Mol Biol Rep (2014) 41:7575–7583 DOI 10.1007/s11033-014-3649-9
Expression, purification, and characterization of recombinant mangrove glutamine synthetase Wei Zhao • Jun Yang • Yongsheng Tian • Xiaoyan Fu • Bo Zhu • Yong Xue • Jianjie Gao Hong-Juan Han • Rihe Peng • Quan-Hong Yao
•
Received: 4 March 2013 / Accepted: 27 July 2014 / Published online: 3 August 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract To expand our knowledge about the relationship of nitrogen use efficiency and glutamine synthetase (GS) activity in the mangrove plant, a cytosolic GS gene from Avicennia marina has been heterologously expressed in and purified from Escherichia coli. Synthesis of the mangrove GS enzyme in E. coli was demonstrated by functional genetic complementation of a GS deficient mutant. The subunit molecular mass of GSI was *40 kDa. Optimal conditions for biosynthetic activity were found to be 35 °C at pH 7.5. The Mg2?-dependent biosynthetic activity was strongly inhibited by Ni2?, Zn2?, and Al3?, whereas was enhanced by Co2?. The apparent Km values of AmGLN1 for the substrates in the biosynthetic assay were 3.15 mM for glutamate, and 2.54 mM for ATP, 2.80 mM for NH4? respectively. The low affinity kinetics of AmGLN1 apparently participates in glutamine synthesis under the ammonium excess conditions. Keywords Glutamine synthetase (GS) Ammonium assimilation Mangrove (Avicennia marina) PPTsensitive
W. Zhao Y. Tian X. Fu B. Zhu Y. Xue J. Gao H.-J. Han R. Peng (&) Q.-H. Yao (&) Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Road, Shanghai, People’s Republic of China e-mail: [email protected] Q.-H. Yao e-mail: [email protected] J. Yang College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu, People’s Republic of China
Introduction The glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle has been considered as the major pathway for ammonium assimilation and regulation of nitrogen metabolism in higher plants. GS (EC 6.3.1.2.) catalyzes the formation of glutamine from glutamate in the presence of NH4? ATP and metal cations, according to the following reaction: L-glutamate ? NH4? ? ATP ? L-glutamine ? ADP ? Pi ? H?. Subsequently, GOGAT (EC 1.4.7.1, EC I.1.1.14) catalyzes the formation of two molecules of L-glutamate from L-glutamine and 2-oxoglutarate. Since all of the N in a plant, whether derived from fertilizer, nitrate reduction, photorespiration, and numerous other sources including catabolic release of NH4? during senescence is channeled through the reactions catalyzed by GS, this reaction has been identified as the ratelimiting step in the control of N-assimilation and N-recycling during growth and development of plants [1, 2]. In higher plants, GS genes can be divided into two distinct families based on their subcellular localization. Typically, GS1 is localized in the cytoplasm and encoded by a small multigene family, whereas, GS2 is founded in the chloroplast and encoded by a single nuclear gene in most species [3, 4]. The existence of a second GS2 gene in Medicago truncatula has been reported recently as an exceptional case [5]. Previous phylogenetic analyses of chloroplast and cytosolic isoenzymes support the hypothesis that the isoenzymes in angiosperms evolved via a gene duplication event that preceded the divergence of monocots and dicots [6]. GS1 is the major form of GS in plant roots, and it plays an important role in the assimilation of external ammonium, the ammonium derived from N2 fixation and other sources of nitrogen, and in the remobilization of nitrogen during senescence [7, 8]. The early study indicated the decamer structure for maize cytosolic GS is
123
7576
composed of two face-to-face pentameric rings of identical subunits, with a total of 10 active sites, each formed between every two neighboring subunits within each ring [9]. GS from plants have been well investigated in terms of the regulation of gene expression and enzymatic characterization. However, most studies have focused on annual plants such as maize [10], rice [11], potato [8] and Arabidopsis [4, 12], thus available information concerning the biochemical properties of GS from woody plant is much more limited [13]. Mangroves are crucial for the coastal areas and nitrogen-use efficiency (NUE) in mangroves is among the highest recorded for angiosperms [14]. Here we report that expression, purification, and characterization of a cytosolic GS isoenzyme (AmGLN1, Q9AXD8) in Escherichia coli. Our research will contribute to expand scientific knowledge about how mangroves can sustain high levels of NUE.
Materials and methods Growth conditions, strains, and plasmids The E. coli Gln-auxotrophic strain JW3841-1 (glnA732::Kan) used for the production of recombinant proteins was obtained from the E. coli Genetic Resource Center (CGSC#10775, Yale University, USA) [15]. The pRARE plasmid (Novagen, USA), which over-expresses tRNAs for Arg, Leu, and Pro codons rarely used by E. coli, was introduced into JW3841-1 to enhance the expression of AmGLN1. pTrcHis-B expression vector was purchased from Invitrogen, USA. Bacteria were routinely grown in Lineweaver–Burk (LB) containing antibiotics (100 lg/ml ampicillin, 20 lg/ml chloramphenicol, 40 lg/ml kanamycin) when necessary. For complementation analysis, strains were grown in M9 minimal medium containing 1 % (wt/ vol) glycerol, thiamine (1 lg/ml), casamino acids [0.66 % (wt/vol)], the appropriate antibiotics, with or without a glutamine supplement (1 mM) at 30 °C. Sequence analysis and synthesis of the AmGLN1 For homology analysis, BLAST searches were conducted using BLASTp programmes (http://www.ncbi.nlm.nih.gov/ BLAST/). The theoretical isoelectric point (pI) and molecular weight (Mw) were computed with the ExPASy ProtParam tool (http://www.expasy.org). Alignment was performed using clustal Omega multiple sequence alignment program [16]. Phylogenetic tree was constructed by MEGA 4.0 software using neighbor joining method with 1,000 times bootstraps. All of the protein sequences were retrieved from the UniProt Database.
123
Mol Biol Rep (2014) 41:7575–7583
DNA coding for full-length AmGLN1 (GenBank accession no. AF338444.1) was synthesized through a simple, high-fidelity, and cost-effective PTDS (PCR-based two-step DNA synthesis) method described previously [17]. Errors in the synthetic gene were identified by DNA sequencing and corrected by overlap extension PCR method. Routine DNA manipulations were performed by standard recombinant method [18]. Purification of recombinant AmGLN1 The synthetic gene was ligated into the pTrcHis-B Expression Vector using the BamHI and SacI linker sites. The resulting construct pTrcHis-B/AmGLN1 was transformed into the E. coli glnA mutant JW3841-1. Several transformants were cultured in 100 ml LB medium at 37 °C until an OD600 of 0.3 was reached, at which time isopropyl-D-thiogalactopyranoside was added at 1 mM to induce gene expression and protein production. After incubated for 12 h at 30 °C on a rotary shaker, the cells collected by centrifugation was re-suspended in 4 ml s of lysis buffer [50 mM Tris–HCl, pH 7.5, 300 mM NaCl, 0.5 mM dithiothreitol (DTT), and 10 mM imidazole]. The His-tagged protein in the soluble fraction was affinitypurified using a nickel-nitrilotriacetic acid agarose column (1.5 ml bed volume, Sigma). Washing was carried out with a buffer containing 50 mM Tris–HCl, pH 7.5, 300 mM NaCl, and 20 mM imidazole. Protein was eluted with the same buffer containing 250 mM imidazole. The GS fractions were then dialyzed against buffer (50 mM Tris–HCl, pH 7.5, 0.1 mM EDTA, and 10 % glycerol). The purified protein was analyzed by 12 % SDS-PAGE and protein concentration was quantified using Bradford assay kit and bovine serum albumin as standard. GS enzyme assay The biosynthetic activities were determined by the methods described by Wellner and Meister [19]. The biosynthetic assay solution contained the following components: 100 mM Tris–HCl (pH 7.5), 5 mM ATP, 50 mM L-glutamate, 30 mM NH4Cl, 20 mM MgCl2, in a total volume of 190 ll. The mixture was equilibrated at 35 °C for 5 min, and the reaction was initiated by adding 10 ll of the enzyme solution. The reaction was terminated after 30 min by the addition of 200 ll of ferric chloride reagent (55 g/l FeCl36H2O, 20 g/l trichloroacetic acid, and 2.1 % concentrated HCl) and the release of c-glutamyl hydroxamate was quantified by measuring the absorbance at 540 nM. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the formation of 1 lmol of c-glutamyl hydroxamate/min.
Mol Biol Rep (2014) 41:7575–7583
pH and temperature optima The pH stability was examined by measuring the activity in Tris–HCl buffer (pH 7.5) after incubating the purified enzyme in the following buffers ranging from pH 2 to 10.0 for 24 h at 4 °C: citric acid–sodium citrate (0.2 M, pH 2.0–6.5), Tris–HCl buffer (0.2 M, pH 7.0–8.0), glycine– NaOH (0.2 M, pH 9.0–10.0). The optimum pH for its maximal activity was obtained by determining the activity with different pH buffers (pH 6.0–10.0). To determine the optimal temperature, enzyme activity was assayed at different temperatures between 20 and 52 °C. Thermostability were determined by measuring the residual activity at 35 °C after incubating the enzyme in Tris–HCl buffer (pH 7.5) at 35, 40 or 45 °C for varying periods of time. Effect of different metal ions (Mn2?, Mg2?, Ca2?, Zn2?, and Ni2?) on AmGLN1 activity was studied by preincubating the enzyme with 10 mM concentration of different metal chlorides at 4 °C for 1 h. Kinetic characterization The kinetic parameters for the different substrates and cofactors were obtained by varying the concentrations of individual reaction components (L-glutamate 2–40 mM, NH4Cl 1–16 mM, ATP 1–8 mM). Unvariable substrates were fixed at the same levels as in standard assay. For Ki value evaluation, the enzyme was assayed in the presence of increasing concentrations of D,L-phosphinothricin (D,LPPT 0.25–5 lM) at varying levels of glutamate (2–40 mM). The enzyme activity was determined as mentioned above and the data were analyzed and plotted using Sigma Plot (version 8.0). Each enzyme-activity assay was replicated independently at least three times.
Results and discussion Sequence alignment and phylogenetic tree To understand the relationship between AmGLN1 and GS1 enzymes from other organisms, we compared the amino acid sequence of AmGLN1 with those from five divergent organisms (Fig. 1). A consensus pattern, [FYWL]-D-G-S– S-X6,8-[DENQSTAK]-[SA]-[DE]-X2-[LIVMFY], which is usually present in the N-terminal section of GS (PROSITE accession no. PS00180) was detected in the N-terminal region of AmGLN1, between residues 55 and 72. Another conserved sequence for ATP-binding region (PROSITE accession no. PS00181), {K–P-[LIVMFYA]-x(3,5)[NPAT]-[GA]-[GSTAN]-[GA]-x–H-x(3)-S}, was also present [20]. The AmGLN1 shared 86–91 % amino acid
7577
sequence identity with other cytosolic GS from Hevea brasiliensis, Cucumis melo, Vitis vinifera, Poplar, and Zea mays. As other cytosolic GS forms, the AmGLN1 lack the characteristic N-terminal and C-terminal amino acid extensions of the chloroplastic polypeptides (data not shown). Ishiyama et al. [21] reported that the existence of Gln49 or Ser174 is sufficient to fulfill the substrate specificities of the high-affinity variants of plant GS1. The amino acid residues at positions 49 and 174 in AmGLN1 were Lys and Ser. This information suggests that AmGLN1 might have low affinity for ammonium. A neighbor-joining tree of GS proteins from mangrove, maize, rice, Arabidopsis and other species was created with the MEGA program. Phylogenetic reconstruction at the molecular level shows the separation of cytosolic (GS1) and chloroplastic (GS2) sequences in angiosperms as two well differentiated clusters (Fig. 2). Figure 2 also showed that the AmGLN1 was closely aligned with the GS1 isoforms of angiosperms but was outside the main subfamilies. Complementation analysis To determine whether the recombinant AmGLN1 was functional in E. coli, the Gln-auxotrophic strain JW3841-1 was transformed with pTrcHis-B/AmGLN1 and as the control the empty pTrcHis-B vector. The transformants were tested for growth on M9 minimal medium containing or lacking glutamine at 30 °C. The results showed that the transformants harboring the pTrcHis-B/AmGLN1 was able to grow on minimal media containing ammonium as the nitrogen source whereas transformants harboring the vector failed to grow on M9 minimal medium except when it was supplemented with glutamine (Fig. 3a). Our results confirmed that AmGLN1 is folded and assembled into an essentially native conformation in E. coli and make it possible to select directly in E. coli for variants, which could be useful in the development of plants with improved nitrogen utilization or herbicide-resistance [22]. The effective complementation by pTrcHis-B/AmGLN1 was observed at temperatures below 37 °C (Fig. 3b). Thus, we considered that lower temperatures favor the native functional conformation of AmGLN1. Similar results have been reported by Bennett and Cullimore [23]. Protein expression and purification The overexpression of functionally active GS in E. coli prevented the inconveniences that interfere with protein extraction procedures from woody plants. It provided a good source of the recombinant GS in unlimited amounts for purification procedures and biochemical studies. As the GS is sensitive to environmental oxidation during
123
7578
Mol Biol Rep (2014) 41:7575–7583
Fig. 1 Sequence alignment of the deduced-amino acid sequences of AmGLN1 with members of the GS1 family from different plants (Hevea brasiliensis, Cucumis melo, Vitis vinifera, Populus canescens, and Zea mays). Single letter amino acid code is used. Asterisks,
colons, and dots indicate identical amino acid residues, conserved substitutions, and semi-conserved substitutions in all sequences used in the alignment, respectively
refolding, the purification was performed in the presence of 0.5 mM of DTT. The recombinant AmGLN1 was produced as soluble protein when expressed in JW3841-1 at 30 °C.
The purified recombinant AmGLN1 showed only single band of *40 kDa (fused with six 9 histidine tag) on the SDS–poly-acrylamide gel, identical to that theoretical
123
Mol Biol Rep (2014) 41:7575–7583
7579
Fig. 2 Phylogenetic comparison of glutamine synthetase protein. Entry names of the sequence in the UniProt Database are given. The sequences are from Alnus glutinosa (AgGLN1), A. thaliana (AtGLN1;1, AtGLN1;2, AtGLN1;3, AtGLN1;4, AtGLN1;5, AtGLN2), A. marina (AmGLN1, AmGLN2), Brassica napus (BnGLN1), Camellia sinensis (CsGLN1), Canavalia lineate (ClGLN1), Datisca glomerata (DgGLN1), Glycine max (GmGLN1), Gossypium raimondii (GrGLN1), H. brasiliensis (HbGLN1), Lotus japonicus (LjGLN1), Medicago truncatula (MtGLN1), Nicotiana attenuata (NaGLN1), Oryza sativa (OsGLN1;1, OsGLN1;2, OsGLN1;3, OsGLN2), Phaseolus vulgaris (PvGLN1), Picea sitchensis (PsGLN1;1, PsGLN1;2), Pinus sylvestris (PsGLN1a, PsGLN1b), Populus trichocarpa (PtGLN1, PtGLN2), Selaginella moellendorffii (SmGLN1), Vigna aconitifolia (VaGLN1), V. vinifera (VvGLN1;1, VvGLN1;2, VvGLN2), and Z. mays (ZmGLN1;1, ZmGLN1;2, ZmGLN1;3, ZmGLN1;4, ZmGLN1;5, ZmGLN2), respectively
123
7580
Mol Biol Rep (2014) 41:7575–7583
Fig. 5 pH optimum and pH stability of AmGLN1. The buffers used, at 100 mM concentration, were citric acid–sodium citrate (0.2 M, pH 2.0–6.5), Tris–HCl buffer (0.2 M, pH 7.0–8.0), glycine–NaOH (0.2 M, pH 9.0–10.0). The values shown represent the mean values from triplicate experiments
Fig. 3 Complementation of the E. coli Gln-auxotrophic strain JW3841-1 by AmGLN1. Transformed cells containing pTrcHis-B as control or including pTrcHis-B/AmGLN1 were plated on M9 minimal medium containing or lacking glutamine, and incubated at 30 °C
Fig. 6 Effect of temperature on activity (a) and thermal sensitivity (b) of AmGLN1. For thermal inactivation, samples of the purified enzyme were incubated in Tris–HCl buffer (pH 7.5) at 35, 40, 45 °C for the indicated times, followed by measurement of the residual GS activity. The values shown represent averages from triplicate experiments (n = 3) Fig. 4 SDS-PAGE analysis of recombinant AmGLN1. The purified AmGLN1 was subjected to SDS-PAGE on 0.1 % SDS-12 % PAGE and stained with Coomassie Brilliant Blue R-250. M protein molecular weight marker (low, Takara), lane 1, crude extract, lane 2 AmGLN1
value of 39.3 kDa (Fig. 4). The 39.3-kDa polypeptide corresponds to the cytosolic isoform form of GS but lower than the plastidic isoform (c. 44–45 kDa) [24].
123
Stability and pH optima In the presence of Mg2?, the enzyme exhibited the maximum activity around pH 7.5, whereas its activity declined steeply towards the acidic pH (Fig. 5). The GS from chlorella [25], rice [26], maize [3], and Camellia sinensis
Mol Biol Rep (2014) 41:7575–7583
7581
Table 1 Effect of metal ions on AmGLN1 activity Metal ion
Control
Ni2?
Mn2?
Zn2?
Co2?
Fe2?
Cu2?
Ca2?
Al3?
Relative activity
100.00
8.92
17.12
9.58
109.09
22.27
22.20
43.86
8.91
The biosynthetic activity assays were performed after incubation the purified enzymes with 10 mM concentration of different metal chlorides and equal volume of water as a control at 4 °C for 1 h
[27] also exhibited optimal activity around the similar pH range. The enzyme was fairly stable at pH 4.0–10.0, while the highest stability was observed at pH 7.0–9.0 (Fig. 5). The remarkable stability of the enzyme under alkaline pH might be an environmental adaptation of Avicennia marina that grew in a highly alkaline environment. The enzyme was optimally active within a narrow range of temperatures from 30 to 42 °C. At higher temperatures ([42 °C) the relative activity rapidly decreased (Fig. 6a). Thermostability study showed that the enzyme was relative stable under 37 °C and sensitive to temperatures above 45 °C (Fig. 6b). The enzymatic activity of AmGLN1 was lost after 30 min of incubation at 45 °C, indicating a lack of functional and structural stability at high temperature. Dissociation of the oligomeric proteins at high temperature, resulting in a loss of activity, is a common phenomenon among GS enzymes from prokaryotes and eukaryotes [28]. It has been reported that the different GS isoforms of pine and maize exhibited distinguishable thermal inactivation profiles [3, 13]. The thermal stability of AmGLN1 was more similar to GS1a from pine and GS1d from maize. Unno et al. [9] has postulated that Ile-161 of GS1a is critical for the stability of GS. Similarly, the thermosensitivity of AmGLN1 can be overcome by replacing of Val161 by Ile through site-directed mutagenesis. Effects of metal ions The effect of different metal ions on the Mg2?-dependent biosynthetic activity of AmGLN1 was shown in Table 1. Compared to the activity determined with Mg2?, the addition of 10 mM Ni2?, Zn2?, and Al3? caused 8.92, 9.58, and 8.91 % loss in activity, respectively. Interestingly, a little improvement of enzyme activity was observed by assaying in the presence of Co2?. Divalent cations (commonly, Mg2?, Mn2?, or Co2?) are absolutely necessary for the activity of all known GSs both in the case of glutamine synthesis and for the transferase reaction catalyzed by the enzyme. A shift of the pH optimum by simultaneous presence of Mg2? and other metal ions in the reaction mixture may resulted in a decrease in over-all activity [29]. The observed increase in enzyme activity with Co2? could be attributed to Co2? has a higher binding constant. This agrees with the result of Antonyuk et al. [30] that the Co2? was more effective than Mg2? in supporting
biosynthetic activity of the metal-ion free GS from Azospirillum brasilense. Kinetic constants for substrates and inhibitor The apparent Km values of AmGLN1 for the substrates in the biosynthetic assay were 3.15 mM for glutamate, 2.54 mM for ATP and 2.80 mM for NH4? respectively. The Km value for glutamate of AmGLN1 were similar to those reported for natural plant GS [3, 26]. However, AmGLN1 exhibited higher affinity for the glutamate than GS1a from pine (Km value 13 mM) [13] and CsGS (Km value 9 mM) [25]. Ishiyama reported that the four isoenzymes of GS1 expressed in Arabidopsis roots exhibited different Km values for ammonium. Similar to GLN1;2 and GLN1;3 (Km values; 2.45 mM for GLN1;2 and 1.21 mM for GLN1;3, respectively) from Arabidopsis, AmGLN1 showed a relative low affinity to ammonium [31]. Recently, it has been shown that up to 99 % of nitrate removal in mangrove sediments is routed through dissimilatory nitrate reduction to ammonium (DNRA) [32]. Ammonium produced by the DNRA process is the primary form of nitrogen in mangrove soils. The high-capacity glutamine synthetic activity of AmGLN1 facilitates the assimilation of ammonium taken up from soils. The low affinity kinetics of AmGLN1 apparently participates in Gln synthesis under the ammonium excess conditions. However, nutrient availability can vary greatly among and within mangrove forests. Many mangrove soils have extremely low N availability. Mangroves perhaps contain other GS1 isoforms with high affinity to ammonium to facilitate rapid conversion of ammonium under nitrogenlimited conditions. In Arabidopsis and rice, the existence of high affinity-type GS1 has been reported previously [11, 31]. The inhibition constants for PPT, determined from the LB plots and the enzyme kinetics module of Sigma Plot (version 8.0), is 0.26 lM. This value is lower than those reported for E. coli (0.6 lM) [33], maize (1.1 lM) [34], barely (3.5 lM) [35], and Aspergillus niger (54 lM) [36]. In this sense, the A. marina GS appears to be relatively more sensitive to PPT inhibition. A current focus in plant improvement is the increase in plant growth, biomass accumulation, and stresses tolerance as a result of overexpression of cytosolic GS. For instance,
123
7582
transgenic poplar overexpressing GS1 has been previously shown to exhibits desirable field performance characteristics, including enhanced NUE [37], enhanced resistance to water stress [38] and enhanced resistance to PPT [39]. AmGLN1 is considered to be a good candidate gene for engineering plants to improve growth phenotype and increase the efficiency of N utilization.
Conclusion Avicennia marina cytosolic GS was synthesized and functional in E. coli based on the observations that AmGLN1 was able to complement the E. coli glnA mutant. Studies on biochemical properties of the purified enzyme revealed thermal instability of AmGLN1. Moreover, kinetic analysis showed that AmGLN1 was highly sensitive to PPT inhibition and had a relative low affinity for ammonium. We propose that AmGLN1 apparently play a role in Gln synthesis under the ammonium excess conditions. The present study, which is the first to biochemically characterize cytosolic GS from mangroves, provides the necessary basis for further investigations into roles of GS1 in nitrogen metabolism during growth and development of mangroves. Further work is required to establish expression profiles and regulation mechanisms of AmGLN1 in vivo and whether other GS1 isoforms with high affinity to ammonium exist in mangroves. Acknowledgments The research was supported by the National Natural Science Foundation of China (31071486), The Key Project Fund of the Shanghai Municipal Committee of Agriculture (Nos. 2009-6-4 and 2011-1-8), and International Scientific and Technological Cooperation (2010DFA62320).
References 1. Martin A, Lee J, Kichey T, Gerentes D, Zivy M, Tatout C, Dubois F, Balliau T, Valot B, Davanture M (2006) Two cytosolic glutamine synthetase isoforms of maize are specifically involved in the control of grain production. Plant Cell 18:3252–3274 2. Miflin BJ, Habash DZ (2002) The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops. J Exp Bot 53:979–987 3. Sakakibara H, Shimizu H, Hase T, Yamazaki Y, Takao T, Shimonishi Y, Sugiyama T (1996) Molecular identification and characterization of cytosolic isoforms of glutamine synthetase in maize roots. J Biol Chem 271:29561–29568 4. Peterman TK, Goodman HM (1991) The glutamine synthetase gene family of Arabidopsis thaliana: light-regulation and differential expression in leaves roots and seeds. Mol Gen Genet 230:145–154 5. Seabra AR, Vieira CP, Cullimore JV, Carvalho HG (2010) Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds. BMC Plant Biol 10:183
123
Mol Biol Rep (2014) 41:7575–7583 6. Biesiadka J, Legocki AB (1997) Evolution of the glutamine synthetase gene in plants. Plant Sci 128:51–58 7. Tabuchi M, Abiko T, Yamaya T (2007) Assimilation of ammonium ions and reutilization of nitrogen in rice (Oryza sativa L.). J Exp Bot 58:2319–2327 8. Teixeira J, Pereira S, Ca´novas F, Salema R (2005) Glutamine synthetase of potato (Solanum tuberosum L. cv Desiree) plants: cell- and organ-specific expression and differential developmental regulation reveal specific roles in nitrogen assimilation and mobilization. J Exp Bot 56:663–671 9. Unno H, Uchida T, Sugawara H, Kurisu G, Sugiyama T, Yamaya T, Sakakibara H, Hase T, Kusunoki M (2006) Atomic structure of plant glutamine synthetase: a key enzyme for plant productivity. J Biol Chem 28:29287–29296 10. Chen Q, Silflow CD (1996) Isolation and characterization of glutamine synthetase genes in Chlamydomonas reinhardtii. Plant Physiol 112:987–996 11. Ishiyama K, Inoue E, Tabuchi M, Yamaya T, Takahashi H (2004) Biochemical background and compartmentalized functions of cytosolic glutamine synthetase for active ammonium assimilation in rice roots. Plant Cell Physiol 45:1640–1647 12. Lothier J, Gaufichon L, Sormani R, Lemaıˆtre T, Azzopardi M, Morin H, Chardon F, Reisdorf-Cren M, Avice JC, MasclauxDaubresse C (2011) The cytosolic glutamine synthetase GLN1;2 plays a role in the control of plant growth and ammonium homeostasis in Arabidopsis rosettes when nitrate supply is not limiting. J Exp Bot 62:1375–1390 13. De la Torre F, Garcı´a-Gutie´rrez A, Crespillo R, Canto´n FR, Avila C, Ca´novas FM (2002) Functional expression of two pine glutamine synthetase genes in bacteria reveals that they encode cytosolic holoenzymes with different molecular and catalytic properties. Plant Cell Physiol 43:802–809 14. Reef R, Feller IC, Lovelock CE (2010) Nutrition of mangroves. Tree Physiol 30:1148–1160 15. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:1–11 16. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, So¨ding J (2011) Fast scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:1–6 17. Xiong AS, Yao QH, Peng RH, Li X, Fan HQ, Cheng ZM, Li Y (2004) A simple rapid high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Res 32:e98 18. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Laboratory Press, Cold Spring Harbor 19. Wellner VP, Meister A (1966) Binding of adenosine triphosphate and adenosine diphosphate by glutamine synthetase. Biochemistry 5(3):872–879 20. Kumada Y, Benson DR, Hillemann D, Hosted TJ, Rochefort DA, Thompson CJ, Wohlleben W, Tateno Y (1993) Evolution of the glutamine synthetase gene one of the oldest existing and functioning genes. Proc Natl Acad Sci USA 90:3009–3013 21. Ishiyama K, Inoue E, Yamaya T, Takahashi H (2006) Gln49 and Ser174 residues play critical roles in determining the catalytic efficiencies of plant glutamine synthetase. Plant Cell Physiol 47:299–303 22. DasSarma S, Tischer E, Goodman HM (1986) Plant glutamine synthetase complements a glnA mutation in Escherichia coli. Science 232:1242–1244 23. Bennett M, Cullimore J (1990) Expression of three plant glutamine synthetase cDNA in Escherichia coli. Formation of catalytically active isoenzymes and complementation of a glnA mutant. FEBS Lett 193:319–324
Mol Biol Rep (2014) 41:7575–7583 24. Forde B, Cullimore J (1989) In: Miflin B (ed) The molecular biology of glutamine synthetase in higher plants. Oxford University Press, Oxford, pp 247–296 25. Beudeker ROBF, Tabita FR (1985) Characterization of glutamine synthetase isoforms from chlorella. Plant Physiol 77:791–794 26. Vegetale LDB (1980) Glutamine synthetase in rice. Plant Physiol 66:619–623 27. Rana NK, Mohanpuria P, Yadav SK (2008) Cloning and characterization of a cytosolic glutamine synthetase from Camellia sinensis (L) O Kuntze that is upregulated by ABA SA and H2O2. Mol Biotechnol 39:49–56 28. Nosworthy NJ, Ginsburg A (1997) Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea. Protein Sci 6:2617–2623 29. Mondert C (1965) Metal ion interactions and glutamine synthetase activity. Biochemistry 6:2617–2623 30. Antonyuk LP, Smirnova VE, Kamnev AA, Serebrennikova OB, Vanoni MA, Zanetti G, Kudelina IA, Sokolov OI, Ignatov VV (2001) Influence of divalent cations on the catalytic properties and secondary structure of unadenylylated glutamine synthetase from Azospirillum brasilense. Biometals 14:13–22 31. Ishiyama K, Inoue E, Watanabe-Takahashi A, Obara M, Yamaya T, Takahashi H (2004) Kinetic properties and ammoniumdependent regulation of cytosolic isoenzymes of glutamine synthetase in Arabidopsis. J Biol Chem 279:16598–16605
7583 32. Fernandes SO, Bonin PC, Michotey VD, Garcia N, LokaBharathi PA (2012) Nitrogen-limited mangrove ecosystems conserve N through dissimilatory nitrate reduction to ammonium. Sci Rep 2:1–5 33. Berlicki Ł, Obojska A, Forlani G, Kafarski P (2005) Design synthesis and activity of analogues of phosphinothricin as inhibitors of glutamine synthetase. J Med Chem 48:6340–6349 34. Forlani G, Obojska A, Berlicki Ł, Kafarski P (2006) Phosphinothricin analogues as inhibitors of plant glutamine synthetases. J Agric Food Chem 54:796–802 35. Acaster MA, Weitzman PDJ (1985) Kinetic analysis of glutamine synthetases from various plants. FEBS Lett 189:241–244 36. Ahuja M, Punekar NS (2008) Phosphinothricin resistance in Aspergillus niger and its utility as a selectable transformation marker. Fungal Genet Biol 45:1103–1110 37. Man HM, Boriel R, El-Khatib R, Kirby EG (2005) Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. N Phytol 167:31–39 38. El-Khatib RT, Hamerlynck EP, Gallardo F, Kirby EG (2004) Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine synthetase gene exhibits enhanced tolerance to water stress. Tree Physiol 24:729–736 39. Pascual MB, Jing ZP, Kirby EG, Ca´novas FM, Gallardo F (2008) Response of transgenic poplar overexpressing cytosolic glutamine synthetase to phosphinothricin. Phytochemistry 69:382–389
123
Expression, purification, and characterization of recombinant mangrove glutamine synthetase Wei Zhao • Jun Yang • Yongsheng Tian • Xiaoyan Fu • Bo Zhu • Yong Xue • Jianjie Gao Hong-Juan Han • Rihe Peng • Quan-Hong Yao
•
Received: 4 March 2013 / Accepted: 27 July 2014 / Published online: 3 August 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract To expand our knowledge about the relationship of nitrogen use efficiency and glutamine synthetase (GS) activity in the mangrove plant, a cytosolic GS gene from Avicennia marina has been heterologously expressed in and purified from Escherichia coli. Synthesis of the mangrove GS enzyme in E. coli was demonstrated by functional genetic complementation of a GS deficient mutant. The subunit molecular mass of GSI was *40 kDa. Optimal conditions for biosynthetic activity were found to be 35 °C at pH 7.5. The Mg2?-dependent biosynthetic activity was strongly inhibited by Ni2?, Zn2?, and Al3?, whereas was enhanced by Co2?. The apparent Km values of AmGLN1 for the substrates in the biosynthetic assay were 3.15 mM for glutamate, and 2.54 mM for ATP, 2.80 mM for NH4? respectively. The low affinity kinetics of AmGLN1 apparently participates in glutamine synthesis under the ammonium excess conditions. Keywords Glutamine synthetase (GS) Ammonium assimilation Mangrove (Avicennia marina) PPTsensitive
W. Zhao Y. Tian X. Fu B. Zhu Y. Xue J. Gao H.-J. Han R. Peng (&) Q.-H. Yao (&) Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Road, Shanghai, People’s Republic of China e-mail: [email protected] Q.-H. Yao e-mail: [email protected] J. Yang College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu, People’s Republic of China
Introduction The glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle has been considered as the major pathway for ammonium assimilation and regulation of nitrogen metabolism in higher plants. GS (EC 6.3.1.2.) catalyzes the formation of glutamine from glutamate in the presence of NH4? ATP and metal cations, according to the following reaction: L-glutamate ? NH4? ? ATP ? L-glutamine ? ADP ? Pi ? H?. Subsequently, GOGAT (EC 1.4.7.1, EC I.1.1.14) catalyzes the formation of two molecules of L-glutamate from L-glutamine and 2-oxoglutarate. Since all of the N in a plant, whether derived from fertilizer, nitrate reduction, photorespiration, and numerous other sources including catabolic release of NH4? during senescence is channeled through the reactions catalyzed by GS, this reaction has been identified as the ratelimiting step in the control of N-assimilation and N-recycling during growth and development of plants [1, 2]. In higher plants, GS genes can be divided into two distinct families based on their subcellular localization. Typically, GS1 is localized in the cytoplasm and encoded by a small multigene family, whereas, GS2 is founded in the chloroplast and encoded by a single nuclear gene in most species [3, 4]. The existence of a second GS2 gene in Medicago truncatula has been reported recently as an exceptional case [5]. Previous phylogenetic analyses of chloroplast and cytosolic isoenzymes support the hypothesis that the isoenzymes in angiosperms evolved via a gene duplication event that preceded the divergence of monocots and dicots [6]. GS1 is the major form of GS in plant roots, and it plays an important role in the assimilation of external ammonium, the ammonium derived from N2 fixation and other sources of nitrogen, and in the remobilization of nitrogen during senescence [7, 8]. The early study indicated the decamer structure for maize cytosolic GS is
123
7576
composed of two face-to-face pentameric rings of identical subunits, with a total of 10 active sites, each formed between every two neighboring subunits within each ring [9]. GS from plants have been well investigated in terms of the regulation of gene expression and enzymatic characterization. However, most studies have focused on annual plants such as maize [10], rice [11], potato [8] and Arabidopsis [4, 12], thus available information concerning the biochemical properties of GS from woody plant is much more limited [13]. Mangroves are crucial for the coastal areas and nitrogen-use efficiency (NUE) in mangroves is among the highest recorded for angiosperms [14]. Here we report that expression, purification, and characterization of a cytosolic GS isoenzyme (AmGLN1, Q9AXD8) in Escherichia coli. Our research will contribute to expand scientific knowledge about how mangroves can sustain high levels of NUE.
Materials and methods Growth conditions, strains, and plasmids The E. coli Gln-auxotrophic strain JW3841-1 (glnA732::Kan) used for the production of recombinant proteins was obtained from the E. coli Genetic Resource Center (CGSC#10775, Yale University, USA) [15]. The pRARE plasmid (Novagen, USA), which over-expresses tRNAs for Arg, Leu, and Pro codons rarely used by E. coli, was introduced into JW3841-1 to enhance the expression of AmGLN1. pTrcHis-B expression vector was purchased from Invitrogen, USA. Bacteria were routinely grown in Lineweaver–Burk (LB) containing antibiotics (100 lg/ml ampicillin, 20 lg/ml chloramphenicol, 40 lg/ml kanamycin) when necessary. For complementation analysis, strains were grown in M9 minimal medium containing 1 % (wt/ vol) glycerol, thiamine (1 lg/ml), casamino acids [0.66 % (wt/vol)], the appropriate antibiotics, with or without a glutamine supplement (1 mM) at 30 °C. Sequence analysis and synthesis of the AmGLN1 For homology analysis, BLAST searches were conducted using BLASTp programmes (http://www.ncbi.nlm.nih.gov/ BLAST/). The theoretical isoelectric point (pI) and molecular weight (Mw) were computed with the ExPASy ProtParam tool (http://www.expasy.org). Alignment was performed using clustal Omega multiple sequence alignment program [16]. Phylogenetic tree was constructed by MEGA 4.0 software using neighbor joining method with 1,000 times bootstraps. All of the protein sequences were retrieved from the UniProt Database.
123
Mol Biol Rep (2014) 41:7575–7583
DNA coding for full-length AmGLN1 (GenBank accession no. AF338444.1) was synthesized through a simple, high-fidelity, and cost-effective PTDS (PCR-based two-step DNA synthesis) method described previously [17]. Errors in the synthetic gene were identified by DNA sequencing and corrected by overlap extension PCR method. Routine DNA manipulations were performed by standard recombinant method [18]. Purification of recombinant AmGLN1 The synthetic gene was ligated into the pTrcHis-B Expression Vector using the BamHI and SacI linker sites. The resulting construct pTrcHis-B/AmGLN1 was transformed into the E. coli glnA mutant JW3841-1. Several transformants were cultured in 100 ml LB medium at 37 °C until an OD600 of 0.3 was reached, at which time isopropyl-D-thiogalactopyranoside was added at 1 mM to induce gene expression and protein production. After incubated for 12 h at 30 °C on a rotary shaker, the cells collected by centrifugation was re-suspended in 4 ml s of lysis buffer [50 mM Tris–HCl, pH 7.5, 300 mM NaCl, 0.5 mM dithiothreitol (DTT), and 10 mM imidazole]. The His-tagged protein in the soluble fraction was affinitypurified using a nickel-nitrilotriacetic acid agarose column (1.5 ml bed volume, Sigma). Washing was carried out with a buffer containing 50 mM Tris–HCl, pH 7.5, 300 mM NaCl, and 20 mM imidazole. Protein was eluted with the same buffer containing 250 mM imidazole. The GS fractions were then dialyzed against buffer (50 mM Tris–HCl, pH 7.5, 0.1 mM EDTA, and 10 % glycerol). The purified protein was analyzed by 12 % SDS-PAGE and protein concentration was quantified using Bradford assay kit and bovine serum albumin as standard. GS enzyme assay The biosynthetic activities were determined by the methods described by Wellner and Meister [19]. The biosynthetic assay solution contained the following components: 100 mM Tris–HCl (pH 7.5), 5 mM ATP, 50 mM L-glutamate, 30 mM NH4Cl, 20 mM MgCl2, in a total volume of 190 ll. The mixture was equilibrated at 35 °C for 5 min, and the reaction was initiated by adding 10 ll of the enzyme solution. The reaction was terminated after 30 min by the addition of 200 ll of ferric chloride reagent (55 g/l FeCl36H2O, 20 g/l trichloroacetic acid, and 2.1 % concentrated HCl) and the release of c-glutamyl hydroxamate was quantified by measuring the absorbance at 540 nM. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the formation of 1 lmol of c-glutamyl hydroxamate/min.
Mol Biol Rep (2014) 41:7575–7583
pH and temperature optima The pH stability was examined by measuring the activity in Tris–HCl buffer (pH 7.5) after incubating the purified enzyme in the following buffers ranging from pH 2 to 10.0 for 24 h at 4 °C: citric acid–sodium citrate (0.2 M, pH 2.0–6.5), Tris–HCl buffer (0.2 M, pH 7.0–8.0), glycine– NaOH (0.2 M, pH 9.0–10.0). The optimum pH for its maximal activity was obtained by determining the activity with different pH buffers (pH 6.0–10.0). To determine the optimal temperature, enzyme activity was assayed at different temperatures between 20 and 52 °C. Thermostability were determined by measuring the residual activity at 35 °C after incubating the enzyme in Tris–HCl buffer (pH 7.5) at 35, 40 or 45 °C for varying periods of time. Effect of different metal ions (Mn2?, Mg2?, Ca2?, Zn2?, and Ni2?) on AmGLN1 activity was studied by preincubating the enzyme with 10 mM concentration of different metal chlorides at 4 °C for 1 h. Kinetic characterization The kinetic parameters for the different substrates and cofactors were obtained by varying the concentrations of individual reaction components (L-glutamate 2–40 mM, NH4Cl 1–16 mM, ATP 1–8 mM). Unvariable substrates were fixed at the same levels as in standard assay. For Ki value evaluation, the enzyme was assayed in the presence of increasing concentrations of D,L-phosphinothricin (D,LPPT 0.25–5 lM) at varying levels of glutamate (2–40 mM). The enzyme activity was determined as mentioned above and the data were analyzed and plotted using Sigma Plot (version 8.0). Each enzyme-activity assay was replicated independently at least three times.
Results and discussion Sequence alignment and phylogenetic tree To understand the relationship between AmGLN1 and GS1 enzymes from other organisms, we compared the amino acid sequence of AmGLN1 with those from five divergent organisms (Fig. 1). A consensus pattern, [FYWL]-D-G-S– S-X6,8-[DENQSTAK]-[SA]-[DE]-X2-[LIVMFY], which is usually present in the N-terminal section of GS (PROSITE accession no. PS00180) was detected in the N-terminal region of AmGLN1, between residues 55 and 72. Another conserved sequence for ATP-binding region (PROSITE accession no. PS00181), {K–P-[LIVMFYA]-x(3,5)[NPAT]-[GA]-[GSTAN]-[GA]-x–H-x(3)-S}, was also present [20]. The AmGLN1 shared 86–91 % amino acid
7577
sequence identity with other cytosolic GS from Hevea brasiliensis, Cucumis melo, Vitis vinifera, Poplar, and Zea mays. As other cytosolic GS forms, the AmGLN1 lack the characteristic N-terminal and C-terminal amino acid extensions of the chloroplastic polypeptides (data not shown). Ishiyama et al. [21] reported that the existence of Gln49 or Ser174 is sufficient to fulfill the substrate specificities of the high-affinity variants of plant GS1. The amino acid residues at positions 49 and 174 in AmGLN1 were Lys and Ser. This information suggests that AmGLN1 might have low affinity for ammonium. A neighbor-joining tree of GS proteins from mangrove, maize, rice, Arabidopsis and other species was created with the MEGA program. Phylogenetic reconstruction at the molecular level shows the separation of cytosolic (GS1) and chloroplastic (GS2) sequences in angiosperms as two well differentiated clusters (Fig. 2). Figure 2 also showed that the AmGLN1 was closely aligned with the GS1 isoforms of angiosperms but was outside the main subfamilies. Complementation analysis To determine whether the recombinant AmGLN1 was functional in E. coli, the Gln-auxotrophic strain JW3841-1 was transformed with pTrcHis-B/AmGLN1 and as the control the empty pTrcHis-B vector. The transformants were tested for growth on M9 minimal medium containing or lacking glutamine at 30 °C. The results showed that the transformants harboring the pTrcHis-B/AmGLN1 was able to grow on minimal media containing ammonium as the nitrogen source whereas transformants harboring the vector failed to grow on M9 minimal medium except when it was supplemented with glutamine (Fig. 3a). Our results confirmed that AmGLN1 is folded and assembled into an essentially native conformation in E. coli and make it possible to select directly in E. coli for variants, which could be useful in the development of plants with improved nitrogen utilization or herbicide-resistance [22]. The effective complementation by pTrcHis-B/AmGLN1 was observed at temperatures below 37 °C (Fig. 3b). Thus, we considered that lower temperatures favor the native functional conformation of AmGLN1. Similar results have been reported by Bennett and Cullimore [23]. Protein expression and purification The overexpression of functionally active GS in E. coli prevented the inconveniences that interfere with protein extraction procedures from woody plants. It provided a good source of the recombinant GS in unlimited amounts for purification procedures and biochemical studies. As the GS is sensitive to environmental oxidation during
123
7578
Mol Biol Rep (2014) 41:7575–7583
Fig. 1 Sequence alignment of the deduced-amino acid sequences of AmGLN1 with members of the GS1 family from different plants (Hevea brasiliensis, Cucumis melo, Vitis vinifera, Populus canescens, and Zea mays). Single letter amino acid code is used. Asterisks,
colons, and dots indicate identical amino acid residues, conserved substitutions, and semi-conserved substitutions in all sequences used in the alignment, respectively
refolding, the purification was performed in the presence of 0.5 mM of DTT. The recombinant AmGLN1 was produced as soluble protein when expressed in JW3841-1 at 30 °C.
The purified recombinant AmGLN1 showed only single band of *40 kDa (fused with six 9 histidine tag) on the SDS–poly-acrylamide gel, identical to that theoretical
123
Mol Biol Rep (2014) 41:7575–7583
7579
Fig. 2 Phylogenetic comparison of glutamine synthetase protein. Entry names of the sequence in the UniProt Database are given. The sequences are from Alnus glutinosa (AgGLN1), A. thaliana (AtGLN1;1, AtGLN1;2, AtGLN1;3, AtGLN1;4, AtGLN1;5, AtGLN2), A. marina (AmGLN1, AmGLN2), Brassica napus (BnGLN1), Camellia sinensis (CsGLN1), Canavalia lineate (ClGLN1), Datisca glomerata (DgGLN1), Glycine max (GmGLN1), Gossypium raimondii (GrGLN1), H. brasiliensis (HbGLN1), Lotus japonicus (LjGLN1), Medicago truncatula (MtGLN1), Nicotiana attenuata (NaGLN1), Oryza sativa (OsGLN1;1, OsGLN1;2, OsGLN1;3, OsGLN2), Phaseolus vulgaris (PvGLN1), Picea sitchensis (PsGLN1;1, PsGLN1;2), Pinus sylvestris (PsGLN1a, PsGLN1b), Populus trichocarpa (PtGLN1, PtGLN2), Selaginella moellendorffii (SmGLN1), Vigna aconitifolia (VaGLN1), V. vinifera (VvGLN1;1, VvGLN1;2, VvGLN2), and Z. mays (ZmGLN1;1, ZmGLN1;2, ZmGLN1;3, ZmGLN1;4, ZmGLN1;5, ZmGLN2), respectively
123
7580
Mol Biol Rep (2014) 41:7575–7583
Fig. 5 pH optimum and pH stability of AmGLN1. The buffers used, at 100 mM concentration, were citric acid–sodium citrate (0.2 M, pH 2.0–6.5), Tris–HCl buffer (0.2 M, pH 7.0–8.0), glycine–NaOH (0.2 M, pH 9.0–10.0). The values shown represent the mean values from triplicate experiments
Fig. 3 Complementation of the E. coli Gln-auxotrophic strain JW3841-1 by AmGLN1. Transformed cells containing pTrcHis-B as control or including pTrcHis-B/AmGLN1 were plated on M9 minimal medium containing or lacking glutamine, and incubated at 30 °C
Fig. 6 Effect of temperature on activity (a) and thermal sensitivity (b) of AmGLN1. For thermal inactivation, samples of the purified enzyme were incubated in Tris–HCl buffer (pH 7.5) at 35, 40, 45 °C for the indicated times, followed by measurement of the residual GS activity. The values shown represent averages from triplicate experiments (n = 3) Fig. 4 SDS-PAGE analysis of recombinant AmGLN1. The purified AmGLN1 was subjected to SDS-PAGE on 0.1 % SDS-12 % PAGE and stained with Coomassie Brilliant Blue R-250. M protein molecular weight marker (low, Takara), lane 1, crude extract, lane 2 AmGLN1
value of 39.3 kDa (Fig. 4). The 39.3-kDa polypeptide corresponds to the cytosolic isoform form of GS but lower than the plastidic isoform (c. 44–45 kDa) [24].
123
Stability and pH optima In the presence of Mg2?, the enzyme exhibited the maximum activity around pH 7.5, whereas its activity declined steeply towards the acidic pH (Fig. 5). The GS from chlorella [25], rice [26], maize [3], and Camellia sinensis
Mol Biol Rep (2014) 41:7575–7583
7581
Table 1 Effect of metal ions on AmGLN1 activity Metal ion
Control
Ni2?
Mn2?
Zn2?
Co2?
Fe2?
Cu2?
Ca2?
Al3?
Relative activity
100.00
8.92
17.12
9.58
109.09
22.27
22.20
43.86
8.91
The biosynthetic activity assays were performed after incubation the purified enzymes with 10 mM concentration of different metal chlorides and equal volume of water as a control at 4 °C for 1 h
[27] also exhibited optimal activity around the similar pH range. The enzyme was fairly stable at pH 4.0–10.0, while the highest stability was observed at pH 7.0–9.0 (Fig. 5). The remarkable stability of the enzyme under alkaline pH might be an environmental adaptation of Avicennia marina that grew in a highly alkaline environment. The enzyme was optimally active within a narrow range of temperatures from 30 to 42 °C. At higher temperatures ([42 °C) the relative activity rapidly decreased (Fig. 6a). Thermostability study showed that the enzyme was relative stable under 37 °C and sensitive to temperatures above 45 °C (Fig. 6b). The enzymatic activity of AmGLN1 was lost after 30 min of incubation at 45 °C, indicating a lack of functional and structural stability at high temperature. Dissociation of the oligomeric proteins at high temperature, resulting in a loss of activity, is a common phenomenon among GS enzymes from prokaryotes and eukaryotes [28]. It has been reported that the different GS isoforms of pine and maize exhibited distinguishable thermal inactivation profiles [3, 13]. The thermal stability of AmGLN1 was more similar to GS1a from pine and GS1d from maize. Unno et al. [9] has postulated that Ile-161 of GS1a is critical for the stability of GS. Similarly, the thermosensitivity of AmGLN1 can be overcome by replacing of Val161 by Ile through site-directed mutagenesis. Effects of metal ions The effect of different metal ions on the Mg2?-dependent biosynthetic activity of AmGLN1 was shown in Table 1. Compared to the activity determined with Mg2?, the addition of 10 mM Ni2?, Zn2?, and Al3? caused 8.92, 9.58, and 8.91 % loss in activity, respectively. Interestingly, a little improvement of enzyme activity was observed by assaying in the presence of Co2?. Divalent cations (commonly, Mg2?, Mn2?, or Co2?) are absolutely necessary for the activity of all known GSs both in the case of glutamine synthesis and for the transferase reaction catalyzed by the enzyme. A shift of the pH optimum by simultaneous presence of Mg2? and other metal ions in the reaction mixture may resulted in a decrease in over-all activity [29]. The observed increase in enzyme activity with Co2? could be attributed to Co2? has a higher binding constant. This agrees with the result of Antonyuk et al. [30] that the Co2? was more effective than Mg2? in supporting
biosynthetic activity of the metal-ion free GS from Azospirillum brasilense. Kinetic constants for substrates and inhibitor The apparent Km values of AmGLN1 for the substrates in the biosynthetic assay were 3.15 mM for glutamate, 2.54 mM for ATP and 2.80 mM for NH4? respectively. The Km value for glutamate of AmGLN1 were similar to those reported for natural plant GS [3, 26]. However, AmGLN1 exhibited higher affinity for the glutamate than GS1a from pine (Km value 13 mM) [13] and CsGS (Km value 9 mM) [25]. Ishiyama reported that the four isoenzymes of GS1 expressed in Arabidopsis roots exhibited different Km values for ammonium. Similar to GLN1;2 and GLN1;3 (Km values; 2.45 mM for GLN1;2 and 1.21 mM for GLN1;3, respectively) from Arabidopsis, AmGLN1 showed a relative low affinity to ammonium [31]. Recently, it has been shown that up to 99 % of nitrate removal in mangrove sediments is routed through dissimilatory nitrate reduction to ammonium (DNRA) [32]. Ammonium produced by the DNRA process is the primary form of nitrogen in mangrove soils. The high-capacity glutamine synthetic activity of AmGLN1 facilitates the assimilation of ammonium taken up from soils. The low affinity kinetics of AmGLN1 apparently participates in Gln synthesis under the ammonium excess conditions. However, nutrient availability can vary greatly among and within mangrove forests. Many mangrove soils have extremely low N availability. Mangroves perhaps contain other GS1 isoforms with high affinity to ammonium to facilitate rapid conversion of ammonium under nitrogenlimited conditions. In Arabidopsis and rice, the existence of high affinity-type GS1 has been reported previously [11, 31]. The inhibition constants for PPT, determined from the LB plots and the enzyme kinetics module of Sigma Plot (version 8.0), is 0.26 lM. This value is lower than those reported for E. coli (0.6 lM) [33], maize (1.1 lM) [34], barely (3.5 lM) [35], and Aspergillus niger (54 lM) [36]. In this sense, the A. marina GS appears to be relatively more sensitive to PPT inhibition. A current focus in plant improvement is the increase in plant growth, biomass accumulation, and stresses tolerance as a result of overexpression of cytosolic GS. For instance,
123
7582
transgenic poplar overexpressing GS1 has been previously shown to exhibits desirable field performance characteristics, including enhanced NUE [37], enhanced resistance to water stress [38] and enhanced resistance to PPT [39]. AmGLN1 is considered to be a good candidate gene for engineering plants to improve growth phenotype and increase the efficiency of N utilization.
Conclusion Avicennia marina cytosolic GS was synthesized and functional in E. coli based on the observations that AmGLN1 was able to complement the E. coli glnA mutant. Studies on biochemical properties of the purified enzyme revealed thermal instability of AmGLN1. Moreover, kinetic analysis showed that AmGLN1 was highly sensitive to PPT inhibition and had a relative low affinity for ammonium. We propose that AmGLN1 apparently play a role in Gln synthesis under the ammonium excess conditions. The present study, which is the first to biochemically characterize cytosolic GS from mangroves, provides the necessary basis for further investigations into roles of GS1 in nitrogen metabolism during growth and development of mangroves. Further work is required to establish expression profiles and regulation mechanisms of AmGLN1 in vivo and whether other GS1 isoforms with high affinity to ammonium exist in mangroves. Acknowledgments The research was supported by the National Natural Science Foundation of China (31071486), The Key Project Fund of the Shanghai Municipal Committee of Agriculture (Nos. 2009-6-4 and 2011-1-8), and International Scientific and Technological Cooperation (2010DFA62320).
References 1. Martin A, Lee J, Kichey T, Gerentes D, Zivy M, Tatout C, Dubois F, Balliau T, Valot B, Davanture M (2006) Two cytosolic glutamine synthetase isoforms of maize are specifically involved in the control of grain production. Plant Cell 18:3252–3274 2. Miflin BJ, Habash DZ (2002) The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops. J Exp Bot 53:979–987 3. Sakakibara H, Shimizu H, Hase T, Yamazaki Y, Takao T, Shimonishi Y, Sugiyama T (1996) Molecular identification and characterization of cytosolic isoforms of glutamine synthetase in maize roots. J Biol Chem 271:29561–29568 4. Peterman TK, Goodman HM (1991) The glutamine synthetase gene family of Arabidopsis thaliana: light-regulation and differential expression in leaves roots and seeds. Mol Gen Genet 230:145–154 5. Seabra AR, Vieira CP, Cullimore JV, Carvalho HG (2010) Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds. BMC Plant Biol 10:183
123
Mol Biol Rep (2014) 41:7575–7583 6. Biesiadka J, Legocki AB (1997) Evolution of the glutamine synthetase gene in plants. Plant Sci 128:51–58 7. Tabuchi M, Abiko T, Yamaya T (2007) Assimilation of ammonium ions and reutilization of nitrogen in rice (Oryza sativa L.). J Exp Bot 58:2319–2327 8. Teixeira J, Pereira S, Ca´novas F, Salema R (2005) Glutamine synthetase of potato (Solanum tuberosum L. cv Desiree) plants: cell- and organ-specific expression and differential developmental regulation reveal specific roles in nitrogen assimilation and mobilization. J Exp Bot 56:663–671 9. Unno H, Uchida T, Sugawara H, Kurisu G, Sugiyama T, Yamaya T, Sakakibara H, Hase T, Kusunoki M (2006) Atomic structure of plant glutamine synthetase: a key enzyme for plant productivity. J Biol Chem 28:29287–29296 10. Chen Q, Silflow CD (1996) Isolation and characterization of glutamine synthetase genes in Chlamydomonas reinhardtii. Plant Physiol 112:987–996 11. Ishiyama K, Inoue E, Tabuchi M, Yamaya T, Takahashi H (2004) Biochemical background and compartmentalized functions of cytosolic glutamine synthetase for active ammonium assimilation in rice roots. Plant Cell Physiol 45:1640–1647 12. Lothier J, Gaufichon L, Sormani R, Lemaıˆtre T, Azzopardi M, Morin H, Chardon F, Reisdorf-Cren M, Avice JC, MasclauxDaubresse C (2011) The cytosolic glutamine synthetase GLN1;2 plays a role in the control of plant growth and ammonium homeostasis in Arabidopsis rosettes when nitrate supply is not limiting. J Exp Bot 62:1375–1390 13. De la Torre F, Garcı´a-Gutie´rrez A, Crespillo R, Canto´n FR, Avila C, Ca´novas FM (2002) Functional expression of two pine glutamine synthetase genes in bacteria reveals that they encode cytosolic holoenzymes with different molecular and catalytic properties. Plant Cell Physiol 43:802–809 14. Reef R, Feller IC, Lovelock CE (2010) Nutrition of mangroves. Tree Physiol 30:1148–1160 15. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:1–11 16. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, So¨ding J (2011) Fast scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:1–6 17. Xiong AS, Yao QH, Peng RH, Li X, Fan HQ, Cheng ZM, Li Y (2004) A simple rapid high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Res 32:e98 18. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Laboratory Press, Cold Spring Harbor 19. Wellner VP, Meister A (1966) Binding of adenosine triphosphate and adenosine diphosphate by glutamine synthetase. Biochemistry 5(3):872–879 20. Kumada Y, Benson DR, Hillemann D, Hosted TJ, Rochefort DA, Thompson CJ, Wohlleben W, Tateno Y (1993) Evolution of the glutamine synthetase gene one of the oldest existing and functioning genes. Proc Natl Acad Sci USA 90:3009–3013 21. Ishiyama K, Inoue E, Yamaya T, Takahashi H (2006) Gln49 and Ser174 residues play critical roles in determining the catalytic efficiencies of plant glutamine synthetase. Plant Cell Physiol 47:299–303 22. DasSarma S, Tischer E, Goodman HM (1986) Plant glutamine synthetase complements a glnA mutation in Escherichia coli. Science 232:1242–1244 23. Bennett M, Cullimore J (1990) Expression of three plant glutamine synthetase cDNA in Escherichia coli. Formation of catalytically active isoenzymes and complementation of a glnA mutant. FEBS Lett 193:319–324
Mol Biol Rep (2014) 41:7575–7583 24. Forde B, Cullimore J (1989) In: Miflin B (ed) The molecular biology of glutamine synthetase in higher plants. Oxford University Press, Oxford, pp 247–296 25. Beudeker ROBF, Tabita FR (1985) Characterization of glutamine synthetase isoforms from chlorella. Plant Physiol 77:791–794 26. Vegetale LDB (1980) Glutamine synthetase in rice. Plant Physiol 66:619–623 27. Rana NK, Mohanpuria P, Yadav SK (2008) Cloning and characterization of a cytosolic glutamine synthetase from Camellia sinensis (L) O Kuntze that is upregulated by ABA SA and H2O2. Mol Biotechnol 39:49–56 28. Nosworthy NJ, Ginsburg A (1997) Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea. Protein Sci 6:2617–2623 29. Mondert C (1965) Metal ion interactions and glutamine synthetase activity. Biochemistry 6:2617–2623 30. Antonyuk LP, Smirnova VE, Kamnev AA, Serebrennikova OB, Vanoni MA, Zanetti G, Kudelina IA, Sokolov OI, Ignatov VV (2001) Influence of divalent cations on the catalytic properties and secondary structure of unadenylylated glutamine synthetase from Azospirillum brasilense. Biometals 14:13–22 31. Ishiyama K, Inoue E, Watanabe-Takahashi A, Obara M, Yamaya T, Takahashi H (2004) Kinetic properties and ammoniumdependent regulation of cytosolic isoenzymes of glutamine synthetase in Arabidopsis. J Biol Chem 279:16598–16605
7583 32. Fernandes SO, Bonin PC, Michotey VD, Garcia N, LokaBharathi PA (2012) Nitrogen-limited mangrove ecosystems conserve N through dissimilatory nitrate reduction to ammonium. Sci Rep 2:1–5 33. Berlicki Ł, Obojska A, Forlani G, Kafarski P (2005) Design synthesis and activity of analogues of phosphinothricin as inhibitors of glutamine synthetase. J Med Chem 48:6340–6349 34. Forlani G, Obojska A, Berlicki Ł, Kafarski P (2006) Phosphinothricin analogues as inhibitors of plant glutamine synthetases. J Agric Food Chem 54:796–802 35. Acaster MA, Weitzman PDJ (1985) Kinetic analysis of glutamine synthetases from various plants. FEBS Lett 189:241–244 36. Ahuja M, Punekar NS (2008) Phosphinothricin resistance in Aspergillus niger and its utility as a selectable transformation marker. Fungal Genet Biol 45:1103–1110 37. Man HM, Boriel R, El-Khatib R, Kirby EG (2005) Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. N Phytol 167:31–39 38. El-Khatib RT, Hamerlynck EP, Gallardo F, Kirby EG (2004) Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine synthetase gene exhibits enhanced tolerance to water stress. Tree Physiol 24:729–736 39. Pascual MB, Jing ZP, Kirby EG, Ca´novas FM, Gallardo F (2008) Response of transgenic poplar overexpressing cytosolic glutamine synthetase to phosphinothricin. Phytochemistry 69:382–389
123
