Physiological and biochemical characterization of three nucleoside diphosphate kinase isozymes from rice (Oryza sativa L.)

Article abstract of DOI:10.1271/bbb.110285

Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme that catalyzes the transfer of the γ-phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate. In this study, we examined the subcellular localization, tissue-specific gene expression, and enzymatic characteristics of three rice NDPK isozymes (OsNDPK1-OsNDPK3). Sequence comparison of the three OsNDPKs suggested differential subcellular localization. Transient expression of green fluorescence protein-fused proteins in onion cells indicated that OsNDPK2 and OsNDPK3 are localized to plastid and mitochondria respectively, while OsNDPK1 is localized to the cytosol. Expression analysis indicated that all the OsNDPKs are expressed in the leaf, leaf sheath, and immature seeds, except for OsNDPK1, in the leaf sheath. Recombinant OsNDPK2 and OsNDPK3 showed lower optimum pH and higher stability under acidic pH than OsNDPK1. In ATP formation, all the OsNDPKs displayed lower K_m values for the second substrate, ADP, than for the first substrate, NTP, and showed lowest and highest K _m values for GTP and CTP respectively.

Full text of DOI:10.1271/bbb.110285

Biosci. Biotechnol. Biochem., 75 (9), 1740–1745, 2011
Physiological and Biochemical Characterization
of Three Nucleoside Diphosphate Kinase Isozymes
from Rice (Oryza sativa L.)
1
1;y
1
2
1
Akihiko KIHARA, Wataru SABURI, Shinji WAKUTA, Myung-Hee KIM, Shigeki HAMADA,
1
2
1
Hiroyuki ITO, Ryozo IMAI, and Hirokazu MATSUI
1
Laboratory of Biochemistry, Research Faculty of Agriculture, Hokkaido University,
N-9, W-9, Kita-ku, Sapporo 060-8589, Japan
2
Crop Cold-Tolerance Research Team, National Agricultural Research Center for The Hokkaido Region,
Hitsujigaoka, Toyohira-ku, Sapporo 062-8555, Japan
1
Received April 11, 2011; Accepted May 25, 2011; Online Publication, September 7, 2011
[doi:10.1271/bbb.110285]
Nucleoside diphosphate kinase (NDPK) is a ubiquitous
as a transcription factor that regulates c-myc gene
8
)
enzyme that catalyzes the transfer of the ꢀ-phosphoryl
group from a nucleoside triphosphate to a nucleoside
diphosphate. In this study, we examined the subcellular
localization, tissue-specific gene expression, and enzy-
matic characteristics of three rice NDPK isozymes
expression has been demonstrated for a human NDPK.
Three types of NDPKs have been identified in plants
9
–14)
based on their amino acid sequences.
is a cytosolic protein,
Type I NDPK
while type II and type III
NDPK bear an N-terminal extension likely to be
1
1,14)
1
5–18)
(
OsNDPK1-OsNDPK3). Sequence comparison of the
involved in targeting of the proteins to chloroplasts
1
9,20)
three OsNDPKs suggested differential subcellular local-
ization. Transient expression of green fluorescence
protein-fused proteins in onion cells indicated that
OsNDPK2 and OsNDPK3 are localized to plastid and
mitochondria respectively, while OsNDPK1 is localized
to the cytosol. Expression analysis indicated that all the
OsNDPKs are expressed in the leaf, leaf sheath, and
immature seeds, except for OsNDPK1, in the leaf sheath.
Recombinant OsNDPK2 and OsNDPK3 showed lower
optimum pH and higher stability under acidic pH than
OsNDPK1. In ATP formation, all the OsNDPKs dis-
played lower Km values for the second substrate, ADP,
than for the first substrate, NTP, and showed lowest and
highest Km values for GTP and CTP respectively.
or mitochondria.
Plant NDPKs have also been found
to be multi-functional proteins, and to be strongly
related to signal transduction, differentiation, and devel-
opment. Arabidopsis type II NDPK (AtNDPK2) regu-
2
1)
lates auxin action and phytochrome-mediated signal
9
)
transduction. AtNDPK2 is thought to regulate the
activity of G protein, since AtNDPK2 directly interacts
with pea small G-proteins, Pra2 and Pra3, and controls
2
2)
their activities.
In rice, only type I NDPK (OsNDPK1) has been
characterized to date. The amount of the OsNDPK1
protein in the embryo increases during imbibition, while
2
3)
it decreases in the endosperm. OsNDPK1 has been
implicated in the cell elongation process in coleoptiles.
The activity of NDPK in coleoptiles correlates with their
2
4)
Key words: nucleoside diphosphate kinase; subcellular
localization; ping-pong mechanism; gene
expression; enzyme characteristics
length in NDPK1 knock-down mutants. Furthermore,
NDPK1 expression was strongly induced by a plant
pathogen (Xanthomonas oryzae pv. oryzae), salicylic
2
5)
acid, jasmonic acid, and abscisic acid.
The three-dimensional structure of OsNDPK1 has
Nucleoside diphosphate kinase (NDPK; EC
,2)
26)
2
.7.4.6)1 is a ubiquitous enzyme that catalyzes the
been solved in a multimeric form (two hexamers). The
transfer of the ꢀ-phosphoryl group from nucleoside
triphosphate (NTP) to nucleoside diphosphate (NDP)
through a ping-pong mechanism.3 Since it catalyzes
reversible reactions, it has been considered a house-
keeping enzyme that regulates nucleotide pools within
the cells. However, recent studies have suggested that
NDPKs play more specific roles in signal transduction in
diverse organisms, from bacteria to mammals. For
instance, an NDPK is involved in the regulation of G
protein activity by changing the availability of GTP in
OsNDPK1 monomer consists of a four-stranded anti-
parallel ꢁ-sheet, surrounded by six ꢂ-helices. The
nucleotide-binding site in a cleft is formed by an area
of highly positive potential. Two crucial, conserved
active site residues, Phe58 and His115, are located on
ꢂA and ꢁ4 respectively (the names of structural
elements used here follow a report of Huang et al. ).
In spite of extensive studies on the structure and
physiological functions of OsNDPK1, the enzymatic
properties of rice NDPK isozymes, including
OsNDPK1, have not been elucidated in detail. Here,
we report subcellular localization, gene expression, and
enzymatic properties of three OsNDPKs.
)
2
6)
4
,5)
the vicinity of G protein. Genes encoding NDPK are
6
)
associated with the suppression of tumor metastasis
7
)
and Drosophila development. Furthermore, a function
y
To whom correspondence should be addressed. Tel/Fax: +81-11-706-2508; E-mail: saburiw@chem.agr.hokudai.ac.jp
Abbreviations: NDPK, nucleoside diphosphate kinase; OsNDPK, Oryza sativa NDPK; AtNDPK, Arabidopsis thaliana NDPK; ATPD, ATPase
delta gene from sweet potato; GFP, green fluorescence protein; RT-PCR, reverse transcription PCR; RFP, red fluorescence protein

Characterization of Three Rice NDPK Isozymes
1741
Materials and Methods
Ubiquitin mRNA was used as internal control with a pair of primers:
0
0
0
sense, 5 -CCAGGACAAGATGATCTGCC-3 ; antisense, 5 -CCAGT-
0
CCATGAACCCGGCGAGTGACG-3 .
Plant materials. Rice plants (Oryza sativa L. cv. Kitaake) were
grown in the experimental field of Hokkaido University. Tissues from
rice plants at the grain-filling period were collected and stored at
ꢁ
Construction of expression plasmids for OsNDPKs. Plasmid vector
pET23a (Novagen, Darmstadt, Gemany) was used to produce
recombinant OsNDPKs in E. coli BL21 (DE3) pLys. Recombinant
OsNDPK2 and 3 were produced as N-terminal-truncated forms starting
from the 73th and 88th amino acids respectively. Recombinant
OsNDPK1 was produced in intact form. A histidine tag from the
vector consisting of six histidine residues was attached to the
C-terminal of each recombinant protein and utilized for affinity
chromatography.
ꢀ
80 C until analysis.
Cloning of genes encoding OsNDPKs. Three OsNDPK genes,
OsNDPK1 (Os07g0492000), OsNDPK2 (Os12g0548300), and
OsNDPK3 (Os05g0595400), are predicted in the rice genome accord-
ing to the rice annotation project database. The cDNAs of these genes
were amplified by reverse transcription PCR (RT-PCR) from immature
seeds, as follows: Total RNA was extracted from immature seeds using
ISOGEN (Nippon Gene, Tokyo), and reverse transcription was
performed with BcaBEST RNA PCR Kit (Takara, Shiga, Japan) using
an oligo dT primer (Takara). RT-PCR was performed with PrimeSTAR
HS DNA polymerase (Takara) and the following primers: for
To clone the cDNA of OsNDPKs into pET23a, NheI and XhoI sites
0
0
were introduced into the 5 - and 3 -termini of the cDNA by PCR with
the following primer sets (NheI and XhoI sites underlined): for
0
0
OsNDPK1, 5 -GAGAGATGGCTAGCTCCTTCATCATG-3 (sense)
0
0
0
0
and 5 -GGCGCGAACTCGAGAGACTCATAGATCC-3 (antisense);
OsNDPK1, 5 -ATCCTCCTCATCGCCTCCGCTTCTG-3 (sense) and
0
0
0
0
0
for OsNDPK2, 5 -CGTCGTCGGTTGCTAGCTCCTATATTATG-3
0
5 -CTTGGATGAACCTAGGGAAAGAGCATG-3 (antisense); for
0
OsNDPK2, 5 -TCATCATCTTCTTCTTCTCCGCTCTCC-3 (sense)
0
(sense) and 5 -GCACTGATCTGGGACTCGAGCTCTACAAG-3
0
0
0
(antisense); and for OsNDPK3, 5 -CGCATGCTGCAGCTAGCGAG-
0
and 5 -CACCTCTGCTGGGAGCTATAGTACAAC-3 (antisense);
0
0
CGCACC-3 (sense) and 5 -GAGATGATTCGCCTCGAGGTTGA-
0
and for OsNDPK3, 5 -GTACATACTAGTACAAGACTGGAGGGC-
0
0
0
CCCC-3 (antisense). The amplified DNA fragments and pET23a were
digested with NheI and XhoI and ligated as described above.
3
(sense) and 5 -TGATTACTCAGTGCACGCTCTACAATC-3 (anti-
sense). Amplified DNA fragments were cloned into pBluescript SK II
(
(
Stratagene, La Jolla, CA) using DNA Ligation Kit Mighty Mix
Takara), and were sequenced with an ABI PISM 310 Genetic
Production and purification of recombinant OsNDPKs. Recombi-
nant OsNDPKs were produced in E. coli BL21 (DE3) pLys cells
carrying the expression plasmids. The transformants were cultured at
Analyzer (Applied Biosystems, Foster City, CA) with a Big Dye
Terminator 3.1 Sequencing Kit (Applied Biosystems). Propagation of
plasmid DNA was performed in Escherichia coli DH5ꢂ, and plasmid
DNA was purified by alkaline lysis methods.27)
ꢁ
37 C in 1 L of Luria-Bertani medium with 100 mg/mL ampicillin until
A600 reached 0.8. Then 1 mL of 0.1 M isopropyl ꢁ-thiogalactopyrano-
ꢁ
side was added to the culture, and the cells were grown further at 20 C
for 16 h. The bacterial cells were harvested by centrifugation and
suspended in 50 mL of 20 mM Tris–HCl buffer (pH 7.4) containing
20 mM imidazole and 1 mM phenylmethylsulfonyl fluoride. They were
disrupted by sonication with Sonifier 450 (Branson, Danbury, CT), and
the lysate was centrifuged to remove cell debris. The cell-free extract
obtained was applied to Ni-chelating column chromatography using
Chlating Sepahrose Fast Flow (GE healthcare, Uppsala, Sweden). The
adsorbed protein was eluted with a linear gradient of imidazole (20–
500 mM) in 20 mM Tris–HCl buffer (pH 7.4) with 10% glycerol after
thorough washing with 20 mM Tris–HCl buffer (pH 7.4) containing
Transient expression of OsNDPKs fused with green fluorescent
protein (GFP). By PCR, BamHI and NdeI sites were introduced into
0
0
the 5 - and 3 -termini of the OsNDPK genes using the following
0
primers (BamHI and NdeI sites underlined): for OsNDPK1, 5 -
0
0
GTTCTTTGGATCCATGGAGCAGTC-3 (sense) and 5 -GCGCGAA-
0
CATATGAGACTCATAGATCCAAG-3 (antisense); for OsNDPK2,
0
0
0
5
GCACTGATCTGGGACATATGCTCTACAAG-3 (antisense); and for
-GAGAAGCAGCTGGATCCATGGACGCC-3 (sense) and 5 -
0
0
0
OsNDPK3, 5 -CCCTAGCCAAGGATCCATGAGCAAGC-3 (sense)
0
0
and 5 -GGGAGATGATTCGCCATATGGTTGACCC-3 (antisense).
The GFP gene was isolated from the 35S-sGFP plasmid, the kind gift
of Dr. Yasuo Niwa (University of Shizuoka), by double digestion of
2
0 mM imidazole. The purity of the recombinant OsNDPK isozymes
4)
3
was confirmed by SDS–PAGE. The collected fraction was dialyzed
against 20 mM Tris–HCl buffer (pH 7.4) with 10% glycerol, and then
NdeI and SacI, and was fused to the OsNDPK genes on binary vector
pTH-1, a derivative of pBE2113.28) The plastidial localization marker,
AtCHL27:RFP, was created by inserting Arabidopsis CHL27 cDNA29)
into the pH7RWG2 vector.30) The mitochondrial marker construct,
ꢁ
more glycerol was added to 40% before storage at ꢀ20 C. The protein
concentration of purified enzymes was measured by the Bradford
method,35) and bovine serum albumin F-V (Nakalai Tesque, Kyoto,
Japan) was used as standard.
ATPD:RFP, which utilizes the signal peptide of sweet potato ATPase
delta,31) was also prepared with pH7RWG2.
Introduction of expression vectors into onion epidermal cell layer
was performed by particle bombardment.32) Gold particles coated with
Enzyme assay. NDPK activity was determined based on the initial
velocity of ATP formation from ADP and GTP. A reaction mixture of
100 mL consisting of an appropriate concentration of the enzyme, 2 mM
ADP (Sigma-Aldrich, St. Louis, MO), 2 mM GTP (Wako, Osaka,
Japan), 4 mM Tris–HCl buffer (pH 7.5), 100 mM KCl, and 25 mM
2.5 mg of the respective plasmid(s) were delivered into onion epidermal
cells using a Biolistic PDS-1000/He system (Bio-Rad, Hercules, CA)
with a rupture setting of 1,100 psi. After bombardment, the epidermal
cell layer was incubated on Murashige and Skoog medium33) at 25 C
overnight. Subcellular localization of the GFP-fusion proteins was
visualized by fluorescence microscopy DM6000B (Leica, Solms,
Germany). Images were processed with imaging software FW4000
ꢁ
ꢁ
MgCl was incubated at 25 C for 10 min. The enzymatic reaction was
2
stopped by boiling for 5 min, and the ATP produced was measured by
HPLC under the following conditions: column, Capcell Pak NH2
ꢁ
UG80 (ꢃ4:6 ꢂ 50 mm, Shiseido, Tokyo); column temperature, 40 C;
(Leica).
injection volume, 20 mL; detection, A ; flow rate, 0.8 mL/min;
2
54
elution, linear gradient of 0–300 mM ammonium phosphate dibasic
(pH 3.0). As standard, 0–2.0 mM ATP (Sigma-Aldrich) was used.
The optimum pH values of the OsNDPKs were determined by
measuring ATP-forming velocities at the indicated pH values. Fifty
mM sodium acetate buffer (pH 4.0 and 5.0), 50 mM MES-NaOH buffer
(pH 5.5–7.0), 50 mM glycine-NaOH buffer (pH 10.0 and 11.0) were
used as the reaction buffer.
Expression analysis of OsNDPK genes. The transcription levels
of OsNDPKs in the leaf, leaf sheath, and immature seeds were
examined by RT-PCR. Total RNA from these tissues was extracted,
and first-strand cDNA was synthesized as described above. PCR was
0
conducted with the following primers: for OsNDPK1, 5 -GCCT-
0
0
CCTGAGTCACCATTATAATTCTG-3 (sense) and 5 -CTTGGA-
0
TGAACCTAGGGAAAGAGCATG-3 (antisense); for OsNDPK2,
0
The temperature and pH stabilities of the OsNDPKs were evaluated
based on residual activity after temperature and pH treatments. The
range of pH and temperature, where the enzymes retained more than
90% of original activity after treatment, were considered to be stable
ranges. Temperature treatment was done by incubation of OsNDPK
solution (OsNDPK1, 29 ng/mL; OsNDPK2, 17 ng/mL; and
0
0
5
CACCTCTGCTGGGAGCTATAGTACAAC-3 (antisense); and for
-CCGATTACTCTCTGTTGTCAAATCTCC-3 (sense) and 5 -
0
0
0
OsNDPK3, 5 -CATCTCCCCTGTTTGTTTTGTTTTTTTTTC-3 (sense)
0
0
and 5 -TGATTACTCAGTGCACGCTCTACAATC-3 (antisense).
ꢁ
ꢁ
Twenty-eight, 30, and 27 cycles (98 C for 5 s, 55 C for 5 s, and
ꢁ
ꢁ
7
2 C for 1 min) for OsNDPK1, 2, and 3 respectively were performed.
OsNDPK3, 47 ng/mL) at 10–70 C for 15 min. For pH treatment,

1742
A. KIHARA et al.
AtNDPK4
AtNDPK3
BrNDPK3
PsNDPK3
OsNDPK3
ZmNDPK4
PpNDPK
OsNDPK1
AhNDPK1
ScNDPK1
PsNDPK1
BrNDPK1
AtNDPK1
AlNDPK1
ZmNDPK1
PpNDPK2a
NtNDPK2
OsNDPK2
A
E
F
3
83
Type III
3
29
304
B
C
D
2
67
7
78
4
25
9
41
5
G
Type I
90
7
73
685
998
9
70
5
10
8
12
475
8
98
ZmNDPK2 Type II
PsNDPK2
BrNDPK2
AtNDPK2
AlNDPK2
Fig. 2. Subcellular Localization of OsNDPKs in Onion Epidermal
Cells.
7
77
7
67
A, Fluorescent images of an onion cell expressing
OsNDPK1:GFP. B–D, fluorescent images of an onion cell co-
expressing OsNDPK2:GFP and AtCHL27:RFP. E–G, fluorescence
images of an onion cell co-expressing OsNDPK3:GFP and
ATPD:RFP. B, GFP fluorescence image of OsNDPK2; C, RFP
fluorescence image of AtCHI27; D, overlay image of B and C; E,
GFP fluorescence image of OsNDPK3; F, RFP fluorescence image
of ATPD; G, overlay image of E and F. Scale bars are 20 mm.
9
82
9
21
0
.2
Fig. 1. Phylogenetic Tree of Plant NDPKs.
A phylogenetic tree of plant NDPKs was constructed using the
Clustal X program (ver. 2.0.11), and visualized with TreeView 1.6.6
software. Numbers in the figure indicate bootstrap values. Os, Oryza
sativa; At, Arabidopsis thaliana; Zm, Zea mays; Ps, Pinus sativum;
Ah, Arachis hypogaea; Nt, Nicotiana tabacum; Br, Brassica rapa.
OsNDPK2:GFP and OsNDPK3:GFP proteins were
detected as dot-like subcellular structures (Fig. 2B
and E). Co-expression experiments with marker genes
for plastidial (AtCHL27:RFP) and mitochondrial
OsNDPKs (OsNDPK1, 0.9 mg/mL; OsNDPK2, 1.8 mg/mL; and
OsNDPK3, 1.4 mg/mL) were diluted 10-fold with 0.5 M Britton-
ꢁ
Robinson buffer (pH 2.0–12.0) and incubated at 4 C for 16 h.
The kinetic parameters of the OsNDPKs for ADP and various NTPs
(
ATPD:RFP) targeting indicated localization of
(GTP, TTP, and CTP) were calculated based on the following reaction
equation for the ping-pong mechanism:
OsNDPK2:GFP and OsNDPK3:GFP to plastids and
mitochondria respectively (Fig. 2D and G).
v ¼ kcat ꢃ a ꢃ b=ðKmb ꢃ a þ Kma ꢃ b þ a ꢃ bÞ
where v is the reaction rate, kcat is the molecular activity, a and b are
concentrations of NTP and ADP respectively, and Kma and Kmb are the
affinity constants for NTP and ADP respectively. The initial velocities
of ATP formation at various concentrations of ADP (0.031–0.083 mM)
and NTP (0–1 mM) were measured under the conditions described
above. The reactions toward TTP and CTP were also analyzed with
HPLC as the reaction to GTP.
Expression of the OsNDPK genes in various tissues
Expression of the OsNDPKs was determined in leaf,
leaf sheath, and immature seeds by RT-PCR (Fig. 3).
The expression level of OsNDPK1 was almost the same
in leaf and immature seed, but was fairly low in the leaf
sheath. In the case of OsNDPK2, high transcript levels
were observed in the leaf and leaf sheath, and a lower
level was detected in immature seed. OsNDPK3 was
strongly expressed in every tissue examined, and no
difference in expression level was observed, unlike other
isozymes.
Results
Phylogenetic analysis of the OsNDPKs
Three OsNDPK genes, OsNDPK1–OsNDPK3, are
predicted in the rice genome. A comparison of the amino
acid sequences of these OsNDPKs revealed that in
addition to a highly conserved C-terminal region (about
Production and purification of recombinant
OsNDPKs
5
0% identity), OsNDPK2 and OsNDPK3 contained
Recombinant OsNDPKs were produced as C-terminal
His-tag proteins. For OsNDPK2 and OsNDPK3, the
N-terminal hydrophobic regions (Met1-Ala72 and Met1-
Ser87 respectively) were deleted because the corre-
sponding full-length proteins were predominantly pro-
duced in the insoluble fractions (data not shown).
Recombinant OsNDPKs were purified from E. coli
cell-free extracts to homogeneity by Ni-chelating col-
umn chromatography (Fig. 4). The molecular masses
of OsNDPK1–OsNDPK3, estimated by SDS–PAGE,
corresponded to the theoretical masses calculated from
the amino acid sequences (17.9, 17.4, and 18.0 kDa
respectively).
putative signal sequences at the N-terminus. Phyloge-
netic analysis was done with the sequences of the
conserved regions available for O. sativa, A. thaliana,
Zea mays, Pinus sativum, Arachis hypogaea, Nicotiana
tabacum, and Brassica rapa. The NDPKs divided into
three types, and OsNDPK1–OsNDPK3 fell into types
I–III respectively (Fig. 1).
Subcellular localization of the OsNDPKs
In order to determine the specific functions of the
OsNDPKs, subcellular localization was determined
using GFP-fused proteins. The full-length cDNAs of
the respective NDPKs were amplified by RT-PCR, and
the GFP gene was fused to the C-terminus.
The fusion genes were transiently expressed in onion
epidermal cells. The fluorescence signals of the
OsNDPK1:GFP protein were detected in the nucleus
and the cytoplasm (Fig. 2A). In contrast, the
Enzymatic properties of recombinant OsNDPKs
The recombinant OsNDPKs were characterized based
on ATP-forming activity from ADP and GTP.
ꢁ
OsNDPK1 and OsNDPK2 were stable up to 25 C. On
ꢁ
the other hand, OsNDPK3 was stable up to 30 C

Characterization of Three Rice NDPK Isozymes
1743
Leaf
Leaf sheath
Seed
1
00
NDPK1
NDPK2
NDPK3
80
60
40
20
0
Ubiquitin
Fig. 3. RT-PCR Analysis for Gene Expression of OsNDPKs in Leaf,
Leaf Sheath, and Immature Seed Tissues.
0
20
40
60
Semi-quantitative RT-PCR was performed to evaluate the
expression levels of OsNDPKs in leaf, leaf sheath, and immature
seed tissues. The ubiquitin gene was used as control.
Temperature (ºC)
Fig. 5. Temperature Stability of Recombinant OsNDPKs.
The residual activities of recombinant OsNDPK1–OsNDPK3
were measured after 15 min incubation at indicated temperatures.
Solid circle, hollow circle, and solid triangle indicate OsNDPK1–
OsNDPK3 respectively. Data are the mean ꢄ SD for three inde-
pendent experiments.
M
1
2
3
(
kDa)
9
6
7
6
4
3
2
1
5
1
2
4
1
00
8
6
4
2
0
0
0
0
0
Fig. 4. SDS–PAGE Analysis of Purified Recombinant OsNDPKs.
Purified enzyme (2 mg) was separated on a 15% w/v polyacryl-
amide gel. The molecular masses of standard proteins are shown on
the left. M, size marker; 1, OsNDPK1; 2, OsNDPK2; 3, OsNDPK3.
3
5
7
9
11
pH
Fig. 6. pH Activity Curves of Recombinant OsNDPKs.
The reaction velocities of recombinant OsNDPK1–OsNDPK3 at
indicated pH values were measured. Solid circle, hollow circle, and
solid triangle indicate OsNDPK1–OsNDPK3 respectively. Data are
the mean ꢄ SD for three independent experiments.
(
Fig. 5). OsNDPK1 showed the highest activity at
pH 8.4, while the optimum pH values for both
OsNDPK2 and OsNDPK3 were 6.2 (Fig. 6). The stable
pH ranges for OsNDPK1–OsNDPK3 were different, as
follows: OsNDPK1, 7.0–9.0; OsNDPK2, 6.0–9.0; and
OsNDPK3, 4.0–9.0 (Fig. 7).
The kinetic parameters of the OsNDPKs for ATP
formation from ADP and NTP were determined by
double reciprocal plots (Table 1). Figure 8 shows the
lines obtained from the initial velocities of ATP
formation at various concentrations of GTP and ADP.
The lines for respective ADP concentrations were
parallel in all the OsNDPKs, indicating that NDPKs
obeyed the ping-pong mechanism, as reported for
human NDPK. All the OsNDPKs displayed lower Km
values for the second substrate, ADP, than for the first
substrate, NTP, and showed lowest and highest Km
values for GTP and CTP respectively (Table 1).
OsNDPK1 exhibited higher Km values than OsNDPK2
and OsNDPK 3 for all the substrates examined.
rice, the physiological functions and three-dimensional
2
3–26)
structure of OsNDPK1 have been elucidated.
But
the enzymatic characteristics of plant NDPKs, including
OsNDPKs, are poorly understood. Here, we investigated
the enzymatic properties of three OsNDPKs along with
subcellular localization and gene expression.
Phylogenetic analysis indicated that plant NDPKs are
to be classified into three structural groups (types I–III)
(Fig. 1). Phylogenetic analysis also suggested that these
three groups of NDPKs diverged before monocot and
dicot separation. OsNDPK1–OsNDPK3 represent each
group, suggesting that each OsNDPK has a distinctive
function that is conserved among plant species.
3)
The subcellular localization of type II and type III
NDPKs in plants is controversial. AtNDPK2 was first
reported as a cytosolic protein that interacts with
9
)
Discussion
phytochrome A and several other cytosolic proteins
3
6)
including mitogen-activated protein kinases and sub-
3
7)
Plant NDPKs are involved in multiple signaling steps
in the regulation of differentiation and development. In
group 3 sucrose-nonfermenting-related kinases. How-
ever, a later report confirmed that AtNDPK2 is localized

1744
A. KIHARA et al.
0
.2
A
1
00
0
.15
8
6
4
2
0
0
0
0
0
0
.1
0.05
0
0
1
2
3
4
5
-1
1
/[GTP] (mM )
0
4
8
12
0
.1
0.08
.06
0.04
B
pH
Fig. 7. pH Stability of Recombinant OsNDPKs.
The residual activities of recombinant OsNDPK1–OsNDPK3
ꢁ
were measured after pH treatment at 4 C for 16 h. Solid circle,
0
hollow circle, and solid triangle indicate OsNDPK1–OsNDPK3
respectively. Data are the mean ꢄ SD for three independent experi-
ments.
Table 1. Kinetic Parameters of Recombinant OsNDPKs
0.02
(
a) OsNDPK1
0
kcat
Kmaa
Kmbb
kcat/Kma
kcat/Kmb
0
1
2
3
4
5
Substrate
ꢀ
1)
ꢀ1
ꢀ1
ꢀ1
ꢀ1
(s
(mM)
(mM)
(s mM
)
)
)
(s mM
)
)
)
-1
1
/[GTP] (mM )
CTP
GTP
TTP
229
6.72
0.552
0.707
0.392
34.1
29.2
10.4
415
69.9
140
0
.12
C
49.4 1.69
55.0 5.28
(
b) OsNDPK2
0.09
kcat
Kmaa
Kmbb
kcat/Kma
ꢀ1 ꢀ1
kcat/Kmb
ꢀ1 ꢀ1
Substrate
ꢀ
1)
(s
(mM)
(mM)
(s mM
9.10
61.6
(s mM
67.6
210
0
.06
CTP
GTP
TTP
85.2 9.37
1.26
27.7 0.450 0.138
14.2 0.890 0.0790
16.0
180
0.03
0
(
c) OsNDPK3
kcat
Kmaa
Kmbb
kcat/Kma
kcat/Kmb
Substrate
ꢀ
1)
ꢀ1
ꢀ1
ꢀ1
ꢀ1
(s
(mM)
(mM)
(s mM
(s mM
0
1
2
3
4
5
-
1
CTP
GTP
TTP
89.0 5.28
0.578
16.9
35.1
25.0
154
143
310
1/[GTP] (mM )
17.9 0.510 0.125
33.5 1.34 0.108
Fig. 8. Lineweaver-Burk Plots for the Reaction of Recombinant
OsNDPKs toward ADP and GTP.
aAffinity constant for NTP
bAffinity constant for ADP
The double reciprocal plots were obtained from the initial
velocities of recombinant OsNDPKs to various concentrations of
ADP and GTP. Hollow triangle, solid triangle, hollow circle, and
solid circle indicate the 1=v values for 0.042, 0.05, 0.063, and
0.083 mM ADP respectively. GTP concentrations were 0.25, 0.33,
0.5, and 1.0 mM. A, OsNDPK1; B, OsNDPK2; C, OsNDPK3. Data
are the mean ꢄ SD for three independent experiments.
to the chloroplasts.15) Furthermore, a type II NDPK from
pea was purified from the leaf chloroplast. Local-
ization of AtNDPK3 is also unclear, because three
groups have reported differently that AtNDPK3 is
1
6)
localized to the mitochondria, the chloroplasts, and
both.2
indicated that OsNDPK2 and OsNDPK3 contain a
putative N-terminal targeting sequence, while the
C-terminal of the three OsNDPKs are well conserved
C-terminal GFP fusion proteins, the subcellular local-
izations of OsNDPK2 and OsNDPK3 were clearly
distinguished. OsNDPK2 was localized to the plastids
(Fig. 2B) and OsNDPK3 to the mitochondria (Fig. 2E).
The recombinant OsNDPK2 and OsNDPK3 showed
higher activity and stability at lower pH values than
OsNDPK1 (Figs. 6 and 7). This can be attributed to
adaptation to the localized organelle, plastids, and
mitochondria respectively, in which the pH values are
more labile than that of cytosol due to excess accumu-
lation of protons. Kinetic analysis of the OsNDPKs
revealed that all the isozymes exhibited lowest affinity
constants for GTP among NTP (Table 1). The higher
0,38,39)
Alignment of the OsNDPK sequences
(
data not shown). The N-terminal targeting sequences
appear to have features typical of both plastidial and
mitochondrial localization. Hence, prediction servers
returned different localization results. For example,
TargetP (http://www.cbs.dtu.dk/services/TargetP/) pre-
dicted plastid localization for both OsNDPK2 and
OsNDPK3, while PSORT (http://psort.hgc.jp/) predicted
mitochondrial localization. According to our data for the

Characterization of Three Rice NDPK Isozymes
1745
preference for GTP suggests a possible role of
OsNDPKs in maintaining GTP levels. Such a mecha-
nism might be involved in regulatory processes, as
found for Drosophila, in which NDPK controls GTP
levels in the vicinity of a G protein that regulates cell
signaling.4
18) Peltier JB, Cai Y, Sun Q, Zabrouskov V, Giacomelli L, Rudella
A, Ytterberg AJ, Rutschow H, and van Wijk KJ, Mol. Cell.
Proteomics, 5, 114–133 (2006).
1
2
2
9) Escobar Galvis ML, H a˚ kansson G, Alexciev K, and Knorpp C,
Biochimie, 81, 1089–1096 (1999).
0) Sweetlove LJ, Mowday B, Hebestreit HF, Leaver CJ, and Millar
AH, FEBS Lett., 508, 272–276 (2001).
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1) Choi G, Kim J, Hong S, Shin B, Choi G, Blakeslee JJ, Murphy
AS, Seo YW, Kim K, Koh E, Song P, and Lee H, Plant Cell
Physiol., 46, 1246–1254 (2005).
Acknowledgment
2
2
2) Shen Y, Han Y, Kim J, and Song P, BMB Rep., 41, 645–650
We thank Dr. Yasuo Niwa (University of Shizuoka)
and Dr. Hirokazu Handa (National Institute of Agro-
biological Sciences) for providing p35S-sGFP and
ATPD:GFP plasmids respectively.
(
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