Structure and Molecular Characterization of Diadenosine Polyphosp
Journal of Plant Biochemistry & Physiology

Journal of Plant Biochemistry & Physiology
Open Access

ISSN: 2329-9029

+44 1478 350008

Research Article - (2018) Volume 6, Issue 3

Structure and Molecular Characterization of Diadenosine Polyphosphate Hydrolase in Brachypodium distachyon

Motohiro Tanaka1, Igor Iamshchikov2, Yusuke Kato3, Rushan Sabirov2, Oleg Gusev4, Wataru Sakamoto3 and Manabu Sugimoto1*
1Group of Gene Regulation, Institute of Plant Science and Resources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046, Japan
2Institute of Fundamental Medicine and Biology, Kazan Federal University, 18 Kremlyovskaya St., Kazan 420008, Republic of Tatarstan, Russia
3Plant Light Acclimation Research Group, Institute of Plant Science and Resources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046, Japan
4Preventive Medicine and Diagnosis Innovation Program, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
*Corresponding Author: Manabu Sugimoto, Group of Gene Regulation, Institute of Plant Science and Resources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046, Japan, Tel: +81 86 434 1253 Email:

Keywords: Brachypodium distachyon; Diadenosine polyphosphate; Drought stress; Nudix hydrolase; UV irradiation


Diadenosine polyphosphate (ApnA) is a ubiquitous family of nucleotides in which two nucleoside moieties are linked 5-5’ through a polyphosphate chain containing 3-7 phosphoryl groups [1,2]. Ap4A is implicated in coupling DNA replication to cell division [3,4], initiation of DNA replication [5,6], recovery from stress by modulating protein refolding [2,7,8], and regulation of ATP-sensitive K+ channels [9,10]. Because the Ap4A level is increased in cells exposed to stress conditions such as oxidative, heat, nutritional, and DNA damage [7,8,11-13] and because Ap4A increases the gene expression of phenylalanine ammonia-lyase and 4-coumarate: CoA ligase consisting of phenylpropanoid pathway by heavy metals in Arabidopsis [14], Ap4A has been proposed as an ‘alarmone’. The long-chain (ApnA) (n=5-6) produces cytotoxic effects, although it is also an intracellular and extracellular signaling molecule [2,15-18]. Distributed among humans, bacteria, fungi, and plants, Ap4A hydrolase metabolizes and regulates (ApnA) levels. It is classified into two groups. One group cleaves Ap4A symmetrically to produce two moles of ADP. Its structure is related to serine/threonine protein phosphatase [19-23]. The other group, which shows asymmetrical Ap4A hydration to produce ATP and AMP, belongs to the nudix hydrolase (NUDX) family [24]. Some asymmetrical Ap4A hydrolases catalyze not only Ap4A but also long-chain (ApnA) and other nucleotide polyphosphates, for which specific activities and their products depend on enzyme characteristics [24-27].

In plants, AtNUDX13, AtNUDX25, AtNUDX26, and AtNUDX27 from Arabidopsis thaliana belong to (ApnA) hydrolase of the NUDX family [28-31]. Actually, AtNUDX13 is active toward Ap6A and Ap5A, but it has no activity to Ap4A and other substrates for Ap4A hydrolases. AtNUDX25 hydrolyzes NADH, coenzyme A (CoA), and guanosine-3′, 5′-tetraphosphate (ppGpp), whereas AtNUDX26 hydrolyzes ppGpp, in addition to the activities of AtNUDX25 and 26 toward Ap5A and Ap4A. AtNUDX27 hydrolyzes only Ap5A. These NUDXs have a well-conserved nudix motif, GX5EX7REUXEEXGU, where U is usually Ile, Leu, or Val [24]. AtNUDX25, AtNUDX26, and AtNUDX27 had a tyrosine residue downstream of the nudix motif found in other Ap4A hydrolases and located in chloroplasts, whereas AtNUDX13 had a glycine tripeptide motif downstream of the nudix motif. The subcellular location was in mitochondria [28,29]. These results suggest that enzymatic properties and biological functions differ between (ApnA) hydrolases that have long-chain (ApnA)-specific activity and which have wide substrate specificity, but most studies of enzymatic properties and diversity of Ap4A hydrolases and long-chain (ApnA) hydrolases have scope that is limited to Arabidopsis NUDXs in plants.

Brachypodium distachyon is a model plant of Pooideae subfamily including wheat and barley, which has tractable features such as small genome size with diploid, small plant size, and short life cycle [32]. It is expected to serve as a useful function model for identification of genes and biological functions related to agronomic interest from Triticeae crops. This study identified the putative gene from Brachypodium which encodes the homologue of AtNUDX13 that hydrolyzes Ap6A and Ap5A specifically. Furthermore, this study elucidated the structure, enzymatic properties, subcellular location, and expression profiles under stress conditions.

Materials and Methods

Plant cultivation and stress treatment

Seeds of Brachypodium, Brachypodium distachyon Bd21, were incubated on filter paper kept moist with water at 23°C for 5 days in the dark. Seedlings were selected randomly from the germinated seeds. Three seedlings were planted on one Wagner pot (1/5000 a) filled with soil under a metal halide light (350 μmol/m2/s) with a light/dark cycle of 16 h/8 h in a growth chamber. After 7 days of cultivation, plants were irradiated with 186, 431, and 438 μW/cm2 of 340, 312, and 260 nm of UV light for 6 h to induce UV stress. The plants were pulled up and dehydrated on a paper towel for 6 h to stimulate drought stress, were soaked in a pot with 100 mM NaCl solution for 24 h to stimulate salt stress or were cultivated under metal halide light as a control. After exposure to stress conditions, the shoots were harvested, frozen in liquid nitrogen, and stored at -80°C.

Quantitative RT-PCR analysis

Total RNA was isolated from shoot samples using the RNeasy Plant mini kit (Qiagen Inc., Tokyo, Japan) following the manufacturer’s instructions. Poly(A)+ RNA was purified from total RNA with the Poly (A) Purist MAG (Ambion Inc., Austin, Texas). Then the purified poly(A)+ RNA was dissolved in the RNA storage solution. First-strand cDNA was synthesized from poly(A)+ RNA using a PrimeScript RT Master Mix (Takara Bio Inc., Shiga, Japan). Quantitative RT-PCR was performed in a mixture of 20 μl containing first-strand cDNA, SYBR Premix Ex Taq (Takara Bio Inc.), and 0.2 μmol of each forward primer, 5′-TGCACTGCTGGAGCGGTTAT-3′, and reverse primer, 5′- ATCAGATGTCGTTTGGAGCA-3′ using LightCycler 2.0 (Roche Applied Science, Mannheim, Germany). The thermal cycle profile was 1 cycle of 95°C for 10 s, followed by 40 cycles of 95°C for 5 s and 60°C for 20 s. The cDNA quantities of each gene were calculated using software (LightCycler 4.0; Roche Applied Science) and were normalized with that of the S-adenosylmethionine decarboxylase gene [33]. The expression analysis was conducted three times.

Expression and purification of BraNUDX15

The active form of HvNUDX 15 genes was amplified with firststrand cDNA from control shoots and the primers, 5′- CCATATGAAGAAGGACGAGGGGAACCC-3′, which creates a Nde I site (denoted as underlined), and 5′- CCTCGAGGCACAATGCAACTGCGCC-3′, which creates a Xho I site (denoted as underlined). The PCR product of 525 bp length was cloned into the pGEM-T vector. Then the fragments of the plasmids digested by Nde I and Xho I were subcloned into a pET-20b (+) vector, in which a polyhistidine tag gene is fused upstream from the start codon. The resulting plasmid, pBraNUDX15-ACT, was transformed into E. coli BL21 cells. E. coli cells harboring pBraNUDX15-ACT were grown at 37°C in Luria-Bertani (LB) medium containing 50 μg/ml ampicillin. When the OD600 reached 0.5, isopropyl-μ-Dthiogalactopyranoside (IPTG) was added to the culture at a final concentration of 0.5 mM. After cultivation at 25°C for 18 h, the cells were harvested by centrifugation and were frozen at -80°C for at least 2 h. The frozen cell pellets were suspended in a protein extraction reagent (BugBuster™ HT; Merck, Darmstadt, Germany) according to the manufacturer’s instructions. The resulting recombinant protein, which showed an insoluble form, was dissolved in 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl, 5 mM imidazole, and 6 M guanidine HCl (Buffer A) was purified using an Ni-NTA agarose column (Qiagen Inc.) initially equilibrated in Buffer A. The column was washed with Buffer A, followed by 60 mM imidazole in Buffer A, with the absorbed protein eluted with 200 mM imidazole in Buffer A. The protein solution was dialyzed against 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl, 5 mM imidazole, and 3 M guanidine HCl, followed by 20 mM Tris-HCl buffer (pH 8.0) containing 1 mM DTT, 100 mM NaCl, 0.5% n-dodecyl-μ-D-maltoside, and 10% ethylene glycol. The dialyzed solution was then concentrated (Vivaspin 4; Sartorius, Goettingen, Germany).

Enzyme and protein assays

The hydrolytic activity of BraNUDX15 was assayed according to a method described previously [34]. The reaction mixture (100 μl), DTT, 100 μM substrate, and recombinant protein, was incubated at which consists of 10 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 10 mM 37°C for 30 min. After the reaction was terminated by 17 μl of 100 mM EDTA, the reaction mixture was subjected to HPLC using a column (Cosmosil C18, 4.6 × 250 mm, Nacalai Tesque Inc., Kyoto, Japan) equilibrated with 100 mM phosphate buffer (pH 6.0) and 5% methanol at a flow rate of 0.6 ml/min. The reaction products were detected by absorption at 293 nm for 8-oxo-dGTP, 8-oxo-dGDP, at 260 nm for (ApnA) (n=4–6), ADP-ribose, NADH, UDP-Gal, dATP, ppGpp, and CoA, at 252 nm for dGTP, at 264 nm for dTTP, and at 271 nm for dCTP. Protein concentrations were quantified according to Bradford [35] with bovine serum albumin as the standard.

GFP transient assay

A DNA fragment encoding a putative transit peptide of BraNUDX15 predicted to the residue within the first 45 N-terminal amino acid residues was amplified by PCR with the first-strand cDNA from control shoot and primers, 5′- GGTCGACATGTCCAGCCTCGTTCTCGC-3′, which creates a Sal I site (denoted as underlined), and 5′- TCCATGGTGTACGGGACACACCCTGCA-3′, which creates a Nco I site (denoted as underlined). The PCR product of 144 bp length was cloned into the pGEM-T vector. Then the fragments of the plasmids digested by Sal I and Nco I were subcloned into a plasmid pTH-2, in which a GFP is fused downstream from the transit peptide [36]. The plasmid, pBraNUDX15-SIG+GFP, was transformed into Arabidopsis protoplasts according to the method explained by Miura et al. [37].


Identification of Brachypodium (ApnA) hydrolase genes

The genes, which encode amino acid sequences showing homology with those of 28 Arabidopsis NUDX families (AtNUDX1–27 and AtDCP2) and nudix motif (e<0.0001), were searched using a BLAST program [38,39] and the RIKEN Brachypodium distachyon Full- Length cDNA Clone Database. The full-length cDNAs of 19 putative Brachypodium NUDX genes, BraNUDX1-19, were identified. The deduced amino acid sequences of BraNUDX1-19 showed 71-43% identities with those of AtNUDXs and nudix motif (Table 1).

Gene Gene ID Identity (%)   Subfamily
BraNUDX1 Bradi1g35490.1 AtNUDX2: 55 ADP-ribose/NADH
    AtNUDX10: 50  
    AtNUDX7: 45  
    AtNUDX6: 43  
BraNUDX2 Bradi3g53887.1 AtNUDX3: 66 n.d.
BraNUDX3 Bradi1g44170.1     n.d.
BraNUDX4 Bradi2g37517.1 AtNUDX9: 60 GDP-mannose
BraNUDX5 Bradi2g32550.1     n.d.
BraNUDX6 Bradi5g08460.1     n.d.
BraNUDX7 Bradi1g49810.1 AtNUDX14: 58 ADP-ribose/ADP-glucose
BraNUDX8 Bradi3g56830.1 AtNUDX17: 51 n.d.
    AtNUDX4: 49  
BraNUDX9 Bradi1g51060.1 AtNUDX19: 59 NADPH
BraNUDX10 Bradi4g28030.2 AtNUDX20: 61 Thiamin diphosphate
    AtNUDX24: 58  
BraNUDX11 Bradi4g37360.1 AtNUDX23: 53 FAD
BraNUDX12 Bradi5g26560.2 AtNUDX26: 58 ApnA/ppGpp
    AtNUDX25: 49  
    AtNUDX27: 47  
BraNUDX13 Bradi3g35160.1 AtNUDX22: 55 CoA
    AtNUDX11: 51  
BraNUDX14 Bradi5g17500.1 AtNUDX8: 50 n.d.
BraNUDX15 Bradi3g44460.1 AtNUDX13: 47 ApnA/ppGpp
    AtNUDX12: 46  
BraNUDX16 Bradi3g35150.1 AtNUDX15: 57 CoA
BraNUDX17 Bradi1g54020.1 AtNUDX16: 71 n.d.
BraNUDX18 Bradi3g56830.1 AtNUDX18: 52 n.d.
    AtNUDX21: 47  
BraNUDX19 Bradi3g54700.1 AtDCP2: 58 mRNA cap
n.d., not detected.        

Table 1: Identity of deduced amino acid sequences of BraNUDX genes with those of AtNUDX genes.

Alignment analysis of amino acid sequences of BraNUDX1-19 obtained using the Clustal W program showed that the nudix motif comprising 23 amino acid residues was conserved in the amino acid sequences of Brachypodium NUDXs, except for the insertion of 22 amino acid residues in BraNUDX4 (Figure 1).


Figure 1: Alignment of the deduced amino acid sequences around the nudix motif in putative Brachypodium NUDXs. Gaps, denoted by a dash were introduced into the sequences to maximize the homology. The nudix motif is shown below the sequence. Identical amino acid residues to those of nudix motif are shown as reversed letters.

According to the substrate specificities of Arabidopsis NUDXs, 15 Brachypodium NUDXs are classified into the following subfamilies: BraNUDX1 belongs to ADP-ribose/NADH hydrolase; BraNUDX4 belongs to GDP-mannose hydrolase; BraNUDX7 belongs to ADPribose/ ADP-glucose hydrolase; BraNUDX9 belongs to NADPH hydrolase; BraNUD×10 belongs to thiamin diphosphate hydrolase; BraNUDX11 belongs to FAD hydrolase; BraNUDX12 and 15 belong to (ApnA)/ppGpp hydrolase; BraNUDX13 and 16 belong to CoA hydrolase; BraNUDX19 belongs to mRNA cap; although BraNUDX2, 3, 5, 6, 8, 14, 17, and 18 were not assigned to any established subfamily (Table 1). These results suggest that two (ApnA) hydrolase genes, BraNUDX12 and 15, are present in Brachypodium . BraNUDX12 conserved Tyr downstream of nudix motif as did Arabidopsis (ApnA) hydrolases, AtNUDX25, 26, and 27, whereas BraNUDX15 conserved glycine tripeptide motif, GX2GX6G, as did AtNUDX13, which hydrolyzes Ap6A and Ap5A specifically (Figure 2).


Figure 2: Alignment of the deduced amino acid sequences around the nudix motif in Brachypodium and Arabidopsis ApnA hydrolases. Glycine residues of glycine tripeptide motif and a tyrosine residue downstream from nudix motif are shown respectively by white and black triangles.

Purification and enzymatic characterization of recombinant BraNUDX15

BraNUDX15 protein, which was encoded by the open reading frame of cDNA, was produced in E. coli cells. However, the protein made an inclusion body and could not be made soluble by refolding (data not shown). Results of alignment analysis of the amino acid sequence of BraNUDX15 with that of AtNUDX13, which had the transit peptide [29], suggest that the N-terminal peptide of 46 amino acid residues of BraNUDX15 is the transit peptide (Figure 3).


Figure 3: Alignment of the deduced amino acid sequences of fulllength amino acid sequences of AtNUDX13 and BraNUDX15. The black frame and asterisks respectively denote the transit peptide of AtNUDX13 and identical amino acid residues between AtNUDX13 and BraNUDX15.

The mature form of BraNUDX15 protein, of which the transit peptide at the N-terminus was eliminated, tagged with a His-Tag at its C-terminus, was produced in E. coli cells. An extra protein with a molecular mass of 22 kDa, which is similar to that calculated from the amino acid sequence, was produced as an inclusion body in E. coli cells and was refolded to soluble form (Figure 4).


Figure 4: Analysis of the expression of BraNUDX15 in E. coli cells using SDS-polyacrylamide gel. E. coli cells harboring pBraNUDXACT were harvested after IPTG induction at 25°C for 18 h. The soluble protein (1), insoluble protein (2), and refolded BraNUDX15 purifying with Ni-NTA column (3) were subjected to 12% SDSPAGE with molecular mass marker (M) followed by Coomassie Brilliant Blue R-250 staining.

The purified BraNUDX15 incubated with Ap6A showed production of ATP, of which the specific activity was 1.23 μmol/min/mg. The enzyme activity was inhibited completely by EDTA and was recovered by MgCl2. BraNUDX15 had maximum activity toward Ap6A at pH 8.0 and relative activities of 95% for Ap5A and 76% for Ap4A to Ap6A hydrolyzing activity, with barely any activity toward dCTP (Table 2).

  Specific activity (mmol/min/mg)
Substrate BraNUDX15 AtNUX13 [28] AtNUDX25 [30,31] AtNUDX26 [30,31] AtNUDX27 [30]
Ap3A n.d. n.d. n.d. n.d. n.d.
Ap4A 0.94 ± 0.02 n.d. 0.026 ± 0.02 13.3 ± 0.36 n.d.
Ap5A 1.17 ± 0.11 4.2 0.017 ± 0.001 21.6 ± 0.58 0.22 ± 0.01
Ap6A 1.23 ± 0.03 10.5 - - -
Ap4G 0.72 ± 0.09 - - - -
Gp4G 0.74 ± 0.04 - - - -
ADP-ribose n.d. - n.d. n.d. n.d.
NADH n.d. - 0.016 ± 0.001 n.d. n.d.
CoA n.d. - 0.012 ± 0.001 0.11 ± 0.01 n.d.
UDP-Gal n.d. - n.d. n.d. n.d.
ppGpp n.d. - 0.06 ± 0.01 0.19 ± 0.05 -
8-oxo-dGTP n.d. - n.d. 0.02 ± 0.01 n.d.
dGTP n.d. - n.d. 0.05 ± 0.01 n.d.
dATP n.d. - n.d. 0.07 ± 0.01 n.d.
dTTP n.d. - n.d. 0.05 ± 0.01 n.d.
dCTP 0.04 ± 0.001 - n.d. 0.07 ± 0.01 n.d.

n.d., not detected; -, not reported.

Table 2: Substrate specificities of Brachypodium and Arabidopsis ApnA hydrolases.

Expression of BraNUDX15 gene under abiotic stress

Brachypodium was cultivated under UV irradiation, drought, and salt conditions to evaluate the response of BraNUDX15 gene to environmental stresses (Figure 5).


Figure 5: Expression profiles of BraNUDX15 gene in Brachypodium under abiotic stress. Total RNAs isolated from shoots of Brachypodium under UV-A, UV-B, UV-C, drought, and 150 mM NaCl conditions were subjected to quantitative RT-PCR. Expression levels were normalized with that of S-adenosylmethionine decarboxylase gene as an internal control. The error bar represents the standard error of the mean for three experiments.

The expression level of BraNUDX15 gene was up-regulated considerably: 2.5, 4.8, and 3.7-fold, respectively, by UV-A, UV-B, and UV-C irradiation. Drought stress reduced the expression level to about half. The expression level was unchanged by salt stresses, which increased it about 10%.

Subcellular localization of BraNUDX15 protein

A DNA fragment corresponding to predicted transit peptide from BraNUDX15 cDNA sequence was fused in frame with GFP at the Cterminus and was expressed in protoplasts under the control of the CaMV 35S promoter. The GFP fusion protein fluorescence in the transgenic cells was colocalized with the surface of chloroplasts (Figure 6).


Figure 6: Subcellular localization of BraNUDX15. Arabidopsis cells were transformed with either pBraNUDX15-SIG+GFP or pTH-2 (GFP control). GFP fluorescence (GFP) and chlorophyll autofluorescence (Chlorophyll) signals were merged (Merged).

The subcellular localization of BraNUDX15 was also predicted in chloroplasts using WoLF PSORT server.


Genes encoding homology with (ApnA) hydrolase were searched from Brachypodium. Of 19 putative NUDX genes, BraNUDX12 and 15 genes showed homology with Arabidopsis (ApnA) hydrolases NUDXs. BraNUDX12 showed identity with AtNUDX25, 26, and 27, which conserved the tyrosine residue found in (ApnA) hydrolases and hydrolyzed Ap4A and/or Ap5A, whereas BraNUDX15 showed identity with AtNUDX13, which had the glycine tripeptide motif and hydrolyzed Ap6A and Ap5A but not Ap4A [28,29]. These results suggest BraNUDX15 as the long-chain (ApnA) specific hydrolase.

The purified BraNUDX15, of which the predicted transit peptide was eliminated, required Mg2+ for hydrolyzing (ApnA), as did other (ApnA) hydrolases. The enzyme had the highest activity toward Ap6A, with relative activities of 95% for Ap5A and 76% for Ap4A to Ap6A hydrolyzing activity. It produced ATP from these substrates, whereas Arabidopsis long-chain (ApnA) hydrolase, AtNUDX13, showed activity toward Ap6A, preferentially toward Ap6A, and relative activity of 40% for Ap5A to Ap6A hydrolyzing activity. However, it showed no activity toward Ap4A. It produced ADP+p4A from Ap6A and AMP +p4A from Ap5A [28]. AtNUDX25 and 26 showed activity not only toward Ap4A and Ap5A but also toward NADH, CoA, 8-oxo-dGTP, ppGpp, or dNTPs [30,31], which were not hydrolyzed by BraNUDX15 except for slight activity toward CoA. These results indicate that BraNUDX15 is a unique (ApnA) hydrolase that has different substrate specificity from Arabidopsis (ApnA) hydrolases and indicate that glycine tripeptide motif is necessary for hydrolyzing long-chain (ApnA).

In plant cells, AtNUDX13 was localized in mitochondria; AtNUDX26 and 27 were localized in chloroplasts [28,30]. Reportedly, AtNUDX26 hydrolyzed ppGpp, of which the level in chloroplasts was increased under environmental stress. Moreover, the expression level of the gene increased under drought stress, suggesting that AtNUDX26 regulates the ppGpp level in chloroplasts [31]. Tomato Ap4A hydrolase gene decreased by CdCl2 [40]; Ap4A increased the gene expression of phenylalanine ammonia-lyase and 4-coumarate: CoA ligase consisting of phenylpropanoid pathway by heavy metals in Arabidopsis [14], indicating that the Ap4A level is regulated by Ap4A hydrolase to induce stress tolerance genes as alarmone. An earlier study showed that Ap6A inhibits ATP-sensitive K+ channels [17] and that extracellular Ap6A and Ap5A influence cytosolic free Ca2+ concentrations [18]. The accumulation of long-chain (ApnA) can produce cytotoxic effects through the inhibition of various kinases [15,16]. Our result demonstrated that BraNUDX15 was localized around the chloroplast surface. The gene expression level was induced under UV-A, -B, and - C exposure, but it was reduced by drought stress. Taken together, the evidence shows that BraNUDX15 can be expected to play a role in accumulating Ap4A to induce drought-stress-relieving genes under drought stress and decreasing long-chain (ApnA) before attaining a potentially toxic concentration under UV irradiation in chloroplasts.


Results of this study demonstrated that Brachypodium (ApnA) hydrolase BraNUDX15, which showed homology with Arabidopsis long-chain (ApnA) hydrolase and conserved glycine tripeptide motif, was a unique (ApnA) hydrolase that has different substrate specificity from those of Arabidopsis (ApnA) hydrolases. The expression level of BraNUDX15 gene was increased by UV irradiation and decreased by drought stress. Moreover, the protein was localized in chloroplasts.

These results suggest that BraNUDX15 is a unique (ApnA) hydrolase with different substrate specificity from those of Arabidopsis (ApnA) hydrolases. It might play a role in regulating (ApnA) levels in chloroplasts under drought stress and UV irradiation.


This research was partially supported by the Ohara Foundation of Kurashiki, Japan.


  1. McLennan AG (2000) Dinucleoside polyphosphates-friend or foe? Pharmacol Ther 87: 73-89
  2. McLennan AG, Barnes LD, Blackburn GM, Brenner C, Gura-nowski A, et al. (2001) Recent progress in the study of the intracellular functions of diadenosine polyphosphates. Drug Dev Res 52: 249-259.
  3. Nishimura A, Moriya S, Ukai H, Nagai K, Wachi M, et al. (1997) Diadenosine 5’, 5’’’-P1, P4-tetraphosphate (Ap4A) controls the timing of cell division in Escherichia coli. Genes Cells 2: 401-413.
  4. Nishimura A (1998) The timing of cell division: Ap4A as a signal. Trends Biochem Sci 23: 157-159
  5. Baril EF, Coughlin SA, Zamecnik PC (1985) 5’, 5’’’-P1, P4- diadenosine tetraphosphate (Ap4A): a putative initiator of DNA replication. Cancer Invest 3: 465-471.
  6. Bambara RA, Crute JJ, Wahl AF (2009) Is Ap4A an Activator of Eukaryotic DNA Replication? Cancer Investigation 3: 473-479.
  7. Johnstone DB, Farr SB (1991) AppppA binds to several proteins in Escherichia coli, including the heat shock and oxidative stress proteins DnaK, GroEL, E89, C45 and C40. EMBO J 10: 3897-3904.
  8. Fuge EK, Farr SB (1993) AppppA-binding protein E89 Is the Escherichia coli heat shock protein ClpB. J Bacteriol 175: 2321-2326
  9. Jovanovic A, Alekseev AE, Terzic A (1997) Intracellular diadenosine polyphosphates – a novel family of inhibitory ligands of the ATP-sensitive K+ channel. Biochem Pharmacol 54: 219-225.
  10. Martin F, Pintor J, Rovira JM, Ripoll C, Miras-Portugal MT, et al. (1998) Intracellular diadenosine polyphosphates: a novel second messenger in stimulus-secretion coupling. FASEB J 12: 1499-1506.
  11. Lee PC, Bochner BR, Ames BN (1983) AppppA, heat-shock stress, and cell oxidation. Proc Natl Acad Sci USA 80: 7496-7500.
  12. Bochner BR, Lee PC, Wilson SW, Cutler CW, Ames BN (1984) AppppA and related adenylated nucleotides are synthesized as a consequence of oxidation stress. Cell 37: 225-232.
  13. Plateau P, Fromant M, Blanquet S (1987) Heat shock and hydro- gen peroxide responses of Escherichia coli are not changed by dinucleoside tetraphosphate hydrolase overproduction. J Bacteriol 169: 3817-3820.
  14. Pietrowska-Borek M, Nuc K, Zielezinska M, Guranowski A (2011) Diadenosine polyphosphates (Ap3A and Ap4A) behave as alarmones triggering the synthesis of enzymes of the phenylpropanoid pathway in Arabidopsis thaliana. FEBS Bio 1: 1-6.
  15. Bone R, Cheng YC, Wolfenden R (1986) Inhibition of adenosine and thymidylate kinases by bisubstrate analogs. J Biol Chem 261: 16410-16413.
  16. Shoyah M (1985) Inhibition of protein kinase activity of phorboid and ingenoid receptor by di(adenosine-5) oligophosphate. Arch Biochem Biophys 236: 441-444.
  17. Jovanovic A, Terzic A (1995) Diadenosine-hexaphosphate is an inhibitory ligand of myocardial ATP-sensitive K+ channels. Eur J Pharmacol 286: R1-R2.
  18. Tepel M, Lowe S, Nofer JR, Assmann G, Schulter H, et al. (1996) Diadenosine polyphosphates regulate cytosolic calcium in human fibroblast cells by interaction with P2x purinoreceptors coupled to phospholipase C. Biochim Biophys Acta 1312: 145-150.
  19. Guranowski A, Jakubowski H, Holler E (1983) Catabolism of di- adenosine 5’, 5’’’-P1, P4-tetraphosphate in procaryotes. Purification and properties of a diadenosine 5’, 5’’’- P1, P4-tetraphosphate (symmetrical) pyrophosphohydrolase from Escherichia coli K12. J Biol Chem 258: 14784-14789.
  20. Guranowski A (2000) Specific and nonspecific enzymes involved in the catabolism of mononucleoside and dinucleoside polyphosphates. Pharmacol Ther 87: 117-139.
  21. Ismail T, Hart CA, McLennan AG (2003) Regulation of dinucleoside polyphosphate pools by the YgdP and ApaH hydrolases is essential for the ability of Salmonella enterica serovar Typhimurium to invade cultured mammalian cells. J Biol Chem 278: 32602-32607.
  22. Barton GJ, Cohen PTW, Barford D (1994) Conservation analysis and structure prediction of the protein serine/threonine phosphatases. Sequence similarity with diadenosine tetraphosphatase from Escherichia coli suggests homology to the protein phosphatases. Eur J Biochem 220: 225-237.
  23. Lohse DL, Denu JM, Dixon JE (1995) Insights derived from the structures of the Ser/Thr phosphatases calcineurin and protein phosphatase 1. Structure 3: 987-990
  24. Bessman MJ, Frick DN, O’Handley SF (1996) The MutT proteins or “Nudix” hydrolases, a family of versatile, widely distributed “house-cleaning” enzymes. J Biol Chem 271: 25059-25062.
  25. McLennan AG (2006) The Nudix hydrolase superfamily. Cell Mol Life Sci 63: 123-142.
  26. Kraszewska E (2008) The plant Nudix hydrolase family. Acta Biochi Pol 55: 663-671.
  27. Xu W, Gauss P, Shen JY, Dunn CA, Bessman MJ (2006) Three new nudix hydrolases from Escherichia coli. J Biol Chem 281: 22794-22798.
  28. Olejnik K, Murcha MW, Whelan J, Kraszewska E (2007) Cloning and characterization of AtNUDT13, a novel mitochondrial Arabidopsis thaliana Nudix hydrolase specific for long-chain diadenosine polyphosphates. FEBS J 274: 4877-4885.
  29. Yoshimura K, Shigeoka S (2015) Versatile physiological functions of the Nudix hydrolase family in Arabidopsis. Biosci Biotech Biochem 79: 354-366.
  30. Ogawa T, Yoshimura K, Miyake H, Ishikawa K, Ito D, et al. (2008) Molecular characterization of organelle-type nudix hydrolases in Arabidopsis. Plant Physiol 148: 1412-1424
  31. Ito D, Kato T, Murata T, Tamoi M, Yoshimura K, et al. (2012) Enzymatic and molecular characterization of Arabidopsis ppGpp pyrophosphohydrolase, AtNUDX26. Biosci Biotechnol Biochem 76: 2236-2241.
  32. Draper J, Mur LAJ, Jenkins G, Ghosh-Biswas GC, Bablak P, et al. (2001) Brachypodium  distachyon. A new model system for functional genomics in grasses. Plant Physiol 127: 1539-1555.
  33. Hong SY, Seo PJ, Yang MS, Xiang F, Park CM (2008) Exploring valid reference genes for gene expression studies in Brachypodium  distachyon by real-time PCR. BMC Plant Biol 8: 112.
  34. Tanaka S, Kihara M, Sugimoto M (2015) Structure and molecular characterization of barley nudix hydrolase genes. Biosci Biotech Biochem 79: 394-401.
  35. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254
  36. Niwa Y (2003) A synthetic green fluorescent protein gene for plant biotechnology. Plant Biotech 20: 1-11.
  37. Miura E, Kato Y, Matsushima R, Albrecht V, Laalami S, et al. (2007) The balance between protein synthesis and degradation in chloroplasts determines leaf variegation in Arabidopsis yellow variegated mutants. Plant Cell 19: 1313-1328.
  38. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403-410.
  39. Karlin S, Altschul SF (1990) Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proc Natl Acad Sci USA 87: 2264-2268.
  40. Feussner K, Guranowski A, Kostka S, Waternack C (1996) Diadenosine 5’, 5’’’-P1, P2-tetraphosphate (Ap4A) hydrolase from Tomato (Lycopersicon esculentum cv. Lukullus) – purification, biochemical properties and behavior during stress. Zeitschrift fur Naturforschung C. 51: 477-486.
Citation: Tanaka M, Iamshchikov I, Kato Y, Sabirov R, Gusev O, et al. (2018) Structure and Molecular Characterization of Diadenosine Polyphosphate Hydrolase in Brachypodium distachyon. J Plant Biochem Physiol 6: 220.

Copyright: © 2018 Tanaka M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.