Download Euglena gracilis ascorbate peroxidase forms an intramolecular

Biochem. J. (2010) 426, 125–134 (Printed in Great Britain)
125
doi:10.1042/BJ20091406
Euglena gracilis ascorbate peroxidase forms an intramolecular dimeric
structure: its unique molecular characterization
Takahiro ISHIKAWA*1 , Naoko TAJIMA*, Hitoshi NISHIKAWA*, Yongshun GAO*, Madhusudhan RAPOLU*2 , Hitoshi SHIBATA*,
Yoshihiro SAWA* and Shigeru SHIGEOKA†
*Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan, and
†Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan
Euglena gracilis lacks a catalase and contains a single APX
(ascorbate peroxidase) and enzymes related to the redox cycle
of ascorbate in the cytosol. In the present study, a full-length
cDNA clone encoding the Euglena APX was isolated and found
to contain an open reading frame encoding a protein of 649 amino
acids with a calculated molecular mass of 70.5 kDa. Interestingly,
the enzyme consisted of two entirely homologous catalytic
domains, designated APX-N and APX-C, and an 102 amino acid
extension in the N-terminal region, which had a typical class II
signal proposed for plastid targeting in Euglena. A computerassisted analysis indicated a novel protein structure with an
intramolecular dimeric structure. The analysis of cell fractionation
showed that the APX protein is distributed in the cytosol,
but not the plastids, suggesting that Euglena APX becomes
mature in the cytosol after processing of the precursor. The
INTRODUCTION
APX (ascorbate peroxidase, EC 1.11.1.11) is widely distributed
in plants, eukaryotic algae and protozoa that have acquired the
ability to synthesize AsA (ascorbate) and catalyses the reduction
of H2 O2 to water with AsA as its specific electron donor [1–3].
APX plays a central role not only in the scavenging of excess
H2 O2 , to protect cells from oxidative damage, but also in the
regulation of cellular redox status, in response to environmental
or physiological conditions, to evoke a redox signal transduction
[1–4].
Genome-wide analyses have indicated that plant APXs belong
to a multigenic family, as there are nine genes in Arabidopsis,
eight in rice and seven in tomato [5–7]. The APX isoenzymes are
further classified into three subfamilies according to subcellular
localization: those in the cytosol, microbodies, stroma and
thylakoid-membranes of chloroplasts. Thus plants have evolved
the strategy of scavenging H2 O2 at sites where it is generated.
Basically, membrane-binding types of APX, i.e. the microbodyand thylakoid-membrane-bound forms, have a very similar
primary structure to the soluble APX, the cytosolic and stromal
forms respectively, except they also have a C-terminal extension
with a hydrophobic anchor region for association with membranes
[1–3]. A native cytosolic APX is typically a homodimer consisting
of a 28 kDa subunit, whereas a stromal APX is monomeric with
kinetics of the recombinant mature FL (full-length)-APX and the
APX-N and APX-C domains with ascorbate and H2 O2 were
almost the same as that of the native enzyme. However, the substrate specificity of the mature FL-APX and the native enzyme was
different from that of APX-N and APX-C. The mature FL-APX,
but not the truncated forms, could reduce alkyl hydroperoxides,
suggesting that the dimeric structure is correlated with substrate
recognition. In Euglena cells transfected with double-stranded
RNA, the silencing of APX expression resulted in a significant
increase in the cellular level of H2 O2 , indicating the physiological
importance of APX to the metabolism of H2 O2 .
Key words: ascorbate peroxidase, Euglena gracilis Z, full-length
cDNA cloning, intramolecular dimer structure, recombinant
expression.
a size of 34 kDa [1–3]. The catalytic properties of cytosolic APX
are similar to those of stromal APX with respect to specificity
for H2 O2 with similar K m values (up to 50 μM) and a failure to
reduce alkyl hydroperoxides, such as t-butyl hydroperoxide and
cumene hydroperoxide [1–3].
In contrast with the APX proteins of higher plants, algal
APXs are quite limited in number and distribution. The APX
in Euglena gracilis is restricted to the cytosol with no isoform
found in chloroplasts or other organelles [8]. Furthermore,
the AsA–glutathione cycle involving monodehydroascorbate
reductase, dehydroascorbate reductase and glutathione reductase
was exclusively found to reside in the cytosol [9]. Similarly,
the APXs of Chlorella and Galdieria are distributed only in the
cytosol [10,11], whereas the Chlamydomonas APX occurs only
in the stroma of chloroplasts [12]. These findings concerning the
number and subcellular localization of APX in eukaryotic algae
suggest a cellular metabolism of H2 O2 different from that in higher
plants. Moreover, a few studies have examined the molecular
characterization and physiological importance of algal APXs.
We previously purified and characterized Euglena APX [13].
The native enzyme was monomeric with a molecular mass of
58 kDa, nearly twice as large as cytosolic APX proteins in higher
plants. Another interesting aspect of Euglena APX was that, apart
from H2 O2 , the enzyme can also reduce alkyl hydroperoxides,
suggesting that like the glutathione peroxidase of animals, the
Abbreviations used: APX, ascorbate peroxidase; APX-C, C-terminal catalytic domain of Euglena APX; APX-N, N-terminal catalytic domain of Euglena
APX; AsA, ascorbate; BES-H2 O2 -Ac, H2 O2 -specific BES (benzenesulfonyl) derivative of a fluorescein probe; dsRNA, double-stranded RNA; EF1-α,
elongation factor 1-α; ER, endoplasmic reticulum; EST, expressed sequence tag; FL-APX, full-length APX; ORF, open reading frame; RNAi, RNA interference;
RT, reverse transcription; TMHMM, transmembrane helices based on a hidden Markov model.
1
To whom correspondence should be addressed (email [email protected]).
2
Present address: Department of Molecular Biosciences and Bioengineering, University of Hawaii, 1955 East–West Road, Honolulu, HI 96822, U.S.A.
The nucleotide sequence data reported for Euglena gracilis ascorbate peroxidase will appear in the DDBJ, EMBL, GenBank® and GSDB Nucleotide
Sequence Databases under accession number AB077953.
c The Authors Journal compilation c 2010 Biochemical Society
126
T. Ishikawa and others
algal APX protects cells from lipid hydroperoxidation leading
to membrane damage. However, the molecular structure and
catalytic properties of Euglena APX remained to be clarified.
In the present paper we report the cloning of a full-length
cDNA and the functional characterization of Euglena APX. We
found that the mature FL (full-length)-APX consisted of two
entirely homologous catalytic domains, designated APX-N and
APX-C (N-terminal and C-terminal catalytic domains of Euglena
respectively), and formed a novel and unique intramolecular
dimer. We also studied the enzymological properties of the
recombinant mature FL-APX and the APX-N and APX-C
domains, as well as the effect of gene silencing on the cellular level
of H2 O2. We discuss the contribution of APX to H2 O2 metabolism
in Euglena cells.
EXPERIMENTAL
Cell culture
Euglena gracilis, strain Z, was maintained by regular subculturing
and was grown in Koren–Hutner medium under continuous
illumination (24 μmol·m−2 ·s−1 ) at 26 ◦C for 6 days, by which
time the stationary phase was reached [14].
Cloning of a full-length cDNA encoding Euglena APX
Using the Euglena EST (expressed sequence tag) database from
the Protest EST Program, gene-specific primers were designed to
amplify the missing 5 end of the APX EST (ELL00005171). The
primers were EgAPX-SP1 (5 -GGACACCGTGGGGTACTTC3 ) and EgAPX-SP2 (5 -ACCCACGTCGGCAGCTCC-3 ) and
were used sequentially for 5 -RACE (rapid amplification of cDNA
ends) with a GeneRacer kit (Invitrogen). Then, based on a partial
cDNA sequence identified previously [15], the full-length coding
sequence of Euglena APX was amplified by PCR with EgAPXF (5 -GAGCTGCCGACGTGGGTGCCGGGCTTCGTG-3 ) and
EgAPX-R (5 -GCAGACCGGGGGGAAGGCGGCGGACGCGTG-3 ). PCR amplification was carried out using a GC-rich PCR
system (Roche Diagnostics).
Expression and purification of the recombinant APX
The cDNAs for the mature FL-APX and each truncated version
(APX-N and APX-C) were amplified from the full-length
cDNA as a template by PCR using as primers: EgAPXNF, 5 -ctcgagGAGCTGCCGACGTGGGTGC-3 ; EgAPX-NR,
5 -aagcttctaCAGCTCCGGCACCCCGAGCTC-3 ; EgAPX-CF,
5 -ctcgagTACCAGCGCCTGGCGGAGC-3 ; and EgAPX-CR,
5 -AAGcttGCGGACGCGTGTGTGGCTGGC-3 in order to
introduce XhoI and HindIII sites containing stop codons into the
3 ends of the cDNAs as the need arises (indicated by lowercase
letters). The amplified fragments were cloned into a pGEM-T
easy vector (Promega) to confirm the absence of PCR errors. The
plasmids were digested with XhoI and HindIII, and the resulting
DNA fragments were ligated into a pCold II vector (Takara) to
produce His6 -tagged recombinant proteins. The Escherichia coli
strain BL21 Star (Stratagene) was used as a host for the expression
of recombinant APXs.
Transformed cells were cultivated at 37 ◦C in 100 ml of LB
(Luria–Bertani) medium containing 50 μg·ml−1 ampicillin until
the D600 reached 0.5. Then, isopropyl β-D-thiogalactoside, glucose
and haemin were added to the culture at a concentration of 1 mM,
2 mM and 10 μM respectively, and the cells were grown further
at 15 ◦C for 20 h. The harvested cells (approx. 2 g wet weight)
were suspended in 2 ml of 50 mM potassium phosphate buffer,
c The Authors Journal compilation c 2010 Biochemical Society
pH 7.0, containing 0.3 M NaCl and 1 mM AsA, and disrupted
by sonication (20 kHz for a total of 5 min with four intervals of
1 min each; ultrasonic processor VP-5, Taitec). The cell lysate was
centrifuged at 100 000 g for 30 min and the recombinant enzymes
were purified from the supernatant. The His6 -tagged recombinant
APXs were purified on a column packed with TALON metalaffinity resin (Clontech). The enzyme was eluted with 50 mM
sodium acetate buffer, pH 5.0, containing 1 mM AsA. The pooled
enzyme fractions were subsequently concentrated and exchanged
into 50 mM potassium phosphate buffer, pH 7.0, containing 1 mM
AsA using a Centricon® ultrafiltration membrane (Millipore) to
adjust to neutral pH.
Assay of APX activity
APX activity was measured as described previously [13].
Briefly, the reaction mixture contained 50 mM potassium
phosphate buffer, pH 7.0, 1 mM EDTA, 0.4 mM AsA and
0.1 mM H2 O2 . The oxidation of AsA was followed by a
decrease in absorbance at 290 nm (ε = 2.80 mM−1 ·cm−1 ).
The specificity of APX for alternate electron donors was
measured in the same assay mixture except that the AsA was
replaced by 20 mM pyrogallol (ε430 = 2.47 mM−1 ·cm−1 ), 10 mM
guaiacol (ε470 = 22.6 mM−1 ·cm−1 ), 0.4 mM D-isoAsA (ε290 = 3.30
mM−1 ·cm−1 ), 0.15 mM NADPH (ε340 = 6.22 mM−1 ·cm−1 ) or
40 μM reduced cytochrome c (ε550 =19.0 mM−1 ·cm−1 ). The
activity of APX with organic peroxides was also assayed using
the same reaction mixture, but H2 O2 was replaced with 0.1 mM
t-butyl hydroperoxide or 0.1 mM cumene hydroperoxide. The
protein concentration was measured with the Coomassie Blue
protein assay reagent (Bio-Rad Laboratories).
Gel-filtration analysis
The recombinant APX-N and APX-C enzymes were mixed
and incubated with an equal amount of protein (0.4 mg)
at 4 ◦C for 1 h. The samples were then subjected to chromatography on a Superdex 200 column (1.0 cm × 30 cm; GE
Healthcare) equilibrated with 10 mM potassium phosphate
buffer, pH 7.0, containing 1 mM AsA and 150 mM NaCl.
The column was calibrated with Blue Dextran 2000, albumin,
ovalbumin, chymotrypsinogen A and ribonuclease A standards
(GE Healthcare). Fractions (0.5 ml) were collected, and the
positions of the APX elution were determined by a direct assay
of the enzyme activity.
Immunoblot analysis
Proteins were separated by SDS/PAGE (12.5 % gel) and blotted
on to an Immobilon PVDF membrane (Millipore), with transfer
buffer as described previously [16] using a semi-dry electroblot
apparatus (Taitec). The blot was incubated with a monoclonal
antibody (EAP1; [13]) raised against the purified Euglena
APX, which was then detected using a secondary horseradish
peroxidase-conjugated goat anti-(mouse IgG) antibody (Cappel)
and Western Lighting Chemiluminescence Reagent Plus
(PerkinElmer) using a luminescence imager (AE-6962C, ATTO).
Genomic Southern blotting analysis
Genomic DNA was isolated from Euglena using a standard
method, as described previously [17]. The DNA (20 μg) was
digested overnight with HindIII or SacI and separated on a
0.8 % agarose gel. The gel was blotted on to a Hybond
N+ membrane (Amersham Pharmacia Biotech) and hybridized
with [α-32 P]dCTP-labelled probes. The probes were prepared
Molecular characterization of Euglena ascorbate peroxidase
127
from cDNAs for the mature FL-APX, the truncated APX-N or
APX-C, by PCR, as described above. The labelling was performed
by random priming using BcaBESTTM polymerase (Takara).
Hybridization was carried out in Hybri-Max (Ambion) at 60 ◦C for
16 h. The hybridized membrane was then washed with 0.1 × SSC
(1 × SSC is 0.15 M NaCl/0.015 M sodium citrate) containing
0.1 % SDS at 60 ◦C for 1 h and the autoradiography was carried
out with a multi bioimager (STORM 825; Amersham Pharmacia
Biotech).
kit (Takara) with an oligo(dT) primer. The PCR was performed using specific primers for APX (APX-CF, 5 -CTCGAGTACCAGCGCCTGGCGGAGC-3 ; APX-CR, 5 -AAGcttGCGGACGCGTGTGTGGCTGGC-3 ), and a polypeptide chain EF1-α
(elongation factor 1-α) from Euglena (accession no. X16890;
EF1α-F, 5 -ACAGATTGGGAACGGGTACGC-3 ; EF1α-R, 5 CGCAGTTTCCCTTCACCATCG-3 ) as a normalizer.
PCR with genomic DNA
The intercellular production of H2 O2 was measured using an
H2 O2 -specific BES (benzenesulfonyl) derivative of a fluorescein
probe, BES-H2 O2 -Ac (Wako). BES-H2 O2 -Ac was added to
the cells at a final concentration of 10 μM. After a 30-min
incubation at room temperature, the cells were collected by
microcentrifugation (1000 g for 5 min) and the supernatant
removed. Fluorescence was observed under a fluorescence
microscope (BX51; Olympus) with excitation and emission
wavelengths set at 485 and 530 nm respectively.
A partial Euglena APX gene inserted between the 3 region of APX-N and 5 -region of APX-C was amplified
from the genomic DNA by PCR using primers EgAPX-GF
(5 -GTATTTCCGGGACTTCGCC-3 ) and EgAPX-GR (5 -GCGCAGCCCTTCTCCGCCACG-3 ). The PCR product was cloned
into the pGEM-T easy vector and the resulting plasmid was
sequenced using a capillary DNA sequencer (ABI PRISM 3100Avant; Applied Biosystems).
Cell fractionation
A cell homogenate was obtained by a partial trypsin digestion
of the pellicle followed by mild mechanical disruption, and
subcellular fractionation by differential centrifugation was
performed as described previously [9].
Estimation of chlorophyll content
Chlorophyll was extracted from Euglena cells with 80 % (v/v)
acetone and its level estimated using the formula: total chlorophyll (μg·ml−1 )=chlorophyll a [12.7 × (A630 −2.69) × A645 ]+
chlorophyll b [22.9 × (A645 −4.68) × A663 ].
RNAi (RNA interference) experiments
Silencing of APX by RNAi was performed as described
previously [18]. An approx. 450-bp partial Euglena APX
cDNA was PCR-amplified with the addition of the T7 RNA
polymerase promoter sequence (underlined in the primer
sequences below) at one end. The primers were EgAPX/
RNAi-F (5 -TAATACGACTCACTATAGGGGACGAGGAGATCGTGGC-3 ) and EgAPX/RNAi-R (5 -TAATACGACTCACTATAGGGGCAAACACGCCTGGAAAGATATG-3 ). The sense
and antisense RNAs were synthesized using the PCR products as
templates (MEGAscript RNAi Kit; Ambion). After purification
of the transcribed RNA, with DNase I digestion followed
by ethanol precipitation, dsRNA (double-stranded RNA) was
made by annealing equimolar amounts of the sense and
antisense RNAs. Euglena cells from 2-day-old cultures were
collected and resuspended in culture medium containing 4.2 mM
calcium nitrate, 3.7 mM monopotassium phosphate and 2.1 mM
magnesium sulfate. The cell suspension (150 μl; approx. 1 × 106
cells) was transferred to a 0.4-cm-gap cuvette and electroporated
with 5 μl of RNA solution (15 μg of APX-dsRNA in 50 mM
Tris/HCl, pH 7.5, containing 1 mM EDTA) using Gene Pulser
II (Bio-Rad Laboratories) at 0.5 kV and 25 μF. After a 30 min
incubation at room temperature (25 ◦C), the cell suspension was
diluted with fresh Koren–Hutner medium and cultured at 26 ◦C
for restoration.
RT (reverse transcription)–PCR
Total RNA was prepared from wild-type and dsRNA-introduced
Euglena cells using RNAiso reagent (Takara). The first strand
cDNA was synthesized using a PrimeScript II first strand cDNA
Detection of cellular H2 O2
RESULTS
Euglena APX contains two homologous catalytic domains, forming
an intramolecular dimeric structure
To isolate a full-length cDNA encoding Euglena APX, we
adopted a PCR-based oligo-capped method utilizing primers
corresponding to sequences of an EST clone and a partial cDNA
isolated previously [19]. The final clone containing the full-length
cDNA, as indicated by the presence of a 5 -TTTTTTTTCG-3
spliced leader at the 5 end [20], was 2403 bp long with a
coding region of 1644 nucleotides and encoded a protein of
649 amino acid residues (see Supplementary Figure S1 available at http://www.BiochemJ.org/bj/426/bj4260125add.htm). The
cDNA obtained had an exceptionally high GC content of
70.8 %. The N-terminal and internal peptide sequences that were
identified previously in the purified Euglena APX could be
found in the deduced amino acid sequence of the cDNA [13].
The exact matching of these amino acid sequences confirmed
the authenticity of the cDNA. As judged from the N-terminal
sequence of the purified APX, the deduced amino acid sequence
had an N-terminal extension of 102 residues. A detailed analysis
of this extension will be described below. Therefore the predicted
mature FL-APX consisted of 547 amino acids with a molecular
mass of 60075 Da, which approximately agreed with the value
obtained previously by SDS/PAGE for the purified native enzyme
[13].
Interestingly, the primary structure of the Euglena APX reveals
that it contained two homologous catalytic domains (designated
APX-N and APX-C), which were 68.9 % identical with each
other (Figure 1). Both domains had conserved distal and proximal
histidine residues and contained all of the residues known to be
essential for catalysis among the APX proteins in higher plants
[2]. The Euglena enzyme is the only APX known to have two
catalytic domains. Computer-assisted comparison of the deduced
amino acid sequences of APX-N and APX-C with sequences in the
database revealed approx. 38–54 % sequence identity with APX
proteins from various species. The phylogenetic relationships of
various APX proteins, including Euglena APX, were determined
by constructing an unrooted tree using the ClustalW program
(Figure 2). This suggested that the two Euglena APX domains
are closest to that of Trypanosoma cruzi and Leishmania major,
separating into their own protozoa clade. In this clade, there is a
common feature distinguishing the enzymes from other plant-type
APX families; the presence of a 16-amino-acid insertion near the
C-terminal region (Figure 1B).
c The Authors Journal compilation c 2010 Biochemical Society
128
Figure 1
T. Ishikawa and others
Sequence alignment of two Euglena APX domains with APXs from pea, soybean and Leishmania
(A) Schematic of the Euglena FL-APX. (B) Comparison of the predicted amino acid sequences of the APX-N and APX-C domains from Euglena with APXs from Leishmania major (L.major; TrEMBL
CAJ07706), Glycine max (Soybean; TrEMBL L10292) and Pisum sativum (Pea; SwissProt CAA43992). The putative linker region between the N-domain and C-domain is underlined. Letters shown
in white on a black background represent conserved amino acids, which are correlated with active sites. The residues involved in AsA-binding are shown in black letters on a grey background. The
boxes show the conserved amino acid residues surrounding distal and proximal histidine-containing regions. ·, homologous residues; ∗, conserved residues.
To gain information on the structure of the Euglena APX,
harbouring the two homologous domains, a computer model for
the three-dimensional structure was constructed. Pea cytosolic
APX (PDB code 1APX), having 54 % and 49 % sequence
identity with the APX-N and APX-C domains respectively, was
selected as the template for modelling, which was performed
with the Molecular Operating Environment molecular graphics
package (Chemical Computing Group). The best model was
chosen using the energy function included with the software.
As shown in Figure 3, the superimposed model indicated that
the overall structure of each domain matched well with that of
the pea cytosolic APX, suggesting that the Euglena APX forms
an intramolecular dimer, hinged by the middle of the structure
including residues 377–385.
Subcellular localization of APX in Euglena
As described above, the deduced Euglena APX had an Nterminal extension of 102 amino acid residues. The TMHMM
(transmembrane helices based on a hidden Markov model)
program [20a] for the estimation of potential membrane-spanning
regions showed the presence of one significant hydrophobic
region in the extension (see Supplementary Figure S2 available
at http://www.BiochemJ.org/bj/426/bj4260125add.htm), a typical
c The Authors Journal compilation c 2010 Biochemical Society
feature of Euglena class II plastid signals proposed by Durnford
and Gray [21]. Therefore to re-assess the cellular localization
of APX in Euglena, we carried out subcellular fractionation by
differential centrifugation. The activity and protein of Euglena
APX were found only in the cytosol, not in organelles including
chloroplasts (Figure 4).
Genomic Southern blot and PCR analysis of the APX gene
We next performed genomic Southern hybridization. The genomic
DNA was digested with HindIII and SacI. The former enzyme
does not digest within the ORF (open reading frame) of APX
and the latter recognizes the border region between APX-N
and APX-C. When the cDNA fragments prepared for the mature
FL-APX, APX-N and APX-C were used as the probe, several
hybridization signals were detected with almost the same pattern
(Figure 5). These observations suggest that the Euglena genome
has multiple copies of the APX gene. Then, PCR of the genomic
DNA, utilizing primers from the 3 - and 5 -terminal sites of
the APX-N and APX-C domains respectively was performed,
yielding a single band. Sequencing revealed that both the APX-N
and APX-C domains were in the same segment of the gene
and in a cis-configuration, separated by an intron containing
536 nucleotides (see Supplementary Figure S3 available at
http://www.BiochemJ.org/bj/426/bj4260125add.htm).
Molecular characterization of Euglena ascorbate peroxidase
Figure 2
129
Phylogenic tree for Euglena APX and other orthologous APX proteins
The phylogenic tree was constructed using the ClustalW program and visualized with TreeView. For the analysis, the amino acid regions 103–376 and 377–649 were used as the template of APX-N
and APX-C respectively. The abbreviations and UniProt accession numbers for the APX orthologues are as follows: At, Arabidopsis thaliana (At.APX1, X59600; At.sAPX, X98925; At.tAPX, X98926);
Cka, Cucurbita cv. Kurokawa Amakuri (Cka.mAPX, AB070626; Cka.tAPX, D83656); Gm, Glycine max (Gm.APX1, L10292); Hv, Hordeum vulgare (Hv.APX1, AB063117); Mc, Mesembryanthemum
crystallinum (Mc.cAPX2, U43561; Mc.mAPX, AF139190; Mc.tAPX, AF069315); Nt, Nicotiana tabacum (Nt.APX, D85912; Nt.tAPX, AB022273); Os, Oryza sativa (Os.APX1, D45423; Os.APX2,
AB053297; Os.APX3, AY382617); So, Spinacia oleracea (So.APX1, D85864; So.APX2, D49679; So.mAPX, D84104; So.tAPX, D77997); Ps, Pisum sativum (Ps.APX, X62077); W80, Chlamydomonas
sp. W80 (W80.sAPX, AB009084); Cr, Chlamydomonas reinhardtii (Cr.sAPX, AJ223325); Tc, Trypanosoma cruzi (Tc.APX, AJ457987); Lm, Leishmania major (Lm.APX, XM_001686044); and Yl,
Yarrowia lipolytica (Yl.APX, XM_503271).
Figure 3
Hypothetical structure model of Euglena APX
The model was generated based on pea cytosolic APX (PDB code 1APX). Euglena APX and pea cytosolic APX are shown in blue and yellow green respectively. The haem molecules are indicated as
a stick format.
c The Authors Journal compilation c 2010 Biochemical Society
130
Figure 4
T. Ishikawa and others
Subcellular localization of APX in Euglena
Subcellular fractionation of lysate from Euglena cells was performed as described in the
Experimental section. Aliquots of fractions containing an equal amount of protein (20 μg)
were subjected to SDS/PAGE (12.5 % gel) and analysed by immunoblotting using the EAP1
monoclonal antibody. The amount of chlorophyll and the level of APX activity in each fraction
are also shown. sup, supernatant; ppt, pellet.
Enzymological properties of the recombinant Euglena APX
We prepared the recombinant mature FL-APX and the APX-N
and APX-C domains, and then compared their catalytic activities.
As shown in Figure 6(A), the recombinant proteins expressed
in E. coli were purified to homogeneity by SDS/PAGE, and
had a size of 60 kDa and 30 kDa for the mature FL-APX
and the individual domains respectively, in good agreement
with the molecular mass calculated from the predicted amino
acid sequence. The recombinant enzymes showed an absorption
spectrum characteristic of the high-spin ferric state of haem
proteins. Soret absorption peaks for recombinant FL-APX,
APX-N and APX-C were found at 410, 413 and 411 nm
respectively and shifted to 430, 431 and 435 nm after reduction of
each protein by the addition of dithionate (Figure 6B). In addition,
cyanide complexes of recombinant FL-APX, APX-N and
APX-C exhibited peaks at 421, 421 and 424 nm respectively
and additional peaks at 542, 542 and 540 nm. Table 1 shows
a comparison of the enzymatic properties of recombinant and
native APXs. The recombinant enzymes reduced H2 O2 using AsA,
obeying Michaelis–Menten kinetics, with apparent K m values
for both AsA and H2 O2 comparable with those of the native
Euglena APX. Furthermore, the specific activity of the purified
recombinant FL-APX, APX-N and APX-C was 330, 371 and
Figure 5
306 μmol·min−1 ·mg−1 of protein respectively, close to that of
the purified native enzyme. Therefore both the turnover rate (kcat )
and catalytic efficiency (kcat /K m ) of the recombinant FL-APX for
AsA and H2 O2 were comparable with those of the native enzyme;
however, APX-N and APX-C had kcat and kcat /K m values approx.
2-fold lower than FL-APX and the native APX. Moreover, the
substrate specificity of the mature FL-APX differed from that
of the truncated enzymes. In a similar manner to the native
enzyme, the recombinant FL-APX exhibited significant activity
toward alkyl hydroperoxides, such as t-butyl hydroperoxide and
cumene hydroperoxide, whereas neither APX-N nor APX-C
had any activity. The purified APX-N and APX-C domains
mixed in a 1:1 molar ratio also showed no activity toward alkyl
hydroperoxides (results not shown). No dimerization of APX
on the simple incubation of APX-N and APX-C was observed;
a protein peak at approx. 60 kDa was not found during sizeexclusion chromatography with the recombinant protein mixture
(results not shown).
Effect of suppressing APX on the cellular H2 O2 level
To determine the physiological role of APX in the metabolism
of H2 O2 in Euglena cells, we temporarily suppressed APX
expression using RNAi. A dsRNA synthesized from part of
the Euglena APX sequence, corresponding to the APX-C
domain, was introduced into Euglena cells by electroporation.
No significant difference was observed in phenotype, including
cell growth and cell shape, between dsRNA-containing cells
and control cells electroporated with buffer only (results not
shown). RT–PCR of the dsRNA-containing cells showed only
a faint amplified band for the target APX, indicating that
endogenous APX mRNA is degraded after introduction of the
dsRNA (Figure 7A). The APX activity of dsRNA-containing
cells decreased to approx. 20 % of the control value (Figure 7B).
To determine intracellular H2 O2 levels in control and dsRNAcontaining cells, we used a chemical fluorescent probe, BESH2 O2 -Ac, recently developed for the non-invasive measurement of
intercellular H2 O2 in vivo [22]. As shown in Figure 7(C), dsRNAcontaining cells showed a prominent increase in fluorescence of
the probe, indicating an accumulation of cellular H2 O2 upon the
suppression of APX expression.
Genomic Southern blot analysis of the Euglena APX gene
Samples of genomic DNA from Euglena (20 μg/lane) were digested with HindIII and SacI, electrophoresed in agarose gels and transferred on to blotting membranes. Probes corresponding to coding
regions of mature FL-APX, APX-N and APX-C were labelled with 32 P and hybridized to the blots under conditions of moderate stringency.
c The Authors Journal compilation c 2010 Biochemical Society
Molecular characterization of Euglena ascorbate peroxidase
Table 1
131
Comparison of substrate specificity and kinetic parameters of the recombinant Euglena APX with those of the native and soybean enzyme
Donor and peroxide specificities were determined using 0.1 mM H2 O2 and 0.4 mM AsA respectively, and are the mean of three determinations. The kinetics values are estimated from the data of three
replicate experiments and are means +
− S.D.. Statistical analysis by Student’s t test indicated that all values in each row were significantly different at P < 0.05 except the pairs indicated by a, b, c
and d. CumOOH, cumene hydroperoxide; n.d., not determined; t-BOOH, t-butyl hydroperoxide; –, not tested.
Figure 6
Parameter
rFL-APX
rAPX-N
rAPX-C
Native [13,44]
Soybean [45,46]
Molecular mass (kDa)
Donor specificity (%)
AsA
D-IsoAsA
Cytochrome c
NADPH
Pyrogallol
Guaiacol
Peroxide specificity (%)
H2 O2
t-BOOH
CumOOH
Linoleic acid hydroperoxide
K m (μM)
AsA
H 2 O2
k cat (s−1 )
AsA
H2 O2
k cat /K m (s−1 ·μM−1 )
AsA
H2 O2
60
31
30
58
28.3
100
60.3
–
0
90.1
0
100
57.5
–
0
77.8
0
100
71.8
–
0
70.2
0
100
64.5
0
0
200
0
100
–
–
0
372
95.6
100
17.4
8.0
n.d.
100
n.d.
n.d.
n.d.
100
n.d.
n.d.
n.d.
100
68.1
52.1
–
493 +
− 17.4
35 +
− 8.2
458 +
− 15.3
30 +
− 1.2
492 +
− 13.5
43 +
− 1.3
410
56
389 +
− 64
–
754 +
− 83
577 +
− 42
a
361 +
− 41b
177 +
20
−
a
365 +
− 47b
170 +
16
−
460
460
272 +
− 32
–
1.53
16.5
0.78c
5.90d
0.74c
3.95d
1.12
8.21
0.69
–
SDS/PAGE and absorption spectra of purified recombinant Euglena APX proteins
(A) The recombinant His6 -tagged proteins were extracted from E. coli BL21 Star cells transformed with pColdII containing the mature FL-APX (rFL-APX), in which the first 102 amino acids had
been removed and the truncated APX, APX-N (rAPX-N) and APX-C domains (rAPX-C) were then purified in a Ni-NTA (Ni2+ -nitrilotriacetate) column. The samples were separated by SDS/PAGE and
visualized with Coomassie Brilliant Blue. (B) The absorption spectrum of each APX (1 μM) was observed in 50 mM potassium phosphate buffer, pH 7.0, containing 1 mM AsA and 1 mM EDTA. The
reduced and CN− -bound forms were measured by the addition of 1 mM dithionite (+dithionate) and 1 mM KCN (+KCN) respectively.
c The Authors Journal compilation c 2010 Biochemical Society
132
Figure 7
T. Ishikawa and others
Suppressing APX expression promotes H2 O2 accumulation in Euglena cells
(A) RT–PCR analysis of APX and EF1-α (for normalization) mRNA levels using total RNA from Euglena cells transfected with (+ dsRNA) and without (− dsRNA) dsRNA. Fragments corresponding
to APX and EF1-α were amplified by PCR using the cDNA preparations as templates. (B) APX activity in crude extracts prepared from Euglena cells. (C) Detection of cellular H2 O2 by fluorescence
microscopy. Cellular H2 O2 was measured using a H2 O2 -specific fluorescence probe, BES-H2 O2 -Ac. Cultures of representative cells were photographed 1 week after the dsRNA was introduced.
DISCUSSION
Euglena APX has unique biochemical properties among the
APX isoenzymes reported to date [13]. The molecular mass of
the enzyme is twice that of plant APXs. Moreover, the plant
isoenzymes are highly specific for H2 O2 as an electron acceptor,
whereas Euglena APX shows a broad specificity, recognizing
alkyl hydroperoxide in addition to H2 O2 [13]. On the basis of
the results in the present study, we draw three conclusions that
are relevant to the function and physiological role of APX in
Euglena. First, sequencing revealed that Euglena APX consists
of two homologous domains (APX-N and APX-C) and hence it
has a unique intramolecular dimeric structure, which is highly
relevant to its ability to recognize substrates. Secondly, Euglena
APX was distributed only in the cytosol after cleavage of the Nterminal extension typical of a class II plastid signal in Euglena.
Thirdly, the suppression of APX expression via the introduction
of dsRNA confirmed that Euglena APX plays a significant role in
the metabolism of H2 O2 .
Molecular and enzymatic properties of Euglena APX
The primary structure of Euglena APX proved to be unique
because of the presence of two nearly identical domains, APX-N
and APX-C, and the 102-amino-acid extension in the N-terminal
region (Figure 1). It is worth noting that the dimerized form of
the mature FL-APX ‘intramolecular dimer’ is a novel structure,
with tandem homologous domains in a single polypeptide. A
comparison of the deduced amino acid sequence of the two
domains of Euglena APX with those from known APX proteins
revealed that the domains are similar to almost all the catalytic
regions of the plant isoenzymes, and are more closely related to
protozoan-type APX families than plant APXs (Figures 1 and 2).
Previous crystallographic studies of pea and soybean cytosolic
APX have identified several amino acid residues involved in
substrate recognition, the reaction mechanism and stabilizing
the enzyme [23–25]. Curiously, most of those key residues are
conserved in the Euglena APX. APX is classified as a haem
c The Authors Journal compilation c 2010 Biochemical Society
containing Class I peroxidase [26]. This class shares a common
feature, the presence of distal and proximal histidine-containing
regions for binding a haem ligand. From sequence alignments, as
shown in Figure 1, the conserved distal and proximal histidine
residues of APX-N and APX-C are His-159 and His-417, and
His-283 and His-540 respectively. Additionally, these residues
are flanked by catalytic residues analogous to Arg-38, Trp-41,
Asp-208 and Trp-179 of the pea cytosolic APX. The two domains
of Euglena APX also contained Cys-32 of the pea APX, which
is situated close to the AsA-binding site and is conserved in all
known isoforms of APX [2,3]. Arg-172 of pea APX plays an
important role in the utilization of AsA to form Compound II
[27]. Moreover, the crystal structure of the soybean APX and
AsA complex indicates that Lys-30 contributes to the substrate
binding [24]. These residues are conserved in both APX-N and
APX-C of Euglena APX. The sequences also contained the metalbinding five-residue K+ site (comprising Asp-187, Thr-164, Thr180, Asn-182 and Ile-185) [28], although Ile-185 was replaced
by a lysine residue and a valine residue in APX-N and APX-C
respectively. A comparison of cytosolic APX from soybean with
cytochrome c peroxidase showed that APX does not contain an
additional loop structure which blocks the AsA-binding site of
cytochrome c peroxidase (residues 34–41) [24]. The sequence
alignment (Figure 1) and structure model (Figure 3) indicated
that the loop structure of Euglena APX was missing in both
the APX-N and APX-C domains, as well as in cytosolic APX
from soybean and pea. Therefore, consistent with the idea that
the truncated recombinant enzymes would be catalytically active,
we found the kinetics of the recombinant APX-N and APX-C
to be comparable with that of the plant cytosolic APX and the
native Euglena APX with AsA and H2 O2 (Table 1). However,
in contrast with the native enzyme, the recombinant APX-N
and APX-C domains did not show any activity towards alkyl
hydroperoxide (Table 1). On the other hand, the recombinant FLAPX exhibited catalytic activity towards alkyl hydroperoxide,
like the native enzyme, indicating that the intramolecular dimeric
structure is correlated with substrate recognition. Badyal et al.
[29] have recently reported that cytosolic APX potentially has a
Molecular characterization of Euglena ascorbate peroxidase
binding ability with t-butyl hydroperoxide, but does not form
the Compound I intermediate. Although it is difficult to explain the mechanism for substrate recognition for Euglena APX,
the conformational alteration of the mature APX by the formation
of the intramolecular dimer may facilitate its activity toward alkyl
hydroperoxide.
In euglenoids, trans-splicing has been described as a
mechanism for the generation of mature mRNAs [30]. It is
apparent that the 5 end of mRNA is replaced by a short spliced
leader exon from a small spliced leader RNA, as can be seen at the
5 end of the full-length cDNA of Euglena APX (Supplementary
Figure S1). We suspected that the mature APX mRNA, except its
5 end, was generated by trans-splicing or cis-splicing machinery.
A genomic Southern analysis using individual probes from APXN and APX-C produced similar signalling patterns (Figure 5).
In addition, a partial analysis of the genomic sequence indicated
that both domains definitely exist in the same segment of the gene
(Supplementary Figure S3). The border sequences of exon/introns
in APX-N and APX-C did not follow the canonical GT/AG rule,
but non-conventional introns have been observed in other genes
in Euglena [31–33]. Accordingly, we concluded that the APX
mRNA is generated by a cis-splicing mechanism. Presumably,
Euglena has incidentally acquired its unique APX gene through
the tandem repetition of a nearly identical sequence during
evolution.
Surprisingly, the primary structure of Euglena APX contained
an N-terminal extension of 102 amino acid residues, which
was very similar to a plastid signal of class II-type in Euglena
(Supplementary Figure S2; [21]), although the mature protein
was localized to the cytosol (Figure 4). As far as we know, this
is the first case of a protein that requires processing in order
to distribute in the cytosol. It is worth considering a route via
the ER (endoplasmic reticulum) for transporting APX into the
cytosol, because this kind of signal in Euglena, unlike other known
photosynthesizing organisms, utilizes a transport vesicle and a
hydrophobic region in the signal sequence acts as a stop transfer
sequence to prevent complete transfer into the ER, so that the
mature protein remains in the cytoplasm [34,35].
133
the cytosolic APX to manage the level of cellular H2 O2 . We
have shown previously that the expression of Euglena APX
is post-transcriptionally regulated during the development of
proplastids into mature functional chloroplasts in response to
light illumination, also supporting its role in the regulation of
H2 O2 generated in the chloroplasts [19].
In higher plants, the cytosolic APX has many subtle functions
in controlling cellular redox conditions. Among various APX
isoenzymes, the cytosolic form is known to be highly responsive
to oxidative stresses, including high-light exposure [38,39]. A
previous transcriptomic analysis also indicated the susceptibility
of the cytosolic APX to environmental stress [40]. Furthermore,
an Arabidopsis mutant lacking APX1, one of the typical cytosolic
isoenzymes, showed a significant increase in cellular H2 O2 , and
an up-regulation of the expression of various genes, including
those in response to stress [41,42]. We reported previously that
suppression of a cytosolic APX by gene silencing in tobacco
BY-2 cells resulted in a significant increase in cellular H2 O2 ,
and cross-tolerance to heat and salinity stress, accompanied by
the activation of a protein kinase and the up-regulation of stressresponsive gene expression [43]. Thus the cytosolic APX in higher
plants is emerging as a key enzyme for the redox gene network
via H2 O2 metabolism. Although the redox regulation of cellular
function including gene expression in eukaryotic algae is still
largely unknown, the silencing of cytosolic APX in Euglena will
provide a clue as to the redox signalling of eukaryotic algae.
AUTHOR CONTRIBUTION
Takahiro Ishikawa designed the study and wrote the manuscript with inputs from all coauthors. Naoko Tajima, Hitoshi Nishikawa, Yongshun Gao and Madhusudhan Rapolu
performed experiments. Yoshihiro Sawa performed the computational analysis and
generated the enzyme structure model. Hitoshi Shibata supervised data analysis. Shigeru
Shigeoka co-ordinated all aspects of the project and edited the manuscript. All authors
discussed the results and commented on the manuscript.
FUNDING
Contribution of APX to H2 O2 metabolism in Euglena cells
The suppression of APX expression by RNAi indicated that
Euglena APX physiologically contributes to the metabolism
of H2 O2 in cells (Figure 7). We demonstrated previously that
Euglena cells produce H2 O2 during ordinary metabolic processes
and electron transport in chloroplasts and mitochondria [36].
In the case of higher plants, APX isoenzymes distributed in
chloroplasts, the cytosol and microbodies play a key role in the
metabolism of H2 O2 at the site of its generation [1,2]. It is of
interest that the Euglena APX is present only in the cytosol
and leads us to question how it manages the intracellular
level of H2 O2 in various cellular compartments. Chloroplasts
are the major site for the generation of H2 O2 due to an
abundance of O2 produced by photochemical reactions and
concomitant photosynthetic electron transport. Photosynthesis
in higher plants is highly susceptible to H2 O2 , but that
in Euglena is not, due to the resistance of the fructose1,6-/sedoheptulose-1,7-bisphosphatase, NADP+ -glyceraldehyde3-phosphate dehydrogenase and ribulose-5-phosphate kinase
enzymes of the Calvin cycle to H2 O2 at up to 1 mM
[37]. Furthermore, H2 O2 generated in both chloroplasts and
mitochondria in Euglena cells diffused into the cytosol, where
it was decomposed by APX [36]. Taken together, H2 O2 -tolerance
and -diffusion systems in Euglena cells would help to allow
This work was supported in part by the Ministry of Education, Culture, Sports, Science
and Technology of Japan (Grant-in-aid for Scientific Research) [grant numbers 19208031
and 21380207 (to S.S. and T.I.)].
REFERENCES
1 Shigeoka, S., Ishikawa, T., Tamoi, M., Miyagawa, Y., Takeda, T., Yabuta, Y. and Yoshimura,
K. (2002) Regulation and function of ascorbate peroxidase isoenzymes. J. Exp. Bot. 53,
1305–1319
2 Ishikawa, T. and Shigeoka, S. (2008) Recent advances in ascorbate biosynthesis and the
physiological significance of ascorbate peroxidase in photosynthesizing organisms.
Biosci. Biotechnol. Biochem. 72, 1143–1154
3 Mittler, R. and Poulos, T. L. (2005) Ascorbate peroxidase. In Antioxidants and Reactive
Oxygen Species in Plants (Smirnoff, N., ed.), pp. 87–100, Blackwell Publishing, Oxford
4 Mittler, R. (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7,
405–410
5 Chew, O., Whelan, J. and Millar, A. H. (2003) Molecular definition of the
ascorbate-glutathione cycle in Arabidopsis mitochondria reveals dual targeting of
antioxidant defenses in plants. J. Biol. Chem. 278, 46869–46877
6 Teixeira, F. K., Menezes-Benavente, L., Margis, R. and Margis-Pinheiro, M. (2004)
Analysis of the molecular evolutionary history of the ascorbate peroxidase gene family:
inferences from the rice genome. J. Mol. Evol. 59, 761–770
7 Najami, N., Janda, T., Barriah, W., Kayam, G., Tal, M., Guy, M. and Volokita, M. (2008)
Ascorbate peroxidase gene family in tomato: its identification and characterization.
Mol. Genet. Genomics 279, 171–182
8 Shigeoka, S., Nakano, Y. and Kitaoka, S. (1980) Metabolism of hydrogen peroxide in
Euglena gracilis Z by L-ascorbic acid peroxidase. Biochem. J. 186, 377–380
c The Authors Journal compilation c 2010 Biochemical Society
134
T. Ishikawa and others
9 Shigeoka, S., Yasumoto, R., Onishi, T., Nakano, Y. and Kitaoka, S. (1987) Properties of
monodehydroascorbate reductase and dehydroascorbate reductase and their participation
in the regeneration of ascorbate in Euglena gracilis . J. Gen. Microbiol. 133,
227–232
10 Takeda, T., Yoshimura., K., Ishikawa, T. and Shigeoka, S. (1998) Purification and
characterization of ascorbate peroxidase in Chlorella vulgaris . Biochimie 80,
295–301
11 Sano, S., Ueda, M., Kitajima, S., Takeda, T., Shigeoka, S., Kurano, N., Miyachi, S.,
Miyake, C. and Yokota, A. (2001) Characterization of ascorbate peroxidases from
unicellular red alga Galdieria partita . Plant Cell Physiol. 42, 433–440
12 Takeda, T., Ishikawa, T. and Shigeoka, S. (1997) Metabolism of hydrogen peroxide by the
scavenging system in Chlamydomonas reinhardtii . Physiol. Plant. 99, 49–55
13 Ishikawa, T., Takeda, T., Kohno, H. and Shigeoka, S. (1996) Molecular characterization of
Euglena ascorbate peroxidase using monoclonal antibody. Biochim. Biophys. Acta 1290,
69–75
14 Koren, L. E. and Hutner, S. H. (1967) High-yield media for photosynthesizing Euglena
gracilis Z. J. Protozool. 14, 17
15 Madhusudhan, R., Ishikawa, T., Sawa, Y., Shigeoka, S. and Shibata, H. (2003)
Post-transcritional regulation of ascorbate peroxidase during light adaptation of Euglena
gracilis . Plant Sci. 165, 233–238
16 Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc.
Natl. Acad. Sci. U.S.A. 76, 4350–4354
17 Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory
Manual, 2nd edn, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
18 Ishikawa, T., Nishikawa, H., Gao, Y., Sawa, Y., Shibata, H., Yabuta, Y., Maruta, T. and
Shigeoka, S. (2008) The pathway via D-galacturonate/L-galactonate is significant for
ascorbate biosynthesis in Euglena gracilis : identification and functional characterization
of aldonolactonase. J. Biol. Chem. 283, 31133–31141
19 Rapolu, M., Ishikawa, T., Sawa, Y., Shigeoka, S. and Shibata, H. (2003)
Post-transcriptional regulation of ascorbate peroxidase during light adaptation of Euglena
gracilis . Plant Sci. 165, 233–238
20 Tessier, L. H., Keller, M., Chan, R. L., Fournier, R., Weil, J. H. and Imbault, P. (1991) Short
leader sequences may be transferred from small RNAs to pre-mature mRNAs by
trans-splicing in Euglena . EMBO J. 10, 2621–2625
20a Krogh, A., Larsson, B., von Heijne, G. and Sonnhammer, E. L. L. (2001) Predicting
transmembrane protein topology with a hidden Markov model: application to complete
genomes. J. Mol. Biol. 305, 567–580
21 Durnford, D. G. and Gray, M. W. (2006) Analysis of Euglena gracilis plastid-targeted
proteins reveals different classes of transit sequences. Eukaryotic Cell 5, 2079–2091
22 Maeda, H. (2008) Which are you watching, an individual reactive oxygen species or total
oxidative stress? Ann. N.Y. Acad. Sci. 1130, 149–156
23 Raven, E. L. (2000) Peroxidase-catalyzed oxidation of ascorbate: structural, spectroscopic
and mechanistic correlations in ascorbate peroxidase. Subcell. Biochem. 35, 317–349
24 Sharp, K. H., Mewies, M., Moody, P. C. and Raven, E. L. (2003) Crystal structure of the
ascorbate peroxidase–ascorbate complex. Nat. Struct. Biol. 10, 303–307
25 Wada, K., Tada, T., Nakamura, Y., Ishikawa, T., Yabuta, Y., Yoshimura, K., Shigeoka, S. and
Nishimura, K. (2003) Crystal structure of chloroplastic ascorbate peroxidase from
tobacco plants and structural insights into its instability. J. Biochem. 134, 239–244
26 Welinder, K. G. (1992) Superfamily of plant, fungal and bacterial peroxidases. Curr. Opin.
Struct. Biol. 2, 388–393
27 Bursey, E. H. and Poulos, T. L. (2000) Two substrate binding sites in ascorbate
peroxidase: the role of arginine 172. Biochemistry 39, 7374–7379
Received 9 September 2009/16 December 2009; accepted 17 December 2009
Published as BJ Immediate Publication 17 December 2009, doi:10.1042/BJ20091406
c The Authors Journal compilation c 2010 Biochemical Society
28 Cheek, J., Mandelman, D., Poulos, T. L. and Dawson, J. H. (1999) A study of the K+ -site
mutant of ascorbate peroxidase: mutations of protein residues on the proximal side of the
heme cause changes in iron ligation on the distal side. J. Biol. Inorg. Chem. 4, 64–72
29 Badyal, S. K., Eaton, G., Mistry, S., Pipirou, Z., Basran, J., Metcalfe, C. L., Gumiero, A.,
Handa, S., Moody, P. C. and Raven, E. L. (2009) Evidence for heme oxygenase activity in a
heme peroxidase. Biochemistry 48, 4738–4746
30 Frantz, C., Ebel, C., Paulus, F. and Imbault, P. (2000) Characterization of trans-splicing in
Euglenoids. Curr. Genet. 37, 349–355
31 Tessier, L. H., Chan, R. L., Keller, M., Weil, J. H. and Imbault, P. (1992) The Euglena
gracilis rbcS gene contains introns with unusual borders. FEBS Lett. 304, 252–255
32 Muchhal, U. S. and Schwartzbach, S. D. (1994) Characterization of the unique intron-exon
junctions of Euglena gene(s) encoding the polyprotein precursor to the light-harvesting
chlorophyll a /b -binding protein of photosystem II. Nucleic Acids Res. 22, 5737–5744
33 Canaday, J., Tessier, L. H., Imbault, P. and Paulus, F. (2001) Analysis of Euglena gracilis
α-, β and γ -tubulin genes: introns and pre-mRNA maturation. Mol. Genet. Genomics
265, 153–160
34 Sulli, C., Fang, Z., Muchhal, U. and Schwartzbach, S. D. (1999) Topology of Euglena
chloroplast protein precursors within endoplasmic reticulum to Golgi to chloroplast
transport vesicles. J. Biol. Chem. 274, 457–463
35 van Dooren, G. G., Schwartzbach, S. D., Osafune, T. and McFadden, G. I. (2001)
Translocation of proteins across the multiple membranes of complex plastids. Biochim.
Biophys. Acta 1541, 34–53
36 Ishikawa, T., Takeda, T., Shigeoka, S., Hirayama, O. and Mitsunaga, T. (1993) Hydrogen
peroxide generation in organelles of Euglena gracilis . Phytochemistry 33, 1297–1299
37 Takeda, T., Yokota, A. and Shigeoka, S. (1995) Resistance of photosynthesis to hydrogen
peroxide in algae. Plant Cell Physiol. 36, 1089–1095
38 Mittler, R. and Zilinskas, B. A. (1992) Molecular cloning and characterization of a gene
encoding pea cytosolic ascorbate peroxidase. J. Biol. Chem. 267, 21802–21807
39 Yoshimura, K., Yabuta, Y., Ishikawa, T. and Shigeoka, S. (2000) Expression of spinach
ascorbate peroxidase isoenzymes in response to oxidative stresses. Plant Physiol. 123,
223–234
40 Kim, D. W., Shibato, J., Agrawal, G. K., Fujihara, S., Iwahashi, H., Kim, du Hu, Shim, IeS.
and Rakwal, R. (2007) Gene transcription in the leaves of rice undergoing salt-induced
morphological changes (Oryza sativa L.). Mol. Cells 24, 45–59
41 Pnueli, L., Liang, H., Rozenberg, M. and Mittler, R. (2003) Growth suppression, altered
stomatal responses, and augmented induction of heat shock proteins in cytosolic
ascorbate peroxidase (Apx1)-deficient Arabidopsis plants. Plant J. 34, 187–203
42 Davletova, S., Rizhsky, L., Liang, H., Shengqiang, Z., Oliver, D. J., Coutu, J., Shulaev, V.,
Schlauch, K. and Mittler, R. (2005) Cytosolic ascorbate peroxidase 1 is a central
component of the reactive oxygen gene network of Arabidopsis . Plant Cell 17, 268–281
43 Ishikawa, T., Morimoto, Y., Rapolu, M., Sawa, Y., Shibata, H., Yabuta, Y., Nishizawa, A. and
Shigeoka, S. (2005) Acclimation to diverse environmental stresses caused by a
suppression of cytosolic ascorbate peroxidase in tobacco BY-2 cells. Plant Cell Physiol.
46, 1264–1271
44 Shigeoka, S., Nakano, Y. and Kitaoka, S. (1980) Purification and some properties of
L-ascorbic-acid-specific peroxidase in Euglena gracilis Z. Arch. Biochem. Biophys. 201,
121–127
45 Dalton, D. A., Hanus, F. J., Russell, S. A. and Evans, H. J. (1987) Purification, properties,
and distribution of ascorbate peroxidase in legume root nodules. Plant Physiol. 83,
789–794
46 Lad, L., Mewies, M. and Raven, E. L. (2002) Substrate binding and catalytic mechanism
in ascorbate peroxidase: evidence for two ascorbate binding sites. Biochemistry 41,
13774–13781
Biochem. J. (2010) 426, 125–134 (Printed in Great Britain)
doi:10.1042/BJ20091406
SUPPLEMENTARY ONLINE DATA
Euglena gracilis ascorbate peroxidase forms an intramolecular dimeric
structure: its unique molecular characterization
Takahiro ISHIKAWA*1 , Naoko TAJIMA*, Hitoshi NISHIKAWA*, Yongshun GAO*, Madhusudhan RAPOLU*2 , Hitoshi SHIBATA*,
Yoshihiro SAWA* and Shigeru SHIGEOKA†
*Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan, and
†Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan
Figure S1
Full-length cDNA sequence of Euglena APX
The amino acid sequences deduced from the ORF are shown below the nucleotide sequences. The amino acid sequences of N-terminal and proteolytic peptides of purified Euglena APX are underlined
(reference [13] in the main paper). The box indicates the putative linker region between the APX-N and APX-C domains. The double underline shows the spliced leader sequence. The arrowhead
indicates the cleavage site of the N-terminal leader sequence. The distal and proximal histidine residues are shown by 䊉.
1
To whom correspondence should be addressed (email [email protected]).
Present address: Department of Molecular Biosciences and Bioengineering, University of Hawaii, 1955 East–West Road, Honolulu, HI 96822, U.S.A.
The nucleotide sequence data reported for Euglena gracilis ascorbate peroxidase will appear in the DDBJ, EMBL, GenBank® and GSDB Nucleotide
Sequence Databases under accession number AB077953.
2
c The Authors Journal compilation c 2010 Biochemical Society
T. Ishikawa and others
Figure S2 Scatter plot showing TMHMM probability for the class II targeting
signal of the Euglena plastid
Potential membrane-spanning regions were identified using the TMHMM program (reference
[20a] in the main paper). The cluster ID numbers for proteins analysed are as follows: PsbW,
ELL00005545; PsaE, ELL00000098; phosphoribulokinase, ELL0000240; peptide chain release
factor 2, ELL00001542; chloroplast 50S ribosomal protein L15, ELL00001392. The N-terminal
extension of 102 amino acid residues of Euglena APX shows the presence of the hydrophobic
region associated with the common signal sequence in all class II proteins in Euglena . TMH1,
transmembrane helix 1.
Figure S3
Partial sequence of the Euglena APX gene amplified with primers extending over APX-N and APX-C regions
Non-coding regions are in lower case. Arrows indicates the primer sequences used for the amplification. The boxes show the border sequence of exon/introns.
Received 9 September 2009/16 December 2009; accepted 17 December 2009
Published as BJ Immediate Publication 17 December 2009, doi:10.1042/BJ20091406
c The Authors Journal compilation c 2010 Biochemical Society