Functional characterization of D-galacturonic acid reductase, a key enzyme of the ascorbate biosynthesis pathway, from Euglena gracilis

Article abstract of DOI:10.1271/bbb.60327

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38-39 kDa, as judged by SDS-PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62:5 ± 4:5 μM and uronic acids, such as D-galacturonic acid (Km = 3:79 ± 0:5 mM) and D-glucuronic acid (Km = 4:67 ± 0:6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP⁺. The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1mM H²O², suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.

Full text of DOI:10.1271/bbb.60327

Biosci. Biotechnol. Biochem., 70 (11), 2720–2726, 2006
Functional Characterization of D-Galacturonic Acid Reductase,
a Key Enzyme of the Ascorbate Biosynthesis Pathway,
from Euglena gracilis
y
Takahiro ISHIKAWA,1; Ikuko MASUMOTO,1 Naofumi IWASA,1 Hitoshi NISHIKAWA,1
Yoshihiro SAWA,1 Hitoshi SHIBATA,1 Ayana NAKAMURA,2
2
Yukinori YABUTA,2 and Shigeru SHIGEOKA
1Department of Life and Environmental Sciences, Shimane University, 1060 Nishikawatsu,
Matsue, Shimane 690-8504, Japan
2Department of Advanced Bioscience, Faculty of Agriculture, Kinki University,
3327-204 Nakamachi, Nara, Nara 631-8505, Japan
Received June 13, 2006; Accepted July 26, 2006; Online Publication, November 7, 2006
[doi:10.1271/bbb.60327]
D-Galacturonic acid reductase, a key enzyme in
ascorbate biosynthesis, was purified to homogeneity
from Euglena gracilis. The enzyme was a monomer with
a molecular mass of 38–39 kDa, as judged by SDS–
PAGE and gel filtration. Apparently it utilized NADPH
with a Km value of 62:5 ꢀ 4:5 ꢀM and uronic acids,
such as D-galacturonic acid (Km ¼ 3:79 ꢀ 0:5 mM) and
D-glucuronic acid (Km ¼ 4:67 ꢀ 0:6 mM). It failed to
catalyze the reverse reaction with L-galactonic acid
and NADPþ. The optimal pH for the reduction of D-
galacturonic acid was 7.2. The enzyme was activated
45.6% by 0.1 mM H2O2, suggesting that enzyme activity
is regulated by cellular redox status. No feedback
regulation of the enzyme activity by L-galactono-1,4-
lactone or ascorbate was observed. N-terminal amino
acid sequence analysis revealed that the enzyme is
closely related to the malate dehydrogenase families.
to be the predominant pathway according to diverse
analysis of its constituent enzymes using transgenic and
mutant plants. In addition to the D-Man/L-Gal pathway,
some researchers have reported that other alternative
pathways via uronic acid intermediates, such as D-
galacturonic acid and D-glucuronic acid, contribute to
AsA biosynthesis,3,4) but the contribution of these
alternative pathways via uronic acid intermediates to
the AsA biosynthesis is still largely unknown.
Unlike animals and plants, the unicellular alga
Euglena gracilis has developed a unique pathway for
AsA biosynthesis. Shigeoka et al.5) have reported that
Euglena cells accumulate AsA at a high level, and that
its biosynthesis pathway comprises D-galacturonic acid,
L-galactonic acid, and L-GalL, judging from the results
of a radio-tracer experiment (Fig. 1). These facts suggest
that Euglena cells are eukaryotic algae having the great
advantage of elucidating the undefined uronic acid
pathway in plants. D-Galacturonic acid reductase (EC
1.1.1.19) catalyzes the reaction of D-galacturonic acid
to L-galactonic acid, and the gene was first identified
from ripening strawberry fruit, suggesting that the
protein belongs to a novel aldo-keto reductase (AKR)
family.3) Recently, Kuorelahti et al.6) reported enzymat-
ic characterizations of the recombinant D-galacturonic
acid reductase from a mold, Hypocrea jecorina. In
the mold, the enzyme appears to contribute to D-
galacturonic acid catabolism, converting pectin to
pyruvate and D-glyceraldehyde 3-phosphate, but not to
AsA biosynthesis. Overall, the available information
on D-galacturonic acid reductase is quite limited. More-
over, it is an interesting problem to elucidate clearly the
Key words: D-galacturonic acid reductase; ascorbate
biosynthesis; Euglena; aldo-keto reductase
The biosynthetic pathways of ascorbic acid (vitamin
C: AsA) between animals and plants are different. In
animals, AsA is synthesized mainly through D-glucur-
onic acid, L-gulonic acid, and L-gulono-1,4-lactone
as intermediates.1) On the other hand, several types of
biosynthetic pathway of AsA have been proposed in
plants. The D-Mannose/L-galactose (D-Man/L-Gal)
pathway proceeds via GDP-D-Man, GDP-L-Gal, L-Gal,
and L-galactono-1,4-lactone (L-GalL), and most of the
enzymes related to the pathway have been identified and
analyzed in detail.2) The D-Man/L-Gal pathway appears
y
To whom correspondence should be addressed. Tel: +81-852-32-6580; Fax: +81-852-32-6092; E-mail: ishikawa@life.shimane-u.ac.jp
Abbreviations: ARK, aldo-keto reductase; AsA, ascorbic acid; BCS, bathocuproine disulfonate; DTT, dithiothreitol; L-Gal, L-galactose; L-GalL,
L-galactono-1,4-lactone; D-Man, D-mannose; MDH, malate dehydrogenase; NEM, N-ethylmaleimide; pCMB, p-chloromercuribenzoate

D-Galacturonic Acid Reductase from Euglena
2721
D-Glucose
D-Galactose
UDP-D-Glucose
UDP-D-Galactose
1
UDP-D-Galacturonic acid
3
UDP-D-Glucuronic acid
2
D-Galacturonic acid
4
D-Glucuronic acid
L-Gulonic acid
L-Galactonic acid
5
L-Gulono-1,4-lactone
L-Galactono-1,4-lactone
6
6
L-Ascorbic acid
Fig. 1. The Ascorbate Biosynthesis Pathway in Euglena gracilis.
1, UDP-glucose dehydrogenase; 2, UDP-glucose-4-epimerase; 3, UDP-D-galacturonic acid pyrophosphatase; 4, D-galacturonic acid reductase;
5, lactonase (lactone hydroxylase); 6, L-gulono-1,4-lactone(L-galactono-1,4-lactone)dehydrogenase.
relationship between D-galacturonic acid reductase and
a novel AKR family enzyme. In this study, we purified
and characterized D-galacturonic acid reductase from
Euglena. We discuss the physiological role of D-
galacturonic acid reductase in AsA biosynthesis of
Euglena cells which are photosynthetic organisms with
AsA at a high level.
Purification of D-galacturonic acid reductase. Eugle-
na cells were collected by brief centrifugation, suspend-
ed in two volumes of ice-cold 50 mM Tris–HCl buffer
(pH 7.2) containing 1 mM EDTA, and disrupted by
sonication. The cell lysate was centrifuged at 100;000 ꢂ
g at 4 ꢁC for 30 min, and the supernatant was used as a
crude extract for enzyme purification. It was loaded onto
a CM-Sepharose (Amersham-Pharmacia Biotech, Up-
psala, Sweden) column (25 ꢂ 40 mm) equilibrated pre-
viously with 50 mM Tris–HCl (pH 7.2). The column was
washed with at least three column volumes of the buffer
and then eluted with 100 ml of a 0–500 mM linear
gradient of KCl in the buffer. The active fractions were
pooled and applied onto a Butyl-Toyopearl (Tosoh,
Tokyo) column (30 ꢂ 150 mm) equilibrated with Tris–
HCl buffer (pH 7.2), containing 30% (W/V) ammonium
sulphate. The column was developed with a linear
gradient of 30–0% (W/V) ammonium sulphate in Tris–
HCl buffer (pH 7.2). The active fractions were pooled
and dialyzed in an ample volume of Tris–HCl buffer
(pH 7.2) with two changes of the buffer. The fraction
was further purified by affinity chromatography using a
HiTrap Blue HP (Amersham-Pharmacia Biotech) col-
umn (10 ꢂ 50 mm) equilibrated with Tris–HCl buffer
(pH 7.2). The column was washed with 20 ml of Tris–
HCl buffer (pH 7.2) containing 0.8 M KCl, and then
eluted with 10 ml of Tris–HCl buffer (pH 7.2) contain-
ing 1.5 M KCl. The active fractions were pooled,
concentrated, and stored at ꢀ20 ꢁC until use.
Materials and Methods
Strain and culture. Euglena gracilis strain Z was
grown in Koren-Hutner medium under continuous light
conditions at a photosynthetic photon flux density of
24 mmol mꢀ2 sꢀ1 at 26 ꢁC for 6 d, by which time the
stationary phase was reached.7)
Assay of D-galacturonic acid reductase. The assay
mixture (1 ml) contained 50 mM Tris–HCl buffer
(pH 7.2), 5 mM D-galacturonic acid, 0.2 mM NADPH
and the enzyme. The reaction was monitored by the
decrease in absorbance at 340 nm (" ¼ 6:22 mMꢀ1 cmꢀ1
)
at 25 ꢁC. The specificity of the enzyme with alternate
substrates was also assayed using the same reaction
mixture. The protein concentration was measured with
the Bio-Rad Protein Assay Reagent (Bio-Rad, Los
Angeles, CA) using BSA as a standard. To check the
reverse reaction, L-galactonic acid and NADPþ were
used as substitutes for the assay mixture. L-Galactonic
acid was obtained by hydrolysis of L-GalL under basic
conditions. Twenty ml of 0.3 M NaOH was added to
100 ml of 10 mM L-GalL and the mixture was vigorously
agitated by vortexing for 20 s. Then 0.3 M hydrochloric
acid (20 ml) was added to the mixture to neutralize the
solution.
SDS–PAGE. SDS–PAGE was performed in a 12.5%
polyacrylamide slab gel using a Tris/glycine buffer
system, as described by King and Laemmli.8) The gels
were stained with Coomassie Blue G-250.

2722
T. ISHIKAWA et al.
Table 1. Purification of D-Galacturonic Acid Reductase from Euglena gracilis Z
Total protein
(mg)
Total activity
(nmol/min)
Specific activity
(nmol/min/mg protein)
Yield
(%)
Purification step
Fold
Crude extract
354
334
294
7.78
0.16
1498
1797
1040
248.4
104.2
4.22
5.38
1
1.2
100
CM Sepharose
30% (NH4)2SO4
Butyl Toyopearl
HiTrap Blue HP
119.9
69.5
16.6
7.0
3.53
0.8
7.6
31.9
671.0
159.0
HPLC analysis of the enzyme reactant. D-Galactur-
onic acid and the corresponding reactant, L-galactonic
acid, were assayed by HPLC on a Fusion-RP C18
column (150 ꢂ 4:6 mm; Phenomenex, Torrance, CA) at
210 nm with an eluent of dilute phosphoric acid (pH 2.5)
at a flow rate of 1 ml/min.
M
1
3
5
2
4
kDa
94
67
43
38 kDa
Analysis of N-terminal amino acid sequence. Analysis
of the N-terminal amino acid sequence was carried out
with an automated pulse-liquid phase sequencer (Model
492; Applied Biosystems, Foster City, CA).
30
20.1
14.4
Results and Discussion
Purification of D-galacturonic acid reductase from
Euglena
Fig. 2. SDS–PAGE Documenting the Progress of Purification of
Euglena D-Galacturonic Acid Reductase.
A typical result of purification of the enzyme from
Euglena gracilis Z is summarized in Table 1. The crude
extract showed a specific activity of 4.22 nmol/min/mg
protein, which was approximately 40-fold higher than
that of Arabidopsis leaves.3) The elution patterns of all
columns showed only one peak of the enzyme activity
(data not shown). The purified enzyme was obtained in
an overall yield of 7% and exhibited an increase of
approximately 160-fold in specific activity. SDS–PAGE
showed a single protein band with a molecular mass of
38 kDa (Fig. 2). The molecular mass of the Euglena
enzyme was almost the same as those of the enzymes
from the strawberry and the mold H. jecorina.3,6) It was
also approximately 39 kDa based on gel filtration on a
calibrated Superose-200 column (data not shown). These
results indicate that D-galacturonic acid reductase from
Euglena cells exists in a monomer in its native state,
corresponding to the known AKR families.9)
D-Galacturonic acid reductase was purified as described in
‘‘Materials and Methods.’’ The samples were loaded onto a 12.5%
polyacrylamide gel and stained with Coomassie brilliant blue. Lane
M, molecular marker; lane 1, crude extract; lane 2, CM sepharose
chromatography; lane 3, 30% (NH4)2SO4 fraction; lane 4, butyl
Toyopearl chromatography; lane 5, HiTrap Blue HP chromatog-
raphy. The positions of molecular markers are indicated on the left
in kDa.
of the enzyme. The enzyme utilized both NADPH and
NADH in a ratio of 10 to 1.8, indicating that it prefers
NADPH to NADH as an electron donor. The apparent
Km value of the enzyme for NADPH was 62:5 ꢃ 4:5
mM, similar to that of the recombinant enzyme from
H. jecorina,6) and the Vmax value was 266:7 ꢃ 19:2
mmol/min/mg protein. It has been reported that the
recombinant enzymes from strawberry and H. jecorina
are highly specific for NADPH, but not for NADH.3,6)
As shown in Table 2, the Euglena enzyme exhibited
high activity with both D-galacturonic acid and D-
glucuronic acid, and also catalyzed the reduction of D-
xylose, D/L-galactose, D-glucose, L-arabinose, and DL-
glyceraldehyde at appropriate rates, indicating that the
enzyme possesses broad substrate specificity. No activ-
ity of the Euglena enzyme was observed with p-nitro-
benzaldehyde or menadione, which are substrates for
carbonyl reductase, one of the known AKR families. As
shown in Table 3, the Km values for D-galacturonic
acid, D-glucuronic acid and D-xylose were 3:79 ꢃ 0:5,
4:67 ꢃ 0:6 and 8:48 ꢃ 0:8 mM, respectively, but the
enzyme showed higher catalytic efficiency (Kcat/Km)
Identification of the reaction product of Euglena D-
galacturonic acid reductase
As shown in Fig. 3, L-galactonic acid was detected
by the HPLC separation as a product of D-galacturonic
acid reduction after a 10-min incubation of the reaction
mixture at 25 ꢁC, clearly indicating that the enzyme
catalyzed D-galacturonic acid to L-galactonic acid in the
forward direction.
Substrate specificity and kinetic study of Euglena D-
galacturonic acid reductase
Next we studied the substrate specificity and kinetics

D-Galacturonic Acid Reductase from Euglena
2723
Table 3. Michaelis Constants and Catalytic Efficiency for Substrates
of Euglena D-Galacturonic Acid Reductase
80
60
A
2.82
kcat
(sꢀ1
Km
kcat/Km
(mMꢀ1 sꢀ1
)
40
Substrate
)
(mM)
20
0
D-Galacturonic acid
D-Glucuronic acid
D-Xylose
0:19 ꢃ 0:01
0:19 ꢃ 0:01
0:06 ꢃ 0:004
3:79 ꢃ 0:5
4:67 ꢃ 0:6
8:48 ꢃ 0:8
50.95
41.35
6.97
D-GalUA
L-GalA
80
60
2.68
40
20
0
galacturonic acid reductase of the strawberry exhibited
much higher activity with D-galacturonic acid than with
D-glucuronic acid.3) On the other hand, the recombinant
enzyme from H. jecorina showed similar Michaelis-
Menten constants for both D-galacturonic acid (Km ¼
6 mM) and D-glucuronic acid (Km ¼ 11 mM). These
findings indicate that the affinity against uronic acids of
the Euglena enzyme tends to be similar to that of the
mold enzyme, but the mold enzyme did not show any
significant activities with other aldoses, such as D-
glucose, D-xylose, D-galactose, and L-arabinose.6) It is
worth noting that there is an alternative ascorbate
biosynthesis pathway in Euglena that takes D-glucuronic
acid and L-gulono-1,4-lactone, although the flux of the
alternative pathway is much lower than that of the D-
galacturonic acid pathway.5) Therefore, it might be
reasonable to consider that the Euglena enzyme has high
catalytic efficiency with both D-galacturonic acid and D-
glucuronic acid.
3.0
3.5
1.5
2.0
2.5
Retention time ( min )
80
60
B
2.82
D-GalUA
40
20
0
3.13
0 min
80
60
2.82
D-GalUA
40
2.68
L-GalA
20
0
3.13
10 min
3.0
Retention time ( min )
3.5
1.5
2.0
2.5
Fig. 3. Formation of L-Galactonic Acid from D-Galacturonic Acid by
D-Galacturonic Acid Reductase Purified from Euglena.
With respect to substrate specificity, AKR families
are classified into three distinct groups.9) The first group
is aldehyde reductase, which catalyzes the reduction of
various types of aldehyde, including uronic acids and
some ketones. The second is aldose reductase, which
catalyzes the reduction of aldehydes, such as glycol-
aldehydes, but is less active against uronic acids. The
third group is carbonyl reductase, which catalyzes the
reduction of quinines, other ketones, and short-chain
aldehydes. On the basis of the present data, it appears
likely that Euglena D-galacturonic acid reductase be-
longs to the first group of AKR families.
Kuorelahti et al.6) have reported that fungal recombi-
nant D-galacturonic acid reductase exhibits activity in
the reverse direction. When we tested the reverse
reaction of the enzyme, no reaction was observed with
L-galactonic acid and NADPþ as substrates (data not
shown).
The purified enzyme was incubated with 5 mM D-galacturonic acid
and 0.2 mM NADPH at 25 ꢁC for 10 min. The experimental
conditions for HPLC analysis are described in ‘‘Materials and
Methods.’’ A, standard compounds; B, analysis of a reaction
mixture. Arrows indicate the compounds. D-GalUA, D-galacturonic
acid; L-GalA, L-galactonic acid.
Table 2. Substrate Specificity for Purified D-Galacturonic Acid
Reductase from Euglena
Relative
activity (%)
Substrate
Electron donor
NADPH
NADH
Electron acceptor
100.0
17.7
D-Galacturonic acid
100.0
82.4
29.9
18.8
14.9
14.6
14.2
14.2
0.0
D-Glucuronic acid
D-Xylose
L-Galactose
D-Galactose
Effects of pH and various compounds on D-galactur-
onic acid reductase activities
The optimal pH for the reduction of D-galacturonic
acid in Euglena D-galacturonic acid reductase was 7.2,
with 75 and 18.5% of maximum activity at pH 6.5 and
9.0 respectively (Fig. 4).
D-Glucose
L-Arabinose
DL-Glyceraldehyde
p-Nitrobenzaldehyde
Menadione
0.0
Metal ions, except for Cu2þ, reducing and oxidizing
reagents such as dithiothreitol (DTT), N-ethylmaleimide
(NEM), and p-chloromercuribenzoate (pCMB), and
chelators had no effect on enzyme activities (Table 4).
for D-galacturonic acid and D-glucuronic acid than for
D-xylose, indicating that it should dominantly reduce
uronic acids in vivo. The purified recombinant D-

2724
T. ISHIKAWA et al.
Euglena cells.10,11) In addition, we have reported that
100
80
60
40
20
0
H2O2 is generated under high light conditions in
chloroplasts of Euglena.12) Accordingly, it appears
likely that activation of D-galacturonic acid reductase
in the presence of H2O2 is a key regulation mechanism
for the acceleration of AsA biosynthesis in Euglena cells
under oxidative stress conditions. Treatment of the
enzyme with thiol-modification reagents, such as DTT,
pCMB, and NEM, had no effect on activity, suggesting
that the enzyme does not include the SH base in the
reaction center, and that the target site for the redox
regulation of the enzyme activity is another amino acid
residue, besides the cysteine residue. It has been
reported that phenylalanine hydroxylase and dihydrop-
teridine reductase from humans were activated by
oxidation of Trp and Met residues respectively by H2O2
treatment.13,14) These observations might fit in with the
case of the activation mechanism of D-galacturonic acid
reductase by H2O2.
6.5
7
7.5
pH
8
8.5
9
Fig. 4. Effect of pH on the Activity of the Purified D-Galacturonic
Acid Reductase.
The activity of the purified enzyme was measured as described in
‘‘Materials and Methods.’’
Table 4. Effects of Various Compounds on Purified D-Galacturonic
Acid Reductase from Euglena
Feedback inhibition of L-Gal dehydrogenase, the
penultimate enzyme in the D-Man/L-Gal pathway in
higher plants, and GDP-D-Man-3,5-epimerase by AsA,
is known to be an important regulation mechanism in
AsA biosynthesis.15,16) To clarify the possibility of
feedback regulation of Euglena D-galacturonic acid
reductase, we studied the effect of two downstream
compounds of the biosynthesis pathway, AsA and L-
GalL, on enzyme activity. Neither AsA nor L-GalL had
an effect on the activity up to 2 mM (Table 4), indicating
no feedback regulation of the enzyme activity in the
AsA biosynthesis pathway of Euglena.
Concentration
(mM)
Relative
activity (%)
Compound
None
—
5
100
CaCl2
CoCl2
MnCl2
NiSO4
ZnSO4
FeSO4
93.8
5
102.9
98.4
94.8
5
5
5
100.7
104.8
110.2
115.0
135.3
96.7
1
5
CuSO4
1
5
EDTA
BCS
DTT
1
N-terminal amino acid sequence
0.5
5
93.9
98.0
The N-terminal amino acid sequence of Euglena D-
galacturonic acid reductase was determined by Edman
degradation. The first 16 residues of the N-terminal
sequence were FKVAV?GAAAGIGQPL. Surprisingly,
homology of the N-terminal sequence apparently exists
among malate dehydrogenases (MDHs) from various
organisms, but not among known D-galacturonic acid
reductases from strawberry or mold (Fig. 5). Next, we
determined the MDH activity of the purified Euglena D-
galacturonic acid reductase, no dehydrogenase activity
was detected, even at high protein concentrations.
Conversely, MDHs from pigs and yeast were not
determined to have any D-galacturonic acid reducing
activity (data not shown). These results indicate that the
Euglena enzyme is clearly distinct from MDHs from
other organisms. The conserved N-terminal region
among MDH families is known to form an ꢀ-helix and
to play an important role in the subunit–subunit interface
to constitute its dimeric form together with another set
of ꢀ-helix domain.17,18) As described above, Euglena D-
galacturonic acid reductase exists in monomeric form,
indicating that the primary or the secondary structure
should be distinct from that of MDH families although it
has a similar N-terminal region with them. It is an
interesting problem to define the evolutionary relation-
NEM
0.5
1
99.7
84.0
pCMB
H2O2
0.05
0.1
2
107.2
145.6
99.8
L-GalL
AsA
2
100.8
Interestingly, 5 mM Cu2þ and 0.1 mM H2O2 activated the
enzyme activity to 135.3% and 145.6% respectively.
The ratio of direct oxidation of NADPH by H2O2 was at
a negligible level. Treatment of the enzyme with a Cu
chelator, bathocuproine disulfonate (BCS), had no effect
on the activity (Table 4), and the enzyme exhibited no
absorbance spectrum bands for a typical Cu-containing
protein, except for a strong 280 nm peak (data not
shown), indicating that it is not a typical Cu-containing
protein, as reported in other known AKR families.9)
These findings suggest that activation of the enzyme in
the presence of Cu2þ is due to generation of H2O2
caused by auto-oxidation of metal ions, and that the
enzyme activity might be regulated by the redox status
of the Euglena cells. Oxidative stress causes an increase
in the levels of AsA, a functional antioxidant, in

D-Galacturonic Acid Reductase from Euglena
2725
Fig. 5. Alignment of N-Terminal Amino Acid Sequences of D-Galacturonic Acid Reductase (D-GalUAR) and Malate Dehydrogenase (MDH).
The accession numbers for the sequences are as follows: strawberry D-GalUAR, AF039182; H. jecorina D-GalUAR, AY862503;
Trypanosoma mitochondrial MDH (mMDH), AF027739; Oryza glyoxysomal MDH (gMDH), D85763; Arabidopsis gMDH, NM001036783;
stevia MDH, DQ269456; pig heart MDH, M16427; yeast mMDH, J02841.
Biotechnol., 21, 177–181 (2003).
ship between D-galacturonic acid reductase and MDH in
Euglena cells.
4) Lorence, A., Chevone, B. I., Mendes, P., and Nessler, C.
L., myo-inositol oxygenase offers a possible entry point
into plant ascorbate biosynthesis. Plant Physiol., 134,
1200–1205 (2004).
5) Shigeoka, S., Nakano, Y., and Kitaoka, S., The bio-
synthetic pathway of L-ascorbic acid in Euglena gracilis
Z. J. Nutr. Sci. Vitaminol., 25, 299–307 (1979).
6) Kuorelahti, S., Kalkkinen, N., Penttila, M.,
Londesborough, J., and Richard, P., Identification in
the mold Hypocrea jecorina of the first fungal D-
galacturonic acid reductase. Biochemistry, 44, 11234–
11240 (2005).
7) Koren, L. E., and Hutner, S. H., High-yield media for
photosynthesizing Euglena gracilis Z. J. Protozool., 14,
17 (1967).
8) Laemmli, U. K., Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature,
227, 680–685 (1970).
In the present study, we purified and characterized a
D-galacturonic acid reductase from Euglena, the first
enzyme identified from the AsA biosynthesis pathway
proposed in Euglena. The present results clearly indicate
that the substrate specificity and primary structure of
the Euglena enzyme are distinct from those of straw-
berry and mold. We also found that the activity of D-
galacturonic acid reductase is activated by the occur-
rence of H2O2, which is involved in the regulation of
redox homeostasis. More experiments will be necessary
to elucidate the relationship between redox regulation
of the enzyme and AsA levels in Euglena cells under
diverse environmental conditions.
Acknowledgments
9) Wermuth, B., Aldo-keto reductases. In ‘‘Enzymology of
Carbonyl Metabolism 2: Aldehydo Dehydrogenase,
Aldo-Keto Reductase, and Alcohol Dehydrogenase,’’
eds. Flynn, T. G., and Weiner, H., Alan R. Liss, New
York, pp. 209–230 (1985).
10) Shigeoka, S., Yokota, A., Nakano, Y., and Kitaoka, S.,
The effect of illumination on L-ascorbic acid content in
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(1979).
This work was supported in part by Core Research for
Evolutional Science and Technology (CREST), Japan
Science and Technology Agency (JST) and was also
supported by the Academic Frontier Project for Private
Universities of Japan: matching fund subsidy from
Ministry of Education, Culture, Sports, Science and
Technology (MEXT), 2004–2008 (to S.S.).
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spinach. J. Photochem. Photobiol. B, 84, 197–203
(2006).
12) Ishikawa, T., Takeda, T., Shigeoka, S., Hirayama, O.,
and Mitsunaga, T., Hydrogen peroxide generation in
organelles of Euglena gracilis. Phytochemistry, 33,
1297–1299 (1993).
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