A Novel Acyl-CoA Oxidase That Can Oxidize Short-chain Acyl-CoA in Plant Peroxisomes*

Short-chain acyl-CoA oxidases are β-oxidation enzymes that are active on short-chain acyl-CoAs and that appear to be present in higher plant peroxisomes and absent in mammalian peroxisomes. Therefore, plant peroxisomes are capable of performing complete β-oxidation of acyl-CoA chains, whereas mammalian peroxisomes can perform β-oxidation of only those acyl-CoA chains that are larger than octanoyl-CoA (C8). In this report, we have shown that a novel acyl-CoA oxidase can oxidize short-chain acyl-CoA in plant peroxisomes. A peroxisomal short-chain acyl-CoA oxidase from Arabidopsis was purified following the expression of the Arabidopsis cDNA in a baculovirus expression system. The purified enzyme was active on butyryl-CoA (C4), hexanoyl-CoA (C6), and octanoyl-CoA (C8). Cell fractionation and immunocytochemical analysis revealed that the short-chain acyl-CoA oxidase is localized in peroxisomes. The expression pattern of the short-chain acyl-CoA oxidase was similar to that of peroxisomal 3-ketoacyl-CoA thiolase, a marker enzyme of fatty acid β-oxidation, during post-germinative growth. Although the molecular structure and amino acid sequence of the enzyme are similar to those of mammalian mitochondrial acyl-CoA dehydrogenase, the purified enzyme has no activity as acyl-CoA dehydrogenase. These results indicate that the short-chain acyl-CoA oxidases function in fatty acid β-oxidation in plant peroxisomes, and that by the cooperative action of long- and short-chain acyl-CoA oxidases, plant peroxisomes are capable of performing the complete β-oxidation of acyl-CoA.

Oilseed plants convert reserve oil to sucrose after germination. This unique type of gluconeogenesis occurs in the storage tissues of oilseeds, such as endosperms or cotyledons (1). The metabolic pathway involves many enzymes in several subcellular compartments, including lipid bodies, glyoxysomes (a specialized peroxisome), mitochondria, and the cytosol. Within the entire gluconeogenic pathway, the conversion of a fatty acid to succinate takes place within the glyoxysomes, which contain enzymes for fatty acid ␤-oxidation and the glyoxylate cycle.
Glyoxysomes and leaf peroxisomes are members of a group of organelles called peroxisomes (2). In glyoxysomes, fatty acids are first activated to fatty acyl-CoA by fatty acyl-CoA synthetase (3). Fatty acyl-CoA is the substrate for fatty acid ␤-oxidation, which consists of four enzymatic reactions (4). The first reaction is catalyzed by acyl-CoA oxidase. The second and third enzymatic reactions are catalyzed by a single enzyme that possesses enoyl-CoA hydratase and ␤-hydroxyacyl-CoA dehydrogenase activities (5). The fourth reaction is catalyzed by 3-ketoacyl-CoA thiolase (referred to as thiolase below) (6). Acetyl-CoA, an end product of fatty acid ␤-oxidation, is metabolized further to produce succinate by the glyoxylate cycle.
In mammalian cells, both peroxisomes and mitochondria contain a functional fatty acid ␤-oxidation system. In peroxisomes, the first enzyme of fatty acid ␤-oxidation, acyl-CoA oxidase, donates electrons to molecular oxygen, producing hydrogen peroxide (7). Mammalian peroxisomes oxidize longchain fatty acids, but are inactive with fatty acids shorter than octanoic acid (C 8 ). This is mainly the consequence of the exclusive presence of long-chain acyl-CoA oxidases and the absence of acyl-CoA oxidases that are active on short-chain acyl-CoAs. In contrast, mammalian mitochondria are capable of complete oxidization of fatty acids to acetyl-CoA (8); the first step of fatty acid ␤-oxidation is accomplished by long-, medium-, and shortchain acyl-CoA dehydrogenases, and electrons generated by the dehydrogenases are transferred to the mitochondrial respiratory chain. By analogy, Thomas and co-workers (9 -11) have postulated the existence of plant mitochondrial ␤-oxidation, but the presence of acyl-CoA dehydrogenase was not investigated or not detected (12). In contrast, data reported by Gerhardt and co-workers (13)(14)(15) have suggested that glyoxysomes in plants can completely metabolize fatty acids to acetyl-CoA.
We have previously reported the existence of an acyl-CoA oxidase that is active on long-chain acyl-CoA in glyoxysomes (16). In the present study, we report evidence that glyoxysomes contain another acyl-CoA oxidase that can metabolize shortchain acyl-CoA. We also discuss the unique features of fatty acid ␤-oxidation accomplished by these acyl-CoA oxidases in plant cells. doxine, 0.5 g/ml nicotinic acid, 0.5 mg/ml Mes 1 -KOH, pH 5.7, and 0.2% Gellan gum (Wako)) in Petri dishes. Arabidopsis seeds were soaked in growth medium and germinated at 22°C under continuous illumination or under darkness, and some of Arabidopsis seedlings were transferred to light after 4 days of growing in the dark. Some seedlings that were grown under continuous illumination for 2 weeks on growth medium were transferred to a 1:1 mixture of perlite and vermiculite. Plants were grown under continuous illumination at 22°C.
Plasmids-The cDNA clone (GenBank TM accession number T46525) was obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH). DNA sequencing was performed by the method of Sanger et al. (17). DNA sequences were analyzed with GeneWorks Release 2.5 computer software (IntelliGenetics, Mountain View, CA). The BLAST server was utilized for the analysis of homologies among proteins. Alignment of several acyl-CoA oxidases and acyl-CoA dehydrogenases was performed using CLUSTAL W software (18).
Preparation of a Specific Antiserum-The Arabidopsis cDNA was inserted into pET32b vector (Novagen, Madison, WI). A fusion protein between short-chain acyl-CoA oxidase and a histidine tag was synthesized in Escherichia coli cells and purified by column chromatography on Ni 2ϩ resin. The purified protein (ϳ0.5 mg of protein) in 1 ml of sterilized water was emulsified with an equal volume of Freund's complete adjuvant (Difco). The emulsion was injected subcutaneously into the back of a rabbit. Four weeks later, a booster injection (ϳ0.25 mg of protein) was given similarly to the first injection. Blood was taken from a vein in the ear 7 days after the second booster injection. The serum was used for immunoblotting.
Expression of Recombinant Short-chain Acyl-CoA Oxidase from Insect Cells-Short-chain acyl-CoA oxidase was produced employing the baculovirus expression system from Invitrogen (San Diego, CA) following the manufacturer's protocols. The system includes Spodoptera frugiperda (Sf9) as the insect cell line, pBlueBac 4.5 (19) as a transfer vector, and engineered baculoviral Autographa californica multiple polyhedrosis virus (Bac-N-Blue DNA) as an expression vector. In brief, the short-chain acyl-CoA oxidase cDNA was inserted into the pBlueBac 4.5 transfer vector and cotransfected together with linearized baculoviral Bac-N-Blue DNA in insect cells. Recombinant viruses were purified from the transfection supernatant by plaque assay on medium containing 5-bromo-4-chloro-3-indolyl ␤-D-galactopyranoside, and recombinant plaques were verified by polymerase chain reaction. Afterward, a high-titer recombinant viral stock was generated, and following a time course of expression experiment, the optimal expression time was determined. The recombinant protein expression levels were optimized, and a large-scale expression of recombinant protein was performed.
Purification of Recombinant Short-chain Acyl-CoA Oxidase from Insect Cells-Log-phase growing Sf9 cells in 20 75-cm 2 flasks were infected with recombinant viral stock at a multiplicity of infection of 10. Four days after infection, the cells were dislodged from the flask walls and centrifuged at 500 ϫ g for 5 min at 4°C. The cell pellets were washed with phosphate-buffered saline, gently suspended in buffer A (50 mM sodium phosphate, pH 6.7, 10 mM NaCl, 100 g/mg phenylmethylsulfonyl fluoride, 10 M FAD, and 10% glycerol), and lysed by three bursts of sonication (3 ϫ 1 min at 30-min intervals on ice). After centrifugation of the sample at 15,000 ϫ g for 30 min, the supernatant was dialyzed against buffer A and loaded on a HiTrap SP column (Amersham Pharmacia Biotech, Tokyo, Japan). Proteins were eluted with a gradient of 10 -500 mM NaCl in buffer A, and fractions of 0.5 ml were collected. Fractions with high short-chain acyl-CoA oxidase activities were pooled and concentrated using Centricon 30 concentrators (Amicon Inc., Beverly, MA) and then loaded on a Superose 12 HR 10/30 column (Amersham Pharmacia Biotech) equilibrated with buffer B (10 mM sodium phosphate, pH 7.2, 250 mM NaCl, 10 M FAD, and 10% glycerol). Proteins were eluted with buffer B, and fractions of 0.5 ml were collected and analyzed for the presence of acyl-CoA oxidase activity.
Subcellular Fractionation-Four-day-old pumpkin etiolated cotyledons (15 g, fresh weight) were homogenized in a Petri dish by chopping with a razor blade for 5 min in 10 ml of a medium that contained 150 mM Tricine-KOH, pH 7.5, 1 mM EDTA, and 0.5 M sucrose. The homogenate was passed through four layers of cheesecloth. Three ml of the filtrate was layered onto a sucrose gradient that consisted of a 1-ml cushion of 60% (w/w) sucrose and 11 ml of a linear sucrose gradient from 60 to 30% without buffer. The gradient was centrifuged at 21,000 rpm for 3 h in a Beckman SW 28.1 rotor in a Beckman Model XL-90 ultracentrifuge. After centrifugation, fractions of 0.5 ml were collected with a gradient fractionator (Model 185, Isco Inc., Lincoln, NE). All procedures were carried out at 4°C. Subcellular fractionation of Arabidopsis etiolated cotyledons was performed as follows. One-hundred mg of seeds (ϳ5000 seeds) was grown on growth medium for 5 days in darkness at 22°C. Etiolated cotyledons were harvested and chopped with a razor blade in a Petri dish with 2 ml of chopping buffer (150 mM Tricine-KOH, pH 7.5, 1 mM EDTA, 0.5 M sucrose, and 1% bovine serum albumin). The extract was then filtered with a cell strainer (Becton Dickinson Labware, Franklin Lakes, NJ). Two ml of the homogenate was layered directly on top of a 16-ml linear sucrose density gradient (30 -60%, w/w) that contained 1 mM EDTA. Centrifugation was performed in the SW 28.1 rotor at 25,000 rpm for 2.5 h at 4°C. Fractions of 0.5 ml were collected with the gradient fractionator.
Immunoelectron Microscopy-Arabidopsis etiolated cotyledons were harvested after 3 days in darkness. The samples were fixed, dehydrated, and embedded in LR white resin (London Resin, Basingstoke, United Kingdom) as described previously (20,21). Ultrathin sections were cut on a Reichert ultramicrotome (Leica, Heidelberg, Germany) with a diamond knife and mounted on uncoated nickel grids. The protein A-gold labeling procedure was essentially the same as that described (20,21). Ultrathin sections were incubated at 4°C overnight with a solution of catalase antiserum (diluted 1:1000) and then with a 30-fold diluted suspension of protein A-gold (10 nm for catalase; Amersham Pharmacia Biotech) at room temperature for 30 min. A solution of short-chain acyl-CoA oxidase antiserum (diluted 1:1000) was added to a 200-fold diluted biotinylated species-specific whole antibody and incubated at room temperature for 1 h and then with a 20-fold diluted suspension of streptavidin-gold (15 nm for short-chain acyl-CoA oxidase; Amersham Pharmacia Biotech) at room temperature for 30 min. The sections were examined with a transmission electron microscope (1200EX, Joel, Tokyo) at 80 kV.
Western Blot Hybridization-Arabidopsis and pumpkin cotyledons were homogenized in extraction buffer (0.1 M Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 0.1% SDS); the homogenate was centrifuged at 15,000 ϫ g for 20 min; and the supernatant was subjected to SDS-polyacrylamide gel electrophoresis. Immunoblot analysis was then performed essentially following the method of Towbin et al. (27). Immunologic reactions were detected by monitoring horseradish peroxidase activity (ECL system, Amersham Pharmacia Biotech). Thiolase (28), castor bean isocitrate lyase (29), and pumpkin catalase (30) antisera were prepared as described previously. Protein was quantitated with a protein assay kit (Nippon Bio-Rad Laboratories, Tokyo).

RESULTS
Identification of a Short-chain Acyl-CoA Oxidase cDNA-As result of a similarity search with a long-chain acyl-CoA oxidase (16) in a DNA data base, we found a putative Arabidopsis acyl-CoA dehydrogenase cDNA 2 and the availability of another homologous cDNA clone (EBI/GenBank TM accession number T46525, AB017643) in the Arabidopsis Expressed Sequence Tag data base. We received the latter from the Arabidopsis Biological Resource Center and fully sequenced it. The expressed sequence tag clone contained an insert of 1.6 kilobases. The open reading frame encodes a polypeptide of 436 amino acids, which corresponds to a molecular mass of ϳ47 kDa (Fig.  1). Because mammalian acyl-CoA dehydrogenase is a mito-chondrial enzyme, this putative acyl-CoA dehydrogenase was thought to be localized in plant mitochondria. However, we failed to find a mitochondrial targeting signal in the amino acid sequence. Instead, a typical peroxisomal targeting signal (PTS1) was present at the carboxyl terminus (SRL) (Fig. 1, boxed) (31). Therefore, we postulated that this cDNA encodes a second acyl-CoA oxidase with a substrate specificity that is different from that of a known plant acyl-CoA oxidase (16).
Expression, Purification, and Characterization of a Shortchain Acyl-CoA Oxidase-To confirm that the Arabidopsis cDNA actually encodes an acyl-CoA oxidase, we expressed the protein from the cDNA employing a baculovirus expression system. To ascertain whether this expression protein has shortchain acyl-CoA oxidase activity or not, we found that crude homogenates obtained from infected insect cells showed acyl-CoA oxidase activity on hexanoyl-CoA (C 6 ) ( Table I). To purify the protein expressed by the cDNA, the crude homogenates were subjected to cation-exchange chromatography on a Hi-Trap SP column. Fractions containing high acyl-CoA oxidase activity were concentrated by ultrafiltration. The sample was then loaded on a Superose 12 column. The results of the purification are summarized in Table I. Analysis by SDS-polyacrylamide gel electrophoresis showed that the protein expressed in the insect cells and isolated by this purification scheme was pure ( Fig. 2A). Fig. 2B shows an immunoblot analysis of the fractions from each purification step and of an extract prepared from Arabidopsis etiolated cotyledons using antibodies raised against this acyl-CoA oxidase. The immunoblot analysis revealed that the molecular mass (47 kDa; arrowheads) of the purified protein coincided with that of the immunoreactive protein in Arabidopsis etiolated cotyledons.
As shown in Fig. 3, the purified protein showed oxidase activity toward acyl-CoAs from butyryl-CoA (C 4 ) to octanoyl-CoA (C 8 ). The maximum activity was observed when hexanoyl-CoA (C 6 ) was used for the substrate. The K m value for hexanoyl-CoA was estimated at 8.3 M (Table II). No activity was observed employing crotonoyl-CoA (C 4:1 , an unsaturated carboxylic ester) or glutaryl-CoA (a dicarboxylic ester). The enzyme was active on isobutyryl-CoA at a concentration of 67 M (2.5 units/mg). Furthermore, we detected no acyl-CoA dehydrogenase activity when hexanoyl-CoA (C 6 ), decanoyl-CoA (C 10 ), and palmitoyl-CoA (C 16 ) were used as substrates. These data indicated that this Arabidopsis cDNA encodes a short-chain acyl-CoA oxidase.
Gel-filtration chromatography of the short-chain acyl-CoA oxidase on a Superose 12 HR 10/30 column indicated a native molecular mass of ϳ180 kDa (Table II). Because the subunit molecular mass of the short-chain acyl-CoA oxidase is 47 kDa,   the purified enzyme must be a homotetramer. The highest activity was observed between pH 8.5 and 9.0. Table II (32). It is possible that these regions are important for the interaction with the substrates. X-ray crystallography and mutational analyses indicated that the glutamic acid residues of mammalian medium-chain (Glu-376) and short-chain (Glu-368) acyl-CoA dehydrogenases serve as the ␣-proton-abstracting base (33)(34)(35)(36). Both the Arabidopsis short-chain and pumpkin long-chain acyl-CoA oxidases contain a glutamic acid residue in a corresponding position (Fig. 4A, asterisk). To analyze the similarity between acyl-CoA oxidases and acyl-CoA dehydrogenases, we compared amino acid sequences of plant acyl-CoA oxidases with human acyl-CoA oxidases and acyl-CoA dehydrogenases. A phylogenetic tree indicates that the plant short-chain acyl-CoA oxidase is clustered together with mitochondrial acyl-CoA dehydrogenases, whereas it is relatively far from other peroxisomal acyl-CoA oxidases (Fig. 4B).
Subcellular Localization of Short-chain Acyl-CoA Oxidase-To investigate the subcellular localization of the shortchain acyl-CoA oxidase, homogenates from 5-day-old Arabidopsis etiolated cotyledons were subjected to sucrose density gradient centrifugation. Fractions thus obtained were analyzed using an immunoblot technique with antibodies raised against the short-chain acyl-CoA oxidase and catalase. Catalase was used as a glyoxysomal marker enzyme. As shown in Fig. 5A, short-chain acyl-CoA oxidase and catalase were present together in fractions 21-23.
Although these enzymes were detected in the first few fractions (top of the gradient), this may be due to disruption of the glyoxysomes during homogenization and subsequent cell fractionation. We confirmed this result using 5-day-old pumpkin etiolated cotyledons. As is the case with Arabidopsis, a shortchain acyl-CoA oxidase was detected in fractions 21-23 by the immunoblot technique (Fig. 5B). These fractions had shortchain acyl-CoA oxidase as well as catalase activities. In contrast, no short-chain acyl-CoA oxidase activity was detected in fractions 8 -13, which correspond to the activity of a mitochondrial marker enzyme, cytochrome c oxidase. Fig. 6 shows an immunoelectron microscopic observation of short-chain acyl-CoA oxidase and catalase in cotyledon cells of Arabidopsis etiolated seedlings. Double staining by polyclonal antibodies against Arabidopsis short-chain acyl-CoA oxidase (arrow) and pumpkin catalase (arrowhead) revealed that both enzymes are co-localized in glyoxysomes. No signal was detected on other organelles. These results clearly indicated that the short-chain acyl-CoA oxidase is exclusively localized in glyoxysomes.

Developmental Changes in the Level of Short-chain Acyl-CoA
Oxidase- Fig. 7 shows changes in the levels of short-chain acyl-CoA oxidase during the post-germinative growth of the Arabidopsis seedlings. An immunoblot analysis of Arabidopsis seedlings grown in the dark showed that short-chain acyl-CoA oxidase as well as thiolase, another enzyme for fatty acid ␤-oxidation, reached a maximum level after 5-7 days of growth. These enzymes were still present in the seedlings after 9 days of growth in the dark. After illumination of the seedlings was started, the amount of these enzymes decreased, but faint bands were still detectable after 5 days of illumination (Fig. 7,  4D5L). Instead, isocitrate lyase, an enzyme of the glyoxylate cycle, reached a maximum level earlier than short-chain acyl-CoA oxidase (3 days after germination) and completely disappeared after 9 days in the dark.
Presence of Short-chain Acyl-CoA Oxidase in Various Organs-Short-chain acyl-CoA oxidase was particularly abundant in 5-day-old Arabidopsis etiolated cotyledons (Fig. 8, upper panel, lane 1). This enzyme was also present in flowers, roots, and siliques (lanes 4, 5, and 7), whereas it was present at very low levels or not at all in 7-day-old green cotyledons, rosette leaves, and stems (lanes 2, 3, and 6). The expression pattern of the thiolase was essentially similar to that of shortchain acyl-CoA oxidase, except that a band was detected at certain levels in 7-day-old green cotyledons, rosette leaves, and   (32). Multiple sequence alignments of the protein sequences were performed using the CLUSTAL W program. The phylogenetic tree was constructed according to the NJPLOT program. AtSACOX, Arabidopsis short-chain acyl-CoA oxidase (Gen-Bank TM accession number AB017643); PumLACOX, pumpkin longchain acyl-CoA oxidase (accession number AF002016); PhaACOX, Phalaenopsis acyl-CoA oxidase (accession number U66299); HumA-COX, human acyl-CoA oxidase (accession number S69189); Hum-BACOX, human branched-chain acyl-CoA oxidase (accession number X95190); HumVLACDH, human very long-chain acyl-CoA dehydrogenase (accession number D43682); HumLACDH, human long-chain acyl-CoA dehydrogenase (accession number M74096); HumMACDH, human medium-chain acyl-CoA dehydrogenase (accession number M16827); HumSACDH, human short-chain acyl-CoA dehydrogenase (accession number M26393); HumSBACDH, human short/branched-chain acyl-CoA dehydrogenase (accession number U12778). stems (Fig. 8, center panel). In contrast, isocitrate lyase was detected only in extracts from etiolated cotyledons (Fig. 8,  lower panel). DISCUSSION In higher plants with fatty seeds such as pumpkin, the triacylglycerols are stored in lipid bodies. During germination, the fatty acids are liberated by lipase and then degraded by the ␤-oxidation system in the glyoxysomes, and the resulting acetyl-CoA is further metabolized by the glyoxylate cycle. Thus, fatty acids serve as the main source for energy and carbon compounds. Therefore, fatty acid ␤-oxidation plays an important role in metabolism until the etiolated cotyledons turn green during late germination. To use storage lipids efficiently, fatty acids need to be completely converted from acyl-CoA to acetyl-CoA by fatty acid ␤-oxidation. Because most storage lipids are long-chain molecules (C 16 -C 18 ) in higher plants, the first step in fatty acid ␤-oxidation begins with long-chain acyl-CoA oxidase, and for the shorter acyl-CoAs, short-chain acyl-

FIG. 5. Subcellular localization of short-chain acyl-CoA oxidase in Arabidopsis (A) and pumpkin (B) etiolated cotyledons.
Both extracts from 5-day-old etiolated cotyledons were fractionated by sucrose density gradient centrifugation. The arrowheads indicate the bands corresponding to the short-chain acyl-CoA oxidase. A, immunological detection of Arabidopsis short-chain acyl-CoA oxidase (SACOX) and catalase; B, immunological detection of pumpkin short-chain acyl-CoA oxidase and enzyme activities. E, short-chain acyl-CoA oxidase; OE, catalase; f, cytochrome c oxidase; OO, sucrose concentration (w/w). Twenty l (Arabidopsis short-chain acyl-CoA oxidase) and 5 l (pumpkin short-chain acyl-CoA oxidase and catalase) of samples from each odd-numbered fraction were subjected to SDS-polyacrylamide gel electrophoresis (10% acrylamide) and immunoblotting. CoA oxidase takes the place of long-chain acyl-CoA oxidase. Thus, higher plants make efficient use of storage lipids to produce carbon and energy sources. In this study, we characterized an Arabidopsis peroxisomal short-chain acyl-CoA oxidase and its cDNA. The presence of a peroxisomal short-chain acyl-CoA oxidase explains how higher plant peroxisomes are able to completely oxidize fatty acids by a ␤-oxidation system.
In mammalian cells, fatty acid ␤-oxidation is localized both in peroxisomes and in mitochondria. The presence of a shortchain acyl-CoA oxidase distinguishes the peroxisomal ␤-oxidation of higher plants from that of mammals. In fact, mammalian peroxisomes contain three acyl-CoA oxidase isoforms that act on CoA derivatives of fatty acids with chain lengths from C 8 to C 18 and that are inactive in oxidizing acyl-CoA esters with carbon chains shorter than 8 carbons. Short-chain fatty acids (C 4 -C 8 ) that could not be oxidized by these peroxisomal acyl-CoA oxidases are transported to mitochondria (7). The mitochondrial ␤-oxidation system is able to completely degrade fatty acids from long-to short-chain fatty acids (37).
Common features of the amino acid sequences of the Arabidopsis short-chain acyl-CoA oxidase and the mammalian mitochondrial acyl-CoA dehydrogenase are shown in Fig. 4 and can be summarized as follow: (a) the presence of the two acyl-CoA dehydrogenase protein signatures (PS1 and PS2) in both enzymes; (b) a 35% identity between acyl-CoA oxidase and acyl-CoA dehydrogenase; and (c) similar subunit molecular masses. However, the short-chain acyl-CoA oxidase differs from pumpkin long-chain acyl-CoA oxidase (16), not considering the substrate specificity, as follows: (a) a subunit molecular mass of 47 versus 77 kDa (precursor subunit), (b) the presence of a Cterminal peroxisomal targeting signal (PTS1) versus an Nterminal cleavable targeting signal (PTS2), (c) a total identity of only ϳ18%, and (d) a tetrameric structure versus a dimeric one. A phylogenetic tree (Fig. 4B) including some representative acyl-CoA dehydrogenases and acyl-CoA oxidases from mammals and higher plants clearly summarizes the data presented above: the short-chain acyl-CoA oxidase of Arabidopsis is relatively unrelated to the other peroxisomal acyl-CoA oxidases, whereas it is clustered together with mitochondrial acyl-CoA dehydrogenases. The low homology to other acyl-CoA oxidases might suggest that the short-chain acyl-CoA oxidase shares a common ancestor with acyl-CoA dehydrogenases. Short-chain acyl-CoA oxidase could have arisen from a mitochondrial acyl-CoA dehydrogenase that acquired the peroxisomal targeting signal and the new intracellular location during evolution. That allowed plant peroxisomes to host a novel acyl-CoA oxidase ability that distinguishes plant organelles from mammalian peroxisomes.
At least five isoforms of acyl-CoA dehydrogenase are present in mammalian mitochondria: very long-, long-, medium-, short-, and short/branched-chain acyl-CoA dehydrogenases. Except for the very long-chain acyl-CoA dehydrogenase, all the other isoforms are tetrameric enzymes with a subunit of ϳ45 kDa. Very long-chain acyl-CoA dehydrogenase appears to be a dimer of ϳ75 kDa (8). In conclusion, both acyl-CoA dehydrogenases and acyl-CoA oxidases are tetramers or dimers of ϳ45 or 75 kDa. Our analysis revealed the presence of a short-chain acyl-CoA oxidase in plant peroxisomes that shares high homology with mitochondrial acyl-CoA dehydrogenases in mammals. The alignment of the conserved regions of acyl-CoA oxidases and acyl-CoA dehydrogenases (Fig. 4A) revealed that the shortas well as long-chain acyl-CoA oxidases contain amino acids of the typical mammalian acyl-CoA dehydrogenase protein signatures (PS1 and PS2). PS1 has the form (G/A/C)(L/I/V/M)(S/ T)EX 2 (G/S/A/N)GSDX 2 (G/S/A), and PS2 has the form (Q/ E)X 2 G(G/S)XG(L/I/V/M/F/Y)X 2 (D/E/N)X 4 (K/R)X 3 (D/E) (32).
The amino acid sequence of pumpkin long-chain acyl-CoA oxidase also contains 7 of the 9 amino acids of PS1 and 6 of the 8 amino acids of PS2 (Fig. 4A). Therefore, PS1 and PS2 might be unrelated to the functions of the dehydrogenase and the oxidase. The purified short-chain acyl-CoA oxidase was active exclusively against short-chain acyl-CoA (C 4 -C 8 ) substrates and had a reduced affinity for octanoyl-CoA (C 8 ) and a very low activity for branched-chain substrates. This substrate specificity resembles the characteristics of the maize short-chain acyl-CoA oxidase as indicated by Hooks et al. (38). The K m value of 8.3 M is close to the value reported for the maize enzyme (6 M). The optimum pH of 8.5-9.0 is similar to that of the maize enzyme (pH 8.3-8.5). Hooks et al. have reported the purification of medium-and short-chain acyl-CoA oxidases from maize. The former was a monomeric enzyme of 62 kDa, and the latter was a homotetrameric enzyme of 15-kDa subunits. The 15-kDa subunit has one-third of the subunit mass (47 kDa) of the Arabidopsis short-chain acyl-CoA oxidase. Since the maize short-chain acyl-CoA oxidase was not yet cloned, the discrepancy in the subunit molecular mass needs to be further investigated to determine whether there are different families of acyl-CoA oxidases.
Regulation of the expression of short-chain acyl-CoA oxidase seems to be similar to that of other ␤-oxidation enzymes such as thiolase (Figs. 7 and 8). A similar regulatory mechanism was reported for the expression of pumpkin long-chain acyl-CoA oxidase (16). On the contrary, isocitrate lyase, a marker enzyme of the glyoxylate cycle, is differently regulated. This enzyme disappeared very quickly compared with short-chain acyl-CoA oxidase and thiolase. Additionally, the organ-specific expression of short-chain acyl-CoA oxidase and thiolase does not appear to be coordinated with the expression of isocitrate lyase. These results suggest that ␤-oxidation enzymes are present in a wider range of organs than enzymes of the glyoxylate cycle such as isocitrate lyase. Particularly, it seems that ␤-oxidation enzymes are present in significant amounts in flowers, roots, and siliques (Fig. 8, lanes 4, 5, and 7). Our data further support the hypothesis that the ␤-oxidation pathway plays an important role not only during the degradation of stored lipids, but also in normal lipid turnover and senescence (28) and in jasmonic acid synthesis (39). This hypothesis is also supported by the finding that the cDNA of an acyl-CoA oxidase of Phalaenopsis (which is probably a long-chain acyl-CoA oxidase) was isolated by a search for flower senescence-related genes (40). Recent additional evidence has indicated that the expression of a gene for medium-chain acyl-CoA oxidase was induced when a lauroylacyl carrier protein thioesterase was overexpressed in Brassica (41), indicating that expression of the acyl-CoA oxidase gene is regulated by fatty acid biosynthesis or by the amount of fatty acids that are present in the cells. Thus, acyl-CoA oxidase isoforms might have a fundamental role in the control of fatty acid homeostasis in higher plants.