Thus, to detect the unstable fatty acid 2-hydroperoxide, the fatty acid as a substrate was incubated with the purified enzyme on ice and the product was immediately esterified with ADAM. The resulting ADAM esters were analyzed by HPLC. When palmitic acid was incubated with the recombinant purified enzyme on ice, 2-hydroperoxide of palmitic acid was detected as a major product as shown in Fig. 5B. To elucidate the stereochemistry of the hydroperoxy group, the ADAM ester was treated with Noe's reagent and the derivative was separated with reversed phase HPLC analysis (5). As shown in Fig. 5C, the stereochemistry of the hydroperoxide was revealed to be (R)-configuration in almost 100% specificity. This stereochemistry is same as that of the -oxygenation enzyme obtained with various marine algae and tobacco plant. The recombinant enzyme per se has extremely low, if any, peroxidase activity to reduce the hydroperoxide. When palmitic acid 2-hydroperoxide was incubated with the recombinant enzyme under the presence of guaiacol, which is widely used as a co-substrate for peroxidase activity of PGHSs, a little increase in the amount of 2-hydroxide was detected. In the case of PGHSs, their peroxidase activities were intrinsically essential to complete their reaction to form prostaglandin H and also essential to make inactive ferrous form of the enzymes active through the formation of a tyrosyl radical that is needed to abstract a hydrogen atom from substrate fatty acid. Thus, the turnover rates of oxygenase pathway and peroxidase pathway are almost stoichiometrically same. In contrast, in the case of rice -oxygenase, fatty acid 2-hydroperoxide was the major product at least under the reaction condition employed here; therefore, the rice enzyme has little need to provoke peroxidase pathway. Nonetheless, at least one catalytic turnover of the peroxidase pathway must be essential to activate the oxygenase. As the proposed mechanism of PGHS action indicated (29), a single peroxidase turnover to produce the tyrosine radical may be sufficient to initiate several hundred cyclooxygenase turnovers. We failed to detect hydroxide as one of the products formed by the rice enzyme, probably because extremely low number of peroxidase turnover was employed during its catalysis.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   HPLC detection of the products formed by purified -oxygenase. A, the purified enzyme was incubated with linoleic acid as a substrate for 10 min at 25 °C. The product, (8Z,11Z)-heptadecadienal, was converted to the corresponding 2,4-dinitrophenylhydrazone derivative and analyzed by HPLC. B, after the reaction of recombinant -oxygenase with palmitic acid for 15 min on ice, the product formed is analyzed as fluorogenic ADAM derivative. C, 2-hydroperoxyhexadecanoic acid formed by the enzyme action was ADAM-esterified and converted into its peracetal derivative and then separated with reversed-phase HPLC (trace a). Each enantiomer can be separated when racemic 2-hydroperoxide was analyzed (trace b). Under the condition employed here (R)-enantiomer elutes fast and then (S)-enantiomer follows (5). Co-injection of the racemic hydroperoxide and enzymatic product enlarged solely the peak corresponding to (R)-enantiomer (trace c).

View larger version (9K): [in this window] [in a new window]   Scheme 1.   The enantioselective -hydroperoxylation of long-chain fatty acids in plants. Long-chain fatty acids undergo -oxidation to give 2-hydroperoxy fatty acids as intermediates, which then degrade to aldehydes non-enzymatically with releasing CO2.

Within palmitic, stearic, oleic, linoleic, and -linolenic acids, linoleic acid was revealed to be the best substrate for the rice -oxygenase, although the other fatty acids also have substantial levels of reactivities, which indicated that (1Z,4Z)-pentadiene system is not the essential prerequisite to be a substrate. With linoleic acid as a substrate, there is no change in the absorbance between 210 and 350 nm including absorption at 234 nm corresponding to the formation of a conjugated diene hydroperoxy moiety during the reaction.² This finding indicated that rice -oxygenase could not abstract the bisallylic proton to form conjugated diene hydroperoxide but exclusively abstract -proton.

The Reaction Progress Curves of -Oxygenase-- When the reaction of recombinant rice -oxygenase was continuously followed by using an oxygen electrode, the kinetic lag phase was observed as shown in Fig. 6. This finding indicated that the native form of recombinant rice -oxygenase is inactive and needs catalytic activation during its reaction to ensure its maximum activity. This was consistent with the proposed catalytic mechanism of PGHSs in which the ferrous form is a native inactive form (30). The ferrous form is oxidized by hydroperoxides to form species analogous to compound I of horseradish peroxidase, which in turn accelerates the formation of tyrosyl radical that is essential to the abstraction of the hydrogen atom of the substrate arachidonic acid (31). If this is also the case with the rice enzyme, the addition of hydroperoxide must abolish the lag phase of the reaction. As shown with Fig. 6, trace B, the addition of tert-butyl hydroperoxide apparently shortened the lag of the reaction of -oxygenase; however, the maximal rate of oxygen consumption was unaffected by the addition.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Effect of hydroperoxide on the kinetics of -oxygenase activity. The purified recombinant enzyme was preincubated with or without 20 mM tert-butyl hydroperoxide at 25 °C for 2 min, and then the reaction was started by the addition of the substrate, linoleic acid. Trace A, without hydroperoxide; trace B, with the hydroperoxide.

Inhibition of -Oxidation Activity-- As noted with plant -oxidation system (8), imidazole was a potent reversible inhibitor for the rice -oxygenase, and the K_i value was determined to be 0.046 mM (Table I). It is well known that imidazole can bind to Fe in most heme proteins (32) that results in the inactivation of the function of the heme protein. On the contrary, no inhibition of ram seminal PGHS has been reported even at high imidazole levels as much as >500 mM (27), although the addition of imidazole changed the absorption spectrum of PGHS, which indicated that imidazole could bind to the PGHS. This addition was again a big difference in the properties of rice -oxygenase and mammalian PGHSs. Nonsteroidal anti-inflammatory drugs such as aspirin and flurbiprofen are known to be irreversible inactivators of PGHSs in vitro. Ser-530 in sheep PGHS is known to be the target of aspirin to exert its inhibitory activity through acetylation of the hydroxy group (30). This residue is thought to have an important role in binding the substrate of PGHS, arachidonic acid. For example, the estimated K_i value of aspirin was reported to be 14 mM with sheep PGHS, and the preincubation of the enzyme with 1 µM flurbiprofen for 1 min decreased the activity to almost 30% of the initial activity (33). As shown in Table I, aspirin and flurbiprofen were not potent inhibitors for rice -oxygenase activity. This finding may suggest that the conformation of the substrate binding site of rice -oxygenase is somewhat different from that of mammalian PGHSs.

View this table: [in this window] [in a new window]   Table I Inhibition of -oxygenase activity Aspirin, flurbiprofen, and ibuprofen were preincubated with the purified enzyme for 15 min at 25 °C at the given concentration shown in table. The remaining activity then was determined after 36-fold dilution of the mixture under the standard condition described under "Materials and Methods." The effect of imidazole was estimated by determining the activity in the presence of the given concentration. na, not applicable. Means ± S.D. (n = 3) are shown.

Site-directed Mutagenesis-- Through comparison of the rice -oxygenase sequence with those of mammalian PGHSs, we found that most residues reported to be essential to the catalytic activity of PGHSs are conserved in the rice sequence. To determine whether they also function as essential residues to support the -oxidation activity, site-directed mutageneses were carried out. The results are summarized in Fig. 7. Within the five mutated proteins, H158Q, Y380F, and S558A showed no detectable activity with either the assay following oxygen consumption or the formation of fatty aldehydes. His-158 could be the heme ligand of -oxygenase, and the inhibition of -oxygenase activity by imidazole indicates that the residue takes a role as one of the heme ligands. Tyr-380 and Ser-558 were also important residues for the activity. The sequences around the tyrosine residues in PGHS and -oxygenase are highly conserved. Furthermore, hydroperoxides are the primary products of both the enzymes from which we can assume that abstraction of a hydrogen atom from the methylene moiety in the substrate is the first commitment step of the catalysis. The fact that Y380F showed no activity indicates that the tyrosine residue is essential to the oxygenase probably through formation of tyrosyl radical. It is interesting to know that Ser-558 is essential for the activity of rice -oxygenase. As shown above, the residue could not be found at entirely the same position where the serine residue is conserved within mammalian PGHSs (Fig. 3B). Furthermore, NSAIDs, which are thought to interact to the serine residue to exert their inhibitory activities, had only a little effect on the rice -oxygenase activity. However, the region around Ser-558 was highly conserved within plant -oxygenases. These lines of evidence suggested that Ser-558 concomitant with the array surrounding it has an essential function to support catalytic activity of plant -oxygenase.

View larger version (55K): [in this window] [in a new window]   Fig. 7.   Effect of site-directed mutagenesis on -oxygenase activity. Each mutant was purified to apparently homogenous state on SDS-PAGE analysis (A), and the activity was determined by HPLC (B). The error bars represent the mean ± S.D. (n = 3). nd, not detected.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This study showed that rice -oxygenase is a heme enzyme having a structural similarity with mammalian PGHSs. The heme moiety seems to be essential to the activity, because exchange of the histidine residues possibly involved in heme binding abolished the activity. The native form of the enzyme is inactive and needs to be activated by hydroperoxide probably by its own product, fatty acid 2-hydroperoxide. The activation may result in the formation of a tyrosyl radical, which in turn abstracts a hydrogen atom from the methylene unit in the substrate to support stereochemical oxygenation of the carbon. As a result, hydroperoxide group is introduced on the C₂-position with (R)-configuration in a stereochemically unequivocal manner. This catalytic course of oxygenation of fatty acids is similar to that of PGHSs; thus, in this context, the catalytic mechanism proposed and widely accepted for mammalian PGHSs can be applied to the rice oxygenase. However, there still is an outstanding difference between rice oxygenase and PGHSs. PGHS is a bifunctional enzyme and can carry out both the cyclooxygenase and peroxidase activities; on the contrary, little peroxidase activity can be found with rice -oxygenase. In the case of PGHS, tight coordination of both the activities is essential to complete its reaction to form PGH. However, both of the activities function independently because Mn³⁺-protoporphyrin-reconstituted PGHS, for example, lacks appreciable peroxidase activity but still exhibits functional cyclooxygenase activity. Apparently, the structure around the heme moiety, especially at the peroxidase site proposed with PGHS (31) must be different between rice -oxygenase and PGHS. The fact that spectrophotometric features of the reduced form of -oxygenase and PGHS totally different from each other might be a result of this difference in the active site.

Unexpectedly, NSAIDs showed little effect on the rice -oxygenase activity. This low effect may be caused because 1) NSAIDs could not reach the serine residue in the active site or 2) there is no serine residue in the active site. Although not complete, the conserved serine residue can be found in the plant -oxygenases in a position near the active site serine in mammalian PGHSs. Furthermore, site-directed mutagenesis of Ser-558 in rice -oxygenase resulted in an inactive enzyme. Thus, the former possibility seems to be more sensible from which we can assume that the structure of the substrate recognition site is also diverged from that of PGHS. This finding is rational, because rice -oxygenase oxygenize only the C₂ position of the fatty acid and a pentadiene system occurring in polyunsaturated fatty acid is not the prerequisite for the activity. To obtain concrete evidence for the assumption depicted with this study, further extensive study is needed.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AAF64042.

To whom correspondence should be addressed. Tel.: 81-83-933-5850; Fax: 81-83-933-5820; E-mail: matsui@agr.yamaguchi-u.ac.jp.

Published, JBC Papers in Press, March 21, 2002, DOI 10.1074/jbc.M110420200

² T. Koeduka, K. Matsui, Y. Akakabe, and T. Kajiwara, unpublished data.

	ABBREVIATIONS

The abbreviations used are: PIOX, pathogen-inducible oxygenase; HPLC, high performance liquid chromatography; ADAM, 9-anthryldiazomethane; PGHS, prostaglandin H synthases; EST, expression sequence tag; NSAID, nonsteroidal anti-inflammatory drug.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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