A Novel Acyl-CoA Oxidase That Can Oxidize Short-chain Acyl-CoA in
Plant Peroxisomes*
Hiroshi
Hayashi
§,
Luigi
De Bellis
¶,
Adriana
Ciurli¶,
Maki
Kondo
,
Makoto
Hayashi
§, and
Mikio
Nishimura
§
From the
Department of Cell Biology, National
Institute for Basic Biology and the § Department of
Molecular Biomechanics, Graduate University for Advanced Studies,
Okazaki 444-8585, Japan and the ¶ Dipartimento di Biologia delle
Piante Agrarie, via Mariscoglio 34, 56124 Pisa, Italy
 |
ABSTRACT |
Short-chain acyl-CoA oxidases are
-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
-oxidation of acyl-CoA chains, whereas mammalian
peroxisomes can perform
-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
-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
-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
-oxidation of
acyl-CoA.
 |
INTRODUCTION |
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 long-chain fatty acids, but are inactive with fatty
acids shorter than octanoic acid (C8). 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 short-chain
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 (13-15) 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 short-chain acyl-CoA. We also discuss the
unique features of fatty acid
-oxidation accomplished by these
acyl-CoA oxidases in plant cells.
 |
EXPERIMENTAL PROCEDURES |
Plant Materials--
Pumpkin seeds (Cucurbita sp.
Kurokawa Amakuri) were purchased from Aisan Seed Co. (Aichi, Japan).
Pumpkin seeds were soaked in running tap water overnight and germinated
in Rock-Fiber soil (66R, Nitto Boseki, Chiba, Japan) at 25 °C in
darkness. Arabidopsis thaliana ecotype Landsberg
erecta seeds were surface-sterilized in 2% NaClO and 0.02%
Triton X-100 and grown on growth medium (2.3 mg/ml Murashige-Skoog
salts (Wako, Osaka, Japan), 1% sucrose, 100 µg/ml myoinositol, 1 µg/ml thiamine HCl, 0.5 µg/ml pyridoxine, 0.5 µg/ml nicotinic
acid, 0.5 mg/ml Mes1-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 (GenBankTM
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
Ni2+ resin. The purified protein (~0.5 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 (~0.25 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-cm2 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 × 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 µg/mg phenylmethylsulfonyl fluoride, 10 µM FAD, and
10% glycerol), and lysed by three bursts of sonication (3 × 1 min at 30-min intervals on ice). After centrifugation of the sample at
15,000 × 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 µM 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 (~5000 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.
Enzyme Assay and Isoelectric Focusing--
Enzyme activities
were measured at 25 °C in 1 ml of reaction mixture and monitored
with a Beckman DU-7500 spectrophotometer. Acyl-CoA oxidase (EC 1.3.3.6)
was assayed according to the method of Gerhardt (22), with the
concentration of acyl-CoA substrates reduced to 25 µM.
Acyl-CoA dehydrogenase (EC 1.3.99.3) was assayed according to Dommes
and Kunau (23) and Furuta et al. (24). Catalase (EC
1.11.1.6) was assayed according to Aebi (25). Cytochrome c
oxidase (EC 1.9.3.1) was assayed according to Hodges and Leonard (26).
Isoelectric focusing was performed at 15 °C using a Multiphor II
electrophoresis system and Immobiline Dry Strip gels (Amersham
Pharmacia Biotech) according to the manufacturer's instructions.
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 × 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 cDNA2 and
the availability of another homologous cDNA clone
(EBI/GenBankTM 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 ~47 kDa
(Fig. 1). Because mammalian acyl-CoA
dehydrogenase is a mitochondrial 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).

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Fig. 1.
Deduced amino acid sequence of
Arabidopsis short-chain acyl-CoA oxidase. The
peroxisomal targeting signal (PTS1) is boxed. The
GenBankTM accession number for Arabidopsis
short-chain acyl-CoA oxidase is AB017643.
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Expression, Purification, and Characterization of a Short-chain
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 short-chain acyl-CoA
oxidase activity or not, we found that crude homogenates obtained from
infected insect cells showed acyl-CoA oxidase activity on hexanoyl-CoA
(C6) (Table I). To purify the
protein expressed by the cDNA, the crude homogenates were subjected
to cation-exchange chromatography on a HiTrap 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.
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Table I
Purification of recombinant Arabidopsis short-chain acyl-CoA oxidase
expressed employing the baculovirus expression system
Short-chain acyl-CoA oxidase activities were tested with hexanoyl-CoA
as a substrate.
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Fig. 2.
SDS-polyacrylamide gel electrophoresis and
immunoblot analysis of samples taken at various steps during the
purification of Arabidopsis short-chain acyl-CoA
oxidase produced in the baculovirus expression system. A,
SDS-polyacrylamide gel stained with Coomassie Brilliant Blue dye;
B, immunoblot analysis of polyclonal antibodies raised
against recombinant Arabidopsis short-chain acyl-CoA
oxidase. The arrowheads indicate the bands corresponding to
the Arabidopsis short-chain acyl-CoA oxidase. Lane
1, homogenate (15,000 × g supernatant) from
insect cells infected with the wild-type baculovirus; lane
2, homogenate (15,000 × g supernatant) from
insect cells infected with the recombinant baculovirus (harboring the
Arabidopsis short-chain acyl-CoA oxidase cDNA);
lane 3, HiTrap SP column fraction showing
short-chain acyl-CoA oxidase activity; lane 4,
gel-filtration fraction showing short-chain acyl-CoA oxidase activity;
lane 5, homogenate (15,000 × g
supernatant) from 5-day-old dark-grown Arabidopsis etiolated
cotyledons.
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As shown in Fig. 3, the purified protein
showed oxidase activity toward acyl-CoAs from butyryl-CoA
(C4) to octanoyl-CoA (C8). The maximum activity
was observed when hexanoyl-CoA (C6) was used for the
substrate. The Km value for hexanoyl-CoA was estimated at 8.3 µM (Table
II). No activity was observed employing crotonoyl-CoA (C4:1, an unsaturated carboxylic ester) or
glutaryl-CoA (a dicarboxylic ester). The enzyme was active on
isobutyryl-CoA at a concentration of 67 µM (2.5 units/mg). Furthermore, we detected no acyl-CoA dehydrogenase activity
when hexanoyl-CoA (C6), decanoyl-CoA (C10), and
palmitoyl-CoA (C16) were used as substrates. These data
indicated that this Arabidopsis cDNA encodes a
short-chain acyl-CoA oxidase.

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Fig. 3.
Substrate specificity of
Arabidopsis short-chain acyl-CoA oxidase produced in a
baculovirus expression system. The activity was monitored
employing various acyl-CoAs as substrates at a concentration of 25 µM.
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Table II
Properties of Arabidopsis short-chain acyl-CoA oxidase
Km and optimal pH values were determined employing
hexanoyl-CoA as a substrate. Acyl-CoA dehydrogenase activity was tested
with hexanoyl-CoA, decanoyl-CoA, and palmitoyl-CoA as substrates.
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Gel-filtration chromatography of the short-chain acyl-CoA oxidase on a
Superose 12 HR 10/30 column indicated a native molecular mass of ~180
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 summarizes the characteristics of the short-chain acyl-CoA oxidase.
Interestingly, the alignment of the conserved regions of acyl-CoA
oxidases and acyl-CoA dehydrogenases revealed that the short- and
long-chain acyl-CoA oxidases have conserved signatures for mammalian
acyl-CoA dehydrogenase (PS1,
(G/A/C)(L/I/V/M)(S/T)EX2(G/S/A/N)GSDX2(G/S/A); and PS2,
(Q/E)X2G(G/S)XG(L/I/V/M/F/Y)X2(D/E/N)X4(K/R)X3(D/E)) (Fig. 4) (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 (33-36). 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).

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Fig. 4.
Partial alignment of acyl-CoA oxidases and
acyl-CoA dehydrogenases (A) and phylogenetic tree of
acyl-CoA oxidases and acyl-CoA dehydrogenases (B).
White letters indicate corresponding PS1 and PS2 amino
acids. The PS1 and PS2 regions are underlined. PS1 has
the form
(G/A/C)(L/I/V/M)(S/T)EX2(G/S/A/N)GSDX2(G/S/A),
and PS2 has the form
(Q/E)X2G(G/S)XG(L/I/V/M/F/Y)X2(D/E/N)X4(K/R)X3(D/E)
(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
(GenBankTM accession number AB017643); PumLACOX,
pumpkin long-chain acyl-CoA oxidase (accession number AF002016);
PhaACOX, Phalaenopsis acyl-CoA oxidase (accession
number U66299); HumACOX, human acyl-CoA oxidase (accession
number S69189); HumBACOX, 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).
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Subcellular Localization of Short-chain Acyl-CoA Oxidase--
To
investigate the subcellular localization of the short-chain 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.

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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. , short-chain acyl-CoA oxidase; ,
catalase; , cytochrome c oxidase; ------, sucrose
concentration (w/w). Twenty µl (Arabidopsis short-chain
acyl-CoA oxidase) and 5 µl (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.
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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 short-chain acyl-CoA oxidase was
detected in fractions 21-23 by the immunoblot technique (Fig. 5B). These fractions had short-chain 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.

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Fig. 6.
Immunoelectron microscopic analysis of the
localization of Arabidopsis cotyledons of 3-day-old
dark-grown seedlings using polyclonal antibodies against
Arabidopsis short-chain acyl-CoA oxidase and pumpkin
catalase. Mt, mitochondria; G, glyoxysome;
L, lipid body. The arrow indicates short-chain
acyl-CoA oxidase (15-nm gold particles), and the arrowhead
indicates catalase (10-nm gold particles). Bar = 1 µm.
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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.

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Fig. 7.
Developmental changes in the levels of
short-chain acyl-CoA oxidase, thiolase, and isocitrate lyase in
Arabidopsis cotyledons. D indicates
the days of growth in the dark. L indicates the days of
continuous illumination following 4 days in the dark. Each lane was
loaded with 10 µg (short-chain acyl-CoA oxidase) or 5 µg (thiolase,
isocitrate lyase) of total proteins extracted from
Arabidopsis cotyledons. Electrophoresed proteins were
blotted on a nylon membrane, and then the membrane was allowed to
hybridize with polyclonal antibodies raised against recombinant
Arabidopsis short-chain acyl-CoA oxidase (SACOX;
A), pumpkin thiolase (B), and castor bean
isocitrate lyase (ICL; C).
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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
short-chain acyl-CoA oxidase, except that a band was detected at
certain levels in 7-day-old green cotyledons, rosette leaves, and stems
(Fig. 8, center panel). In contrast, isocitrate lyase was
detected only in extracts from etiolated cotyledons (Fig. 8,
lower panel).

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Fig. 8.
Short-chain acyl-CoA oxidase, thiolase, and
isocitrate lyase expression in various Arabidopsis
tissues. Each lane was loaded with 10 µg of total
proteins. The tissues indicated below were excised from 5-week-old
plants, except cotyledons, which were excised 5 or 7 days after sowing.
SACOX, short-chain acyl-CoA oxidase; thi,
thiolase; ICL, isocitrate lyase. Lane 1,
Arabidopsis etiolated cotyledons from plants grown in the
dark for 5 days; lane 2, green
Arabidopsis cotyledons from plants grown in the dark for 4 days, followed by 3 days in the light; lane 3,
rosette leaves; lane 4, flowers; lane
5, roots; lane 6, stems;
lane 7, siliques.
|
|
 |
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
(C16-C18) 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-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 short-chain 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 C8 to C18 and that are inactive in oxidizing acyl-CoA esters with carbon chains shorter than 8 carbons. Short-chain fatty acids (C4-C8) 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 C-terminal peroxisomal targeting signal (PTS1)
versus an N-terminal cleavable targeting signal (PTS2),
(c) a total identity of only ~18%, 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 ~45 kDa. Very long-chain acyl-CoA dehydrogenase appears to be a dimer of ~75 kDa (8). In
conclusion, both acyl-CoA dehydrogenases and acyl-CoA oxidases are
tetramers or dimers of ~45 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 short- as 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)EX2(G/S/A/N)GSDX2(G/S/A), and PS2 has the form
(Q/E)X2G(G/S)XG(L/I/V/M/F/Y)X2(D/E/N)X4(K/R)X3(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 (C4-C8)
substrates and had a reduced affinity for octanoyl-CoA (C8)
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
Km value of 8.3 µM is close to the
value reported for the maize enzyme (6 µM). 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.
 |
ACKNOWLEDGEMENTS |
We are most grateful to Dr. Masayoshi
Maeshima (Nagoya University) for kindly providing the antibody against
castor bean isocitrate lyase and Dr. Roland R. Theimer (Institute for
Physiological Chemistry of Plants, Bergische University, Wuppertal,
Germany) for stimulating discussions and helpful comments on the manuscript.
 |
FOOTNOTES |
*
This work was supported by Research for the Future Program
Grant JSPS-RFTF 96L00407 from the Japan Society for the Promotion of
Science; by Grants-in-aid for Scientific Research 09440271, 09274101, and 09274103 from the Ministry of Education, Science, and Culture of
Japan; by a grant from the Nissan Science Foundation (Tokyo, Japan);
and by the National Institute for Basic Biology Program for Molecular
Mechanisms of Stress Response.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.
To whom correspondence should be addressed. Tel.:
81-564-55-7500; Fax: 81-564-55-7505; E-mail:
mikosome{at}nibb.ac.jp.
2
Grellet, F., Gaubier, P., Wu, H.-J., Laudie, M.,
Berger, C., and Delseny, M. (1996) EBI/GenBankTM accession
number U72505.
 |
ABBREVIATIONS |
The abbreviations used are:
Mes, 4-morpholineethanesulfonic acid;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
PTS, peroxisomal targeting signal;
PS, protein signature.
 |
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