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Binding and catalysis of Humulus lupulus adenylate isopentenyltransferase for the synthesis of isopentenylated diadenosine polyphosphates
FEBS Letters 584 (2010) 4083–4088
journal homepage: www.FEBSLetters.org
Binding and catalysis of Humulus lupulus adenylate isopentenyltransferase for the synthesis of isopentenylated diadenosine polyphosphates Hsing-Mao Chu a,1, Feng-Yuan Chen a,b,1, Tzu-Ping Ko a, Andrew H.-J. Wang a,b,⇑ a b
Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan
a r t i c l e
i n f o
Article history: Received 9 August 2010 Revised 26 August 2010 Accepted 26 August 2010 Available online 31 August 2010 Edited by Ulf-Ingo Flügge Keywords: Prenyltransferase Cytokinin Calorimetry Diadenosine polyphosphates
a b s t r a c t Various plant developmental processes involve phytohormones such as cytokinins. Isopentenyltransferase (IPT) reaction is the key rate-limiting step in cytokinin biosynthesis that transfers the isopentenyl (iP) group from dimethylallyl diphosphate to the N6-amino group of adenine. Here, a series of diadenosine polyphosphates (ApnA) were screened as possible substrates of IPT, among which diadenosine tetraphosphate, diadenosine pentaphosphate and diadenosine hexaphosphate showed higher affinity than did the authentic substrates ADP and ATP. In addition, formation of mono-isopentenyl ApnA and di-isopentenyl ApnA was observed. Judging by the existing biosynthetic and hydrolytic systems for ApnA in plants, ApnA and isopentenyl-ApnA may occur in the plant cells, with functional importance. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Cytokinins (CKs) are important adenine-base phytohormones which promote cell division and their biosynthesis has two pathways depending on different substrates. The first pathway begins with the transfer of isopentenyl (iP) group from dimethylallyl diphosphate (DMAPP) to the N6-amino group of AMP, ADP or ATP by adenylate isopentenyltransferase (AIPT) [1]. In the second pathway, tRNA-IPT catalyzes the isopentenyl transfer reaction at certain adenine base, and CK is produced by subsequent degradation of the prenylated tRNA [2]. In either pathway, the IPT-catalyzed reaction is the key rate-limiting step in cytokinin biosynthesis. Several plant AIPTs from Arabidopsis thaliana, rice, mulberry (Morus alba), and hop (Humulus lupulus L) have been cloned and characterized [3–7]. In higher plants, because the plant
Abbreviations: CKs, cytokinins; DMAPP, dimethylallyl diphosphate; AIPT, adenylate isopentenyltransferase; iP, isopentenyl; ITC, isothermal titration calorimetry; Ap3A, diadenosine triphosphate; Ap4A, diadenosine tetraphosphate; Ap5A, diadenosine pentaphosphate; Ap6A, diadenosine hexaphosphate; ApnA, diadenosine polyphosphates; iso-ApnA, mono-isopentenyl ApnA; di-iso-ApnA, di-isopentenyl ApnA ⇑ Corresponding author at: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan. Fax: +886 2 2788 2043. E-mail address: [email protected] (A.H.-J. Wang). 1 These authors have equal contribution.
AIPT predominantly uses DMAPP and ATP, or ADP, the major initial product is an isopentenyl nucleotide, such as isopentenyl riboside 50 -triphosphate (iPRTP) or isopentenyl riboside 50 -diphosphate (iPRDP) [8]. Although it is well known that the plant AIPT predominantly uses ATP or ADP as the isopentenyl acceptor, it cannot be excluded, however, that other adenosine-related molecules (such as dinucleotide polyphosphates) can serve as acceptors. In general, the adenosine-related dinucleotide molecules are divided into two main groups. The first group has the adenosine linked via a pyrophosphate to a ribosylated nicotinamide (NAD) or ribitolylated flavin adenine (FAD). The second group, diadenosine polyphosphates (ApnA, n = 2–6), are formed by two adenosines joined by a variable number of phosphate groups. ApnA are naturally and ubiquitously occurring molecules and have been identified and found to be stored in a diverse number of biological tissues including platelets, adrenal medullary chromaffin granules and the central nervous system [9–11]. Although the existence of ApnA in plants has yet to be unequivocally demonstrated, their corresponding hydrolytic enzyme systems have been shown to exist according to amino acid sequence homology analysis [12]. Recently, we solved the first plant AIPT from Humulus lupulus (HlAIPT) [13]. In order to further characterize HlAIPT, its binding affinity and catalytic activity for different adenosine-related molecules were measured, and results suggest a series of possible substrates for this enzyme, including ApnA.
0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.08.038
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Table 1 Thermodynamic parameters of interactions between HlAIPT and adenosine derivatives. Ligand
Ka (M1)
DH (cal/mol)
DS (cal mol1 K1)
% Ka
ATP Ap3A Ap4A Ap5A Ap6A NAD NADP FAD
(7.64 ± 0.27) 105 (4.44 ± 0.44) 104 (6.07 ± 0.33) 105 (1.47 ± 0.07) 106 (6.00 ± 0.29) 105 N.D. N.D. N.D.
(4.01 ± 0.03) 104 (2.56 ± 0.30) 104 (1.43 ± 0.01) 105 (1.36 ± 0.19) 104 (5.09 ± 0.05) 104
107 64.6 370 426 144
100 5 79 190 78
Protein and substrate were dissolved in HEPES buffer (20 mM, pH 7.0; 150 mM NaCl, 3 mM DTT). ITC experiments were performed at 25 °C. Ka: association constant; DH: enthalpy changes; DS: entropy changes. Plus/minus (±) signed values represent standard error between the observed data and fitted parameters. N.D. means not detectable.
2. Materials and methods 2.1. Preparation of truncated HlAIPT and mutants All chemicals were purchased from Sigma–Aldrich except mentioned otherwise. The pQE80L plasmid containing the truncated
HlAIPT (aa 27–329), which lacks the N-terminal signal peptide, was used. Affinity and activity measurement were based on this truncated and more stable HlAIPT. We overexpressed the protein containing the N-terminal hexahistidine tag in Escherichia coli BL21 (DE3) (Novagen) following the previous expression and purification procedures [13]. 2.2. Isothermal titration calorimetry All adenosine-related molecules were dissolved in the dialysis buffer. Experiments were conducted at 25 °C. For diadenosine tetraphosphate (Ap4A), diadenosine pentaphosphate (Ap5A), nineteen 2 ll injections of 0.2 mM ligand into a 0.2 ml sample cell containing 16 lM of the truncated HlAIPT (aa 27–329) were made. For diadenosine triphosphate (Ap3A), NAD, NADP and FAD, nineteen 2 ll injections of 0.1 mM ligand were made. In the case of diadenosine hexaphosphate (Ap6A), six 1.5 ll injections of 0.1 mM ligand and subsequent fifteen 2 ll injections were conducted. Injections were made over a period of 4 s with 150 s intervals between injections. The sample cell was stirred at 1000 rpm. Data were acquired on an iTC200 microcalorimeter (Microcal, Northhampton, MA) and analyzed with the ORIGIN v.7.0 software provided with the microcalorimeter.
Fig. 1. The activity of ApnA molecules for HlAIPT. (A) Model of complex structure of (HlAIPT/Ap5A). (B) Surface charge distribution of complex structure (HlAIPT/Ap5A) showing a positively-charged binding cavity. (C) Substrate specificity of HlAIPT.
H.-M. Chu et al. / FEBS Letters 584 (2010) 4083–4088
2.3. HlAIPT activity assay The standard reaction mixture contained 150 lM prenyl acceptor substrate (ATP, Ap3A, Ap4A, Ap5A and Ap6A), 100 lM prenyl donor substrate (DMAPP), and 4 lg of the purified recombinant truncated HlAIPT in a final volume of 100 ll of 20 mM Tris–HCl buffer, pH 5.0, containing 1 mM MgCl2. Incubations were carried out at 25 °C for a series of period, and stopped by adding 10 ll 20% acetic acid. The product was analyzed by reverse-phase HPLC on a hydrosphere C18 column (4.6 150 mm I.D., YMC) with a flow rate of 0.5 ml/min. The isocratic elution was performed with solvent A, 50% CH3CN in 20 mM tetrabutylammonium bromide, 20 mM KH2PO4: 0–30 min. Elutions were monitored by a multichannel UV detector (Waters, Dual Absorbance detector) at 260 nm. 2.4. Mass spectroscopy Enzyme reaction products were characterized by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry. MALDI-TOF data: Ap3A, m/z 757 [M+H]+; isopentenyl Ap3A, m/z 825 [M+H]+; di-isopentenyl Ap3A (di-iso-Ap3A), m/ z 893 [M+H]+; Ap4A, m/z 837 [M+H]+; isopentenyl Ap4A, m/z 905 [M+H]+; di-iso-Ap4A, m/z 973 [M+H]+; Ap5A, m/z 917 [M+H]+; isopentenyl Ap5A, m/z 985 [M+H]+; di-iso-Ap5A, m/z 1053 [M+H]+; Ap6A, m/z 997 [M+H]+; isopentenyl Ap6A, m/z 1065 [M+H]+; diiso-Ap6A, m/z 1133 [M+H]+.These samples were mixed with the same volume of matrix which containing CHCA (a-cyano-4-hydro-
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xy-trans-cinnamic-acid), and were solvated in 50% acetonitrile and 0.1% TFA (trifluoroacetic acid) as detectable materials, and spotted on a MALDI plate. 3. Results 3.1. The affinities of adenosine-related nucleotides with HlAIPT In order to assess the binding affinity between HlAIPT and adenosine-related dinucleotides, we use isothermal titration calorimetry (ITC) to screen a series of adenosine-related dinucleotides including ApnA (P1,P3-di(adenosine-50 )-polyphosphate) from triphosphates to hexaphosphates {Ap3A, Ap4A, Ap5A, and Ap6A}, NADP, NAD and FAD. As shown in Table 1, HlAIPT discriminates the dinucleotides by different Ka terms. A few typical ITC tracings, showing the raw and integrated data, can be found in Supplementary Fig. S1. Interestingly, the results of ITC experiments revealed a length-dependent binding affinity order of Ap5A >ATP Ap6A Ap4A >Ap3A to HlAIPT. For NADP, NAD and FAD, there was no detectable binding to HlAIPT (Table 1). 3.2. The dinucleotides binding to HlAIPT in a broad positively-charged pocket These ApnA show ability to bind HlAIPT and Ap5A displays the highest binding affinity (1.47 106 (M1)) for HlAIPT among them. In order to explain the effect on binding affinity due to different
Fig. 2. Formation of di-isopentenyl Ap4A. (A) Synthesis from Ap4A and DMAPP to its products, mono-isopentenyl Ap4A (iso-Ap4A) and di-isopentenyl Ap4A (di-iso-Ap4A) were monitored by reverse-phase HPLC. (B) Analysis of the concentration of Ap4A, iso-Ap4A, and di-iso-Ap4A versus time. (C) The formations of the iso- and di-iso-ApnAs by enzymatic catalysis.
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length of polyphosphates backbone, a HlAIPT/Ap5A complex model was constructed by computer modeling, based on the complex structure of HlAIPT with ATP. The model showed that the Ap5A molecule adopted an extended conformation and fully occupied the positively charged binding cavity (Fig. 1A and B). Thus, when ApnA molecules bind to HlAIPT, the binding affinity may be enhanced with increasing number of phosphate groups from triphosphates to pentaphosphates. However, the Ap6A molecule show lower binding affinity than Ap5A because its sixth phosphate groups may make the molecule too long to allow docking of the second adenosine moiety onto the protein surface. Taken together, the experimental results are consistent with the observation in the computer model that Ap5A is optimally sized for high binding affinity to HlAIPT among different ApnA. In the case of NADP, NAD and FAD, no detectable binding to HlAIPT was observed, indicating that the linker of dinucleotides bound to the cavity of HlAIPT requires a minimum length of triphosphates in order to accommodate the phosphate backbone and the two dinucleotides at the same time (Fig. 1B).
3.3. Enzyme-catalyzed formation of mono-isopentenyl ApnA Because ApnA consist of two adenosines and different numbers polyphosphates, it was worth investigating whether HlAIPT shows activities towards these compounds or not. Interestingly, analysis of formation of mono-isopentenyl ApnA (iso-ApnA, n = 3–6) molecules by HPLC showed that the HlAIPT activity for Ap3A, Ap4A, Ap5A and Ap6A are 51%, 87%, 76%, and 90%, respectively, relative to that for ATP (100%) (Fig. 1C). The activity for Ap3A was the minimal among all ApnA molecules, whereas significant activities for Ap4A, Ap5A and Ap6A were revealed in vitro that reached 70–90% that for ATP. The activities of HlAIPT for ApnA molecules are generally consistent with the order of binding affinity. 3.4. Formation of di-iso-ApnA Intriguingly, we also observed a peak that indicated the formation of di-iso-Ap4A in the HPLC elution profile and reached 50% turnover within 20 min. Complete formation of di-isopentenyl
Fig. 3. The proposed catalytic reaction of ApnA molecule (Ap6A as an example) to proceed through the several steps.
H.-M. Chu et al. / FEBS Letters 584 (2010) 4083–4088
diadenosine was achieved within 40 min. Besides, we observed that the concentration of iso-Ap4A reached a maximum concentration within 15 min and then gradually decreased to an undetectable amount (Fig. 2A). Hence, we compared the turnover rate of converting Ap4A to iso-Ap4A with that of converting iso- to di-iso-Ap4A, and found a similar formation rate by analyzing the reaction profile of the two products. This may suggest that the isopentenyl side-chain of iso-Ap4A does not interfere with its binding to HlAIPT. Consequently the efficiency of isopentenylation reaction is similar for Ap4A and iso-Ap4A. Furthermore, we also tested other ApnA molecules and found that all of the corresponding di-iso-ApnA were formed when these reaction products (iso- and di-iso-Ap3A, Ap4A, Ap5A and Ap6A) where further subjected to Mass Spectrum analysis. These results were shown in Supplementary Fig. S2. This is the first observation of the formations of iso- and di-iso-ApnA by enzymatic catalysis (Fig. 2B and C).
4. Discussion Based on the binding and activity assays, it was found that these ApnA (n = 4–6) not only showed higher binding affinity to HlAIPT than that of ADP (4.49 105 (M1)), but they were also catalyzed to produce mono- and di-isopentenyl products (Fig. 2A and B). These results indicate that HlAIPT may act on a series of previously unidentified substrates in plant. Herein, based on these results, we proposed a mechanism reaction of Ap6A molecule to proceed through the following steps (Fig. 3). First, the initial binding of Ap6A molecule to active-site pocket is facilitated by the nonspecific interactions between positively-charged surface of the pocket and the negatively-charged phosphate groups of Ap6A molecule. After entry of DMAPP to active site, isopentenyl group was transferred from DMAPP to N6 of the adenine ring of Ap6A molecule, and then iso-Ap6A molecule was released from the cavity. In the further step of reaction, iso-Ap6A molecule reorients itself and the unmodified adenine ring again binds to the active site of HlAIPT while the isopentenylated adenine ring of Ap6A molecule is exposed to outside of the positively-charged the pocket. Finally, when the second isopentenylation reaction of Ap6A molecule is finished, the diiso-Ap6A molecule is released from the active site of HlAIPT. ApnA (n = 3–6) have been detected in rabbit and human tears by HPLC [14,15]. These molecules are ubiquitous nucleotide derivatives existing in micromolar range in cell and may even rise to millimolar concentration under special physiological condition [16]. Previous studies have revealed that ApnA (n = 3–6) molecules can serve as signaling molecules acting intracellularly and extracellularly and should be finely controlled [17]. Especially the Ap4A molecule, which is the best known, has been found involving in a number of physiological process such as the modulation of heat shock protein activity, control of DNA replication and regulation of ATP-sensitive K+ channel [18–21]. Recently, enzymes responsible for the synthesis (such as phenylalanyl-and seryl-tRNA synthetase) and hydrolysis (such as Nudix hydrolase family: AtNUDX13, 26, 27 of Arabidopsis) of ApnA molecules have been identified in plants [11,22–26]. (See Table S1 for a list of the protein names, predicted cellular localizations and catalyzed reactions of these enzymes.) Therefore, although the functions of these ApnA molecules are still unclear in plant, it can be suggested that ApnA molecules may be important to several biological processes in plant. Concerning the high binding affinities and activities of ApnA molecules for HlAIPT in vitro, it is possible that the ApnA molecules are converted to iso- and di-iso-ApnA molecules by HlAIPT in vitro and then hydrolyzed, as a third biosynthetic pathway of CKs or as a new class of signaling molecules.
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The active site of other enzymes, such as the Agrobacterial IPT [27] can also accommodate ApnA (Supplementary Fig. S3), but the substrate affinity may need further investigation to know whether they can potentially utilize ApnA. Some important questions need to be elucidated through enzymatic kinetics, exploring endogenous isopentenyl ApnA molecules and clarifying their roles in the cell. Hopefully, this study could be a pioneering report to encourage further research of the modified ApnA molecules in plant. Acknowledgements We thank Dr. S.-C. Jao (Biophysics Core Facility, Scientific Instrument Center, Academia Sinica) for technical assistance with the ITC experiment. This work was supported by Academia Sinica and Core Facility for Protein Production and X-ray Structural Analysis (Grant NSC97-3112-B-001-035-B4 to A.H.-J.W.).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2010.08.038.
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[19] Jovanovic, S. and Jovanovic, A. (2001) Diadenosine tetraphosphate-gating of cardiac K (ATP) channels requires intact actin cytoskeleton. Naunyn Schmiedebergs Arch. Pharmacol. 364, 276–280. [20] Miras-Portugal, M.T., Pintor, J. and Gualix, J. (2003) Ca2+ signaling in Brain synaptosomes activated by dinucleotides. J. Mem. Biol. 194, 1–10. [21] Tepel, M., Lowe, S., Nofer, J.R., Assmann, G., Schulter, H. and Zidek, W. (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. [22] Guranowski, A. (2000) Specific and nonspecific enzymes involved in the catabolism of mononucleoside and dinucleoside polyphosphates. Pharmaco. Therap. 87, 117–139. [23] Jakubowski, H. (1983) Synthesis of diadenosine 5’, 5’’-P1, P4-tetraphosphate and related compounds by plant (Lupinus luteus) seryl-tRNA and phenylalanyl-tRNA synthetases. Acta Biochim. Pol. 30, 51–69.
[24] Pietrowska-Borek, M., Stuible, H.P., Kombrink, E. and Guranowski, A. (2003) 4Coumarate: coenzyme A ligase has the catalytic capacity to synthesize and reuse various (Di) adenosine polyphosphates. Plant Physiol. 131, 1401–1410. [25] Olejnik, K., Murcha, M.K., Whelan, J. and 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. [26] Guranowski, A., Wojdyla, A.M., Pietrowska-Borek, M., Bieganowski, P., Khurs, E.N., Cliff, M.J., Blackburn, G.M., Blaziak, D. and Stec, W.J. (2008) Fhit proteins can also recognize substrates other than dinucleoside polyphosphates. FEBS Lett. 582, 3152–3158. [27] Sugawara, H., Ueda, N., Kojima, M., Makita, N., Yamaya, T. and Sakakibara, H. (2008) Structural insight into the reaction mechanism and evolution of cytokinin biosynthesis. Proc. Natl Acad. Sci. USA 105, 2734–2739.
journal homepage: www.FEBSLetters.org
Binding and catalysis of Humulus lupulus adenylate isopentenyltransferase for the synthesis of isopentenylated diadenosine polyphosphates Hsing-Mao Chu a,1, Feng-Yuan Chen a,b,1, Tzu-Ping Ko a, Andrew H.-J. Wang a,b,⇑ a b
Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan
a r t i c l e
i n f o
Article history: Received 9 August 2010 Revised 26 August 2010 Accepted 26 August 2010 Available online 31 August 2010 Edited by Ulf-Ingo Flügge Keywords: Prenyltransferase Cytokinin Calorimetry Diadenosine polyphosphates
a b s t r a c t Various plant developmental processes involve phytohormones such as cytokinins. Isopentenyltransferase (IPT) reaction is the key rate-limiting step in cytokinin biosynthesis that transfers the isopentenyl (iP) group from dimethylallyl diphosphate to the N6-amino group of adenine. Here, a series of diadenosine polyphosphates (ApnA) were screened as possible substrates of IPT, among which diadenosine tetraphosphate, diadenosine pentaphosphate and diadenosine hexaphosphate showed higher affinity than did the authentic substrates ADP and ATP. In addition, formation of mono-isopentenyl ApnA and di-isopentenyl ApnA was observed. Judging by the existing biosynthetic and hydrolytic systems for ApnA in plants, ApnA and isopentenyl-ApnA may occur in the plant cells, with functional importance. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Cytokinins (CKs) are important adenine-base phytohormones which promote cell division and their biosynthesis has two pathways depending on different substrates. The first pathway begins with the transfer of isopentenyl (iP) group from dimethylallyl diphosphate (DMAPP) to the N6-amino group of AMP, ADP or ATP by adenylate isopentenyltransferase (AIPT) [1]. In the second pathway, tRNA-IPT catalyzes the isopentenyl transfer reaction at certain adenine base, and CK is produced by subsequent degradation of the prenylated tRNA [2]. In either pathway, the IPT-catalyzed reaction is the key rate-limiting step in cytokinin biosynthesis. Several plant AIPTs from Arabidopsis thaliana, rice, mulberry (Morus alba), and hop (Humulus lupulus L) have been cloned and characterized [3–7]. In higher plants, because the plant
Abbreviations: CKs, cytokinins; DMAPP, dimethylallyl diphosphate; AIPT, adenylate isopentenyltransferase; iP, isopentenyl; ITC, isothermal titration calorimetry; Ap3A, diadenosine triphosphate; Ap4A, diadenosine tetraphosphate; Ap5A, diadenosine pentaphosphate; Ap6A, diadenosine hexaphosphate; ApnA, diadenosine polyphosphates; iso-ApnA, mono-isopentenyl ApnA; di-iso-ApnA, di-isopentenyl ApnA ⇑ Corresponding author at: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan. Fax: +886 2 2788 2043. E-mail address: [email protected] (A.H.-J. Wang). 1 These authors have equal contribution.
AIPT predominantly uses DMAPP and ATP, or ADP, the major initial product is an isopentenyl nucleotide, such as isopentenyl riboside 50 -triphosphate (iPRTP) or isopentenyl riboside 50 -diphosphate (iPRDP) [8]. Although it is well known that the plant AIPT predominantly uses ATP or ADP as the isopentenyl acceptor, it cannot be excluded, however, that other adenosine-related molecules (such as dinucleotide polyphosphates) can serve as acceptors. In general, the adenosine-related dinucleotide molecules are divided into two main groups. The first group has the adenosine linked via a pyrophosphate to a ribosylated nicotinamide (NAD) or ribitolylated flavin adenine (FAD). The second group, diadenosine polyphosphates (ApnA, n = 2–6), are formed by two adenosines joined by a variable number of phosphate groups. ApnA are naturally and ubiquitously occurring molecules and have been identified and found to be stored in a diverse number of biological tissues including platelets, adrenal medullary chromaffin granules and the central nervous system [9–11]. Although the existence of ApnA in plants has yet to be unequivocally demonstrated, their corresponding hydrolytic enzyme systems have been shown to exist according to amino acid sequence homology analysis [12]. Recently, we solved the first plant AIPT from Humulus lupulus (HlAIPT) [13]. In order to further characterize HlAIPT, its binding affinity and catalytic activity for different adenosine-related molecules were measured, and results suggest a series of possible substrates for this enzyme, including ApnA.
0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.08.038
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Table 1 Thermodynamic parameters of interactions between HlAIPT and adenosine derivatives. Ligand
Ka (M1)
DH (cal/mol)
DS (cal mol1 K1)
% Ka
ATP Ap3A Ap4A Ap5A Ap6A NAD NADP FAD
(7.64 ± 0.27) 105 (4.44 ± 0.44) 104 (6.07 ± 0.33) 105 (1.47 ± 0.07) 106 (6.00 ± 0.29) 105 N.D. N.D. N.D.
(4.01 ± 0.03) 104 (2.56 ± 0.30) 104 (1.43 ± 0.01) 105 (1.36 ± 0.19) 104 (5.09 ± 0.05) 104
107 64.6 370 426 144
100 5 79 190 78
Protein and substrate were dissolved in HEPES buffer (20 mM, pH 7.0; 150 mM NaCl, 3 mM DTT). ITC experiments were performed at 25 °C. Ka: association constant; DH: enthalpy changes; DS: entropy changes. Plus/minus (±) signed values represent standard error between the observed data and fitted parameters. N.D. means not detectable.
2. Materials and methods 2.1. Preparation of truncated HlAIPT and mutants All chemicals were purchased from Sigma–Aldrich except mentioned otherwise. The pQE80L plasmid containing the truncated
HlAIPT (aa 27–329), which lacks the N-terminal signal peptide, was used. Affinity and activity measurement were based on this truncated and more stable HlAIPT. We overexpressed the protein containing the N-terminal hexahistidine tag in Escherichia coli BL21 (DE3) (Novagen) following the previous expression and purification procedures [13]. 2.2. Isothermal titration calorimetry All adenosine-related molecules were dissolved in the dialysis buffer. Experiments were conducted at 25 °C. For diadenosine tetraphosphate (Ap4A), diadenosine pentaphosphate (Ap5A), nineteen 2 ll injections of 0.2 mM ligand into a 0.2 ml sample cell containing 16 lM of the truncated HlAIPT (aa 27–329) were made. For diadenosine triphosphate (Ap3A), NAD, NADP and FAD, nineteen 2 ll injections of 0.1 mM ligand were made. In the case of diadenosine hexaphosphate (Ap6A), six 1.5 ll injections of 0.1 mM ligand and subsequent fifteen 2 ll injections were conducted. Injections were made over a period of 4 s with 150 s intervals between injections. The sample cell was stirred at 1000 rpm. Data were acquired on an iTC200 microcalorimeter (Microcal, Northhampton, MA) and analyzed with the ORIGIN v.7.0 software provided with the microcalorimeter.
Fig. 1. The activity of ApnA molecules for HlAIPT. (A) Model of complex structure of (HlAIPT/Ap5A). (B) Surface charge distribution of complex structure (HlAIPT/Ap5A) showing a positively-charged binding cavity. (C) Substrate specificity of HlAIPT.
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2.3. HlAIPT activity assay The standard reaction mixture contained 150 lM prenyl acceptor substrate (ATP, Ap3A, Ap4A, Ap5A and Ap6A), 100 lM prenyl donor substrate (DMAPP), and 4 lg of the purified recombinant truncated HlAIPT in a final volume of 100 ll of 20 mM Tris–HCl buffer, pH 5.0, containing 1 mM MgCl2. Incubations were carried out at 25 °C for a series of period, and stopped by adding 10 ll 20% acetic acid. The product was analyzed by reverse-phase HPLC on a hydrosphere C18 column (4.6 150 mm I.D., YMC) with a flow rate of 0.5 ml/min. The isocratic elution was performed with solvent A, 50% CH3CN in 20 mM tetrabutylammonium bromide, 20 mM KH2PO4: 0–30 min. Elutions were monitored by a multichannel UV detector (Waters, Dual Absorbance detector) at 260 nm. 2.4. Mass spectroscopy Enzyme reaction products were characterized by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry. MALDI-TOF data: Ap3A, m/z 757 [M+H]+; isopentenyl Ap3A, m/z 825 [M+H]+; di-isopentenyl Ap3A (di-iso-Ap3A), m/ z 893 [M+H]+; Ap4A, m/z 837 [M+H]+; isopentenyl Ap4A, m/z 905 [M+H]+; di-iso-Ap4A, m/z 973 [M+H]+; Ap5A, m/z 917 [M+H]+; isopentenyl Ap5A, m/z 985 [M+H]+; di-iso-Ap5A, m/z 1053 [M+H]+; Ap6A, m/z 997 [M+H]+; isopentenyl Ap6A, m/z 1065 [M+H]+; diiso-Ap6A, m/z 1133 [M+H]+.These samples were mixed with the same volume of matrix which containing CHCA (a-cyano-4-hydro-
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xy-trans-cinnamic-acid), and were solvated in 50% acetonitrile and 0.1% TFA (trifluoroacetic acid) as detectable materials, and spotted on a MALDI plate. 3. Results 3.1. The affinities of adenosine-related nucleotides with HlAIPT In order to assess the binding affinity between HlAIPT and adenosine-related dinucleotides, we use isothermal titration calorimetry (ITC) to screen a series of adenosine-related dinucleotides including ApnA (P1,P3-di(adenosine-50 )-polyphosphate) from triphosphates to hexaphosphates {Ap3A, Ap4A, Ap5A, and Ap6A}, NADP, NAD and FAD. As shown in Table 1, HlAIPT discriminates the dinucleotides by different Ka terms. A few typical ITC tracings, showing the raw and integrated data, can be found in Supplementary Fig. S1. Interestingly, the results of ITC experiments revealed a length-dependent binding affinity order of Ap5A >ATP Ap6A Ap4A >Ap3A to HlAIPT. For NADP, NAD and FAD, there was no detectable binding to HlAIPT (Table 1). 3.2. The dinucleotides binding to HlAIPT in a broad positively-charged pocket These ApnA show ability to bind HlAIPT and Ap5A displays the highest binding affinity (1.47 106 (M1)) for HlAIPT among them. In order to explain the effect on binding affinity due to different
Fig. 2. Formation of di-isopentenyl Ap4A. (A) Synthesis from Ap4A and DMAPP to its products, mono-isopentenyl Ap4A (iso-Ap4A) and di-isopentenyl Ap4A (di-iso-Ap4A) were monitored by reverse-phase HPLC. (B) Analysis of the concentration of Ap4A, iso-Ap4A, and di-iso-Ap4A versus time. (C) The formations of the iso- and di-iso-ApnAs by enzymatic catalysis.
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length of polyphosphates backbone, a HlAIPT/Ap5A complex model was constructed by computer modeling, based on the complex structure of HlAIPT with ATP. The model showed that the Ap5A molecule adopted an extended conformation and fully occupied the positively charged binding cavity (Fig. 1A and B). Thus, when ApnA molecules bind to HlAIPT, the binding affinity may be enhanced with increasing number of phosphate groups from triphosphates to pentaphosphates. However, the Ap6A molecule show lower binding affinity than Ap5A because its sixth phosphate groups may make the molecule too long to allow docking of the second adenosine moiety onto the protein surface. Taken together, the experimental results are consistent with the observation in the computer model that Ap5A is optimally sized for high binding affinity to HlAIPT among different ApnA. In the case of NADP, NAD and FAD, no detectable binding to HlAIPT was observed, indicating that the linker of dinucleotides bound to the cavity of HlAIPT requires a minimum length of triphosphates in order to accommodate the phosphate backbone and the two dinucleotides at the same time (Fig. 1B).
3.3. Enzyme-catalyzed formation of mono-isopentenyl ApnA Because ApnA consist of two adenosines and different numbers polyphosphates, it was worth investigating whether HlAIPT shows activities towards these compounds or not. Interestingly, analysis of formation of mono-isopentenyl ApnA (iso-ApnA, n = 3–6) molecules by HPLC showed that the HlAIPT activity for Ap3A, Ap4A, Ap5A and Ap6A are 51%, 87%, 76%, and 90%, respectively, relative to that for ATP (100%) (Fig. 1C). The activity for Ap3A was the minimal among all ApnA molecules, whereas significant activities for Ap4A, Ap5A and Ap6A were revealed in vitro that reached 70–90% that for ATP. The activities of HlAIPT for ApnA molecules are generally consistent with the order of binding affinity. 3.4. Formation of di-iso-ApnA Intriguingly, we also observed a peak that indicated the formation of di-iso-Ap4A in the HPLC elution profile and reached 50% turnover within 20 min. Complete formation of di-isopentenyl
Fig. 3. The proposed catalytic reaction of ApnA molecule (Ap6A as an example) to proceed through the several steps.
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diadenosine was achieved within 40 min. Besides, we observed that the concentration of iso-Ap4A reached a maximum concentration within 15 min and then gradually decreased to an undetectable amount (Fig. 2A). Hence, we compared the turnover rate of converting Ap4A to iso-Ap4A with that of converting iso- to di-iso-Ap4A, and found a similar formation rate by analyzing the reaction profile of the two products. This may suggest that the isopentenyl side-chain of iso-Ap4A does not interfere with its binding to HlAIPT. Consequently the efficiency of isopentenylation reaction is similar for Ap4A and iso-Ap4A. Furthermore, we also tested other ApnA molecules and found that all of the corresponding di-iso-ApnA were formed when these reaction products (iso- and di-iso-Ap3A, Ap4A, Ap5A and Ap6A) where further subjected to Mass Spectrum analysis. These results were shown in Supplementary Fig. S2. This is the first observation of the formations of iso- and di-iso-ApnA by enzymatic catalysis (Fig. 2B and C).
4. Discussion Based on the binding and activity assays, it was found that these ApnA (n = 4–6) not only showed higher binding affinity to HlAIPT than that of ADP (4.49 105 (M1)), but they were also catalyzed to produce mono- and di-isopentenyl products (Fig. 2A and B). These results indicate that HlAIPT may act on a series of previously unidentified substrates in plant. Herein, based on these results, we proposed a mechanism reaction of Ap6A molecule to proceed through the following steps (Fig. 3). First, the initial binding of Ap6A molecule to active-site pocket is facilitated by the nonspecific interactions between positively-charged surface of the pocket and the negatively-charged phosphate groups of Ap6A molecule. After entry of DMAPP to active site, isopentenyl group was transferred from DMAPP to N6 of the adenine ring of Ap6A molecule, and then iso-Ap6A molecule was released from the cavity. In the further step of reaction, iso-Ap6A molecule reorients itself and the unmodified adenine ring again binds to the active site of HlAIPT while the isopentenylated adenine ring of Ap6A molecule is exposed to outside of the positively-charged the pocket. Finally, when the second isopentenylation reaction of Ap6A molecule is finished, the diiso-Ap6A molecule is released from the active site of HlAIPT. ApnA (n = 3–6) have been detected in rabbit and human tears by HPLC [14,15]. These molecules are ubiquitous nucleotide derivatives existing in micromolar range in cell and may even rise to millimolar concentration under special physiological condition [16]. Previous studies have revealed that ApnA (n = 3–6) molecules can serve as signaling molecules acting intracellularly and extracellularly and should be finely controlled [17]. Especially the Ap4A molecule, which is the best known, has been found involving in a number of physiological process such as the modulation of heat shock protein activity, control of DNA replication and regulation of ATP-sensitive K+ channel [18–21]. Recently, enzymes responsible for the synthesis (such as phenylalanyl-and seryl-tRNA synthetase) and hydrolysis (such as Nudix hydrolase family: AtNUDX13, 26, 27 of Arabidopsis) of ApnA molecules have been identified in plants [11,22–26]. (See Table S1 for a list of the protein names, predicted cellular localizations and catalyzed reactions of these enzymes.) Therefore, although the functions of these ApnA molecules are still unclear in plant, it can be suggested that ApnA molecules may be important to several biological processes in plant. Concerning the high binding affinities and activities of ApnA molecules for HlAIPT in vitro, it is possible that the ApnA molecules are converted to iso- and di-iso-ApnA molecules by HlAIPT in vitro and then hydrolyzed, as a third biosynthetic pathway of CKs or as a new class of signaling molecules.
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The active site of other enzymes, such as the Agrobacterial IPT [27] can also accommodate ApnA (Supplementary Fig. S3), but the substrate affinity may need further investigation to know whether they can potentially utilize ApnA. Some important questions need to be elucidated through enzymatic kinetics, exploring endogenous isopentenyl ApnA molecules and clarifying their roles in the cell. Hopefully, this study could be a pioneering report to encourage further research of the modified ApnA molecules in plant. Acknowledgements We thank Dr. S.-C. Jao (Biophysics Core Facility, Scientific Instrument Center, Academia Sinica) for technical assistance with the ITC experiment. This work was supported by Academia Sinica and Core Facility for Protein Production and X-ray Structural Analysis (Grant NSC97-3112-B-001-035-B4 to A.H.-J.W.).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2010.08.038.
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