Cloning and functional characterization of a homoglutathione synthetase from pea nodules

Physiologia Plantarum 115 (1): 69-73 (2002) 3 Cloning and functional characterization of a homoglutathione synthetase from pea nodules Iñaki Iturbe-Ormaetxe, Begoña Heras, Manuel A. Matamoros, Javier Ramos, Jose F. Moran and Manuel Becana* Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, Apdo 202, 50080 Zaragoza, Spain *Corresponding author, e-mail: becana@eead.csic.es Received 15 August 2001; revised 2001 4 The thiol tripeptide glutathione (GSH; γGlu-Cys-Gly) is very abundant in legume nodules where it performs multiple functions that are critical for optimal nitrogen fixation. Some legume nodules contain another tripeptide, homoglutathione (hGSH; γGlu-Cys-βAla), in addition to or instead of GSH. We have isolated from a pea (Pisum sativum L.) nodule library a cDNA, GSHS2, that is expressed in nodules but not in leaves. This cDNA was overexpressed in insect cells and its protein product was identified as a highly active and specific hGSH synthetase. The enzyme, the first of this type to be completely purified, is predicted to be a homodimeric cytosolic protein. It shows a specific activity of 3400 nmol hGSH min-1mg-1 of protein with a standard substrate concentration (5 mM β−alanine) and Km values of 1.9 mM for β−alanine and 104 mM for glycine. The specificity constant (Vmax/Km) shows that the pure enzyme is 57.3-fold more specific for β−alanine than for glycine. Southern blot analysis revealed that the gene is present as a single copy in the pea genome and that there are homologous genes in other legumes. We conclude that the synthesis of hGSH in pea nodules is catalyzed by a specific hGSH synthetase and not by a GSH synthetase with broad substrate specificity. Abbreviations - γEC, γ−glutamylcysteine; γECS, γ−glutamylcysteine synthetase; GSHS, glutathione synthetase; hGSH, homoglutathione; hGSHS, homoglutathione synthetase; ORF, open reading frame. 5 Introduction The thiol tripeptide glutathione (GSH; γGlu-Cys-Gly) is a major antioxidant metabolite in most procaryotic and eucaryotic cells. The synthesis of GSH involves two sequential reactions catalyzed by γ−glutamylcysteine (γEC) synthetase (γECS; EC 6.3.2.2) and GSH synthetase (GSHS; EC 6.3.2.3) (Fig. 1). Both enzymes show a strict requirement for ATP and Mg2+ (Rennenberg 1997). However, plants may contain another thiol tripeptides (Fig. 1), such as hydroxymethylglutathione (γGlu-Cys-Ser), found in cereals, and homoglutathione (hGSH; γGlu-Cys-βAla), found exclusively in legumes (Klapheck 1988, Rennenberg 1997, Matamoros et al. 1999). The pathway of hGSH synthesis is also thought to proceed through two steps, catalyzed respectively by γECS and either a specific hGSH synthetase (hGSHS) or a GSHS with broad substrate specificity (Macnicol 1987). Thiol compounds are particularly abundant in nodules and this may be related to their critical role in the overall protection of nitrogen fixation (Dalton et al. 1986, Matamoros et al. 1999). In previous work on thiol metabolism in pea plants, we found GSHS activity in leaves and nodules, whereas hGSHS activity was only detected in nodules (Matamoros et al. 1999). We subsequently isolated two cDNA clones, GSHS1 and GSHS2, from a pea nodule library. Based on the correlation between activity and expression data, we concluded that GSHS1 and GSHS2 code for GSHS and hGSHS, respectively (Moran et al. 2000). A similar correlative hypothesis was proposed for two partial GSHS clones obtained from a Medicago truncatula root cDNA library (Frendo et al. 1999). Sequence analysis revealed that, in pea, GSHS1 encodes a protein bearing a mitochondrial signal peptide whereas GSHS2 encodes a cytosolic protein (Moran et al. 2000). These data, although predictive, indicate that GSHS enzymes may be localized in at least two subcellular compartments of nodules. 6 In fact, we were able to detect GSHS activity in mitochondria of cowpea nodules (a GSH producing species) but not of bean nodules (a hGSH producing species), suggesting that hGSHS is not present in mitochondria. Up to date a hGSHS enzyme has not been completely purified from any plant (Macnicol 1987) or from any heterologous organism (Frendo et al. 2001). This is probably due to the lability and low abundance of the enzyme in plant tissues (Macnicol 1987; Klapheck et al. 1988) and the low yield of conventional heterologous expression systems (Frendo et al. 2001). The availability of a cDNA that putatively encodes pea hGSHS, the absence of pure enzyme preparations for adequate kinetic analysis and thereby for function assignment, and the presence of GSHS2 transcripts specifically in pea nodules, all prompted us to characterize the GSHS2 cDNA and the corresponding protein product. Materials and methods Plant material Nodulated plants of pea (Pisum sativum L. cv. Lincoln x Rhizobium leguminosarum biovar. viciae strain NLV8) and common bean (Phaseolus vulgaris L. cv. Contender x Rhizobium leguminosarum biovar. phaseoli strain 3622) were grown under controlled environment conditions as described by Gogorcena et al. (1997). Leaves and nodules to be used for extraction of genomic DNA or mRNA were harvested from plants at the vegetative growth period (approximately 30 days of age), immediately frozen in liquid N2, and stored at -80C. 7 Overproduction and purification of recombinant protein The open reading frame (ORF) of GSHS2 was PCR-amplified using cDNA from 3-week-old pea nodules as a template using gene-specific primers (NcoI and NotI sites are underlined in the respective primers): forward 5'-caccatggcta aatcatctcaacagc-3' and reverse 5'-CTAATCGCAGCGGCCGC AATGCTA-3'. The resulting 1.7 kb fragment was gel purified, subcloned into pCRIITOPO (Invitrogen, Groningen, The Netherlands), and transformed into DH5α competent cells. The inserted ORF of GSHS2 was digested out with NcoI and NotI, gel purified, and ligated into pFastBac HTb. This procedure resulted in the GSHS2 cDNA being placed under the transcriptional control of the strong polyhedrin promoter (Autographa californica nuclear polyhedrosis virus) and in the addition of a poly-His tag to the recombinant protein for further detection and purification. DH5α competent cells were then transformed and positive colonies were identified by PCR using pFastBac specific primers. The pFAstBa c::GSHS2 DNA was isolated from an overnight culture and used to transform DH10BAC competent cells following the BAC-to-BAC protocol (Life Technologies, Paisley, UK). White positive colonies were verified by colony PCR. High molecular mass recombinant bacmid DNA was produced overnight in Escherichia coli and used to transfect Sf21 Spodoptera frugiperda insect cells with CellFectin reagent (Life Technologies). Recombinant baculoviruses were harvested 72 h post-transfection and amplified by infecting monolayer cultures of insect cells. These cultures were grown at 27¼C in TC-100 medium supplemented with 10% fetal calf serum and antibiotics, using media and chemicals from Sigma and protocols available from Life Technologies. Recombinant viruses were collected 48 h after infection from the culture supernatant and kept at 4¼C or -20¼C until subsequent infection of fresh cells. To optimize infection conditions and protein yield, confluent Sf21 cell cultures (5 ml of medium) were infected with different amounts of recombinant 8 viruses and cells were collected by centrifugation 24 to 96 h after infection. Cells were resuspended in lysis medium consisting of 10 mM Tris-HCl (pH 7.5), 130 mM NaCl, 10 mM NaF, 10 mM sodium phosphate buffer (pH 7.5), 10 mM sodium pyrophosphate, 1% Triton X-100, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and protease inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany). Cell-free extracts were loaded on a cobalt Talon affinity column (Clontech, Palo Alto, CA, USA) and protein was eluted with 50 mM sodium phosphate buffer (pH 7.0), 300 mM NaCl, and 150 mM imidazole. Western blot analysis Western blots were performed following protocols supplied by the manufacturer (Clontech). Briefly, protein samples were separated in SDS gels, blotted onto nitrocellulose membranes, incubated overnight at 4¡C with blocking solution (0.1% Tween-20, 1% nonfat milk in phosphate-saline buffer), and then for 1 h with the antibodies diluted in blocking solution. The primary antibody (6xHis monoclonal antibody; Clontech) was used at a 1:5000 dilution and the secondary antibody (anti-mouse antibody-alkaline phosphatase conjugate; Sigma) at a 1:2500 dilution. Color was developed with 5-bromo-4-chloro-3indolyl phosphate/nitroblue tetrazolium chloride (Sigma). Functional characterization of the enzyme The assay of GSHS and hGSHS activities of the GSHS2 overexpressed protein was based on the amount of GSH and hGSH synthesized from γEC and Gly or β-Ala, respectively (Matamoros et al. 1999). Thiol tripeptides were derivatized with monobromobimane and quantified by HPLC with fluorescence detection (Fahey and Newton 1987) with minor modifications (Matamoros et al. 1999). The Km and Vm values were calculated from double-reciprocal plots using 0.5 mM γEC and 10 to 150 mM Gly (for GSHS activity) or 0.5 mM γEC and 0.4 to 5 9 mM β−Ala (for hGSHS activity). For comparison, the activity rates (V) of GSHS and hGSHS were also measured using fixed standard concentrations of γEC (0.5 mM) and Gly or β-Ala (5 mM). Southern blot analysis of GSHS2 Genomic DNA was isolated from pea and bean leaves, digested with the restrict ion enzymes stated in Figure 3, fractionated on agarose gels, and transferred to Hybond N+ membranes (Amersham) following standard protocols. Hybridizations were performed at high stringency with 32P-labeled probes prepared from PCR products. For pea, the primers (forward 5'-GCAGTCGCAATCGTTTA CTTCC-3', reverse 5'-CCCACCTTCATCAAATAATGATGG-3') amplified a 594bp fragment within the ORF (GenBank accession no. AF258319). For bean, the primers (forward 5'-GAAAGTGGCTATATGGTGCG-3', reverse 5'-GACACCAT TCAGTAGGAAAAGC-3') amplified a 233-bp fragment including part of the ORF and part of the 3'-untranslated region (GenBank accession no. AF258320). Results and Discussion We attempted initially to overproduce pea nodule GSHS2 using conventional E. coli expression systems but this approach proved unsuccessful. In contrast, we found that large amounts of virtually pure protein could be produced efficiently in insect cells. The yield of GSHS2 protein was optimized by monitoring the amount of baculovirus used to infect the insect cells and the time course of protein production. Western blot analysis demonstrated that the protein was correctly expressed (expected size of approximately 59 kD) in infected cells (Fig. 2). The protein yield was similar between 48 and 96 h after infection but after this time there were significant amounts of smaller degradation products. Therefore, protein production was scaled up by culturing 10 insect cells in 50 ml of medium and by harvesting cells 48 h after infection. The baculovirus expression system allowed us to produce large amounts of highly pure active GSHS2 enzyme suitable for biochemical characterization. Thus, the GSHS and hGSHS activities of GSHS2 were first determined using identical standard concentrations (5 mM) of the substrates, Gly and β−Ala, respectively (Table 1). The specific activity of GSHS2 with β−Ala was 3433 nmol of hGSH produced min-1 mg-1 protein, which is approximately between 100- and 1000-fold higher than the two putative hGSHS activities reported in the leaves of other legumes (Macnicol 1987, Klapheck et al. 1988). This is consistent with the highly purified enzyme preparation that we obtained using the insect expression system. Likewise, the hGSHS/GSHS ratio of activities was 21.7, thus suggesting a higher affinity of GSHS2 for β−Ala than for Gly. The catalytic constants of GSHS2 were then determined using a fixed saturating concentration of γEC and a range of concentrations of Gly or β-Ala (see "Materials and methods"). The enzyme showed saturation kinetics and linear double-reciprocal plots with respect to both substrates. The Km of GSHS2 for β−Ala was 55-fold lower than for Gly but, perhaps most relevant in terms of substrate specificity, the Vmax/Km ratio (specificity constant) for β−Ala was 57-fold higher (Table 1). These kinetic data using virtually pure, recombinant enzyme demonstrate that GSHS2 encodes a genuine hGSHS. Very recently, Frendo et al. (2001) reported the expression, in E.coli, of a cDNA from Medicago truncatula. The enzyme product in bacterial crude extracts showed a specific activity of 0.32 nmol min-1mg-1 protein as hGSHS and of 0.12 nmol min-1mg-1 protein as GSHS. These activities were therefore about 10000- and 1300-fold, respectively, lower than those of our enzyme preparation. These extremely large differences in activities are due to the use of crude extracts instead of purified enzyme and probably also to the fact that the pea GSHS2 protein has been expressed in an eucaryotic system, which can improve the folding and processing of the enzyme. Reliable kinetic analysis 11 requires enzyme purification. Clearly, our highly purified enzyme preparation is more appropriate for kinetic studies and also allows the subsequent structural analysis of the protein. Genomic Southern blot analysis of GSHS2 was performed in pea and common bean using gene-specific probes for each legume species (Fig. 3). Bean was included in this analysis because this plant has hGSHS (but not GSHS) activity and hence a functional GSHS2 gene (Moran et al. 2000). In both legumes, restriction enzymes cutting inside (Xba I, Hind III) or outside (other enzymes) of the ORFs generated single fragments (Fig. 3). This observation, along with the high sequence identity (73%) between pea and bean GSHS2 (Moran et al. 2000), allowed us to conclude that an homologous gene to pea nodule GSHS2 is present in the bean genome, that both pea and bean GSHS2 are present as single copies, and that the pea GSHS2 enzyme is responsible for the hGSH content and hGSHS activity found in nodule extracts (Matamoros et al. 1999). Assuming that the molecular mass of native hGSHS is similar to that of GSHS (113-120 kD) of other plants (Rennenberg 1997), it follows that hGSHS is also present in the nodules as a homodimer. The derived amino acid sequence of hGSHS (GSHS2) is devoid of N-terminal signal peptides or C-terminal motifs, and the enzyme is predicted by several algorithms to be located to the cytosol (Moran et al. 2000). We conclude that thiol biosynthesis in pea nodules proceeds via two genuinely different enzymes (GSHS and hGSHS), rather than two GSHS isozymes. The enzymes are located in two nodule compartments known to generate toxic oxygen species at high rates (Becana et al. 2000). The synthesized GSH and hGSH may fulfil antioxidative and regulatory roles that are important during nodule initiation and senescence. Thus, GSH is involved in the osmotic and oxidative stress tolerance of bacteroids (Riccillo et al. 2000), and both GSH and hGSH are involved in peroxide detoxification in the plant fraction of nodules via the Halliwell-Asada 12 pathway (Moran et al. 2000, Iturbe-Ormaetxe et al. 2001). The two thiols are generally assumed to be functionally interchangeable (Klapheck 1988). While this may be correct, our compartmentation results emphasize that there is at least the potential for specific different functions of GSH and hGSH. With the availability of hGSHS cDNAs (this work) and the use of antisense technology this question may be adequately addressed in future. Acknowledgments We thank David Dalton and an anonymous reviewer for helpful comments on the manuscript. Thanks are also due to Maria R. Clemente for help with Figure 2. M.A.M. was the recipient of a predoctoral fellowship from the Basque Government (Spain). References Becana M, Dalton DA, Moran JF, Iturbe-Ormaetxe I, Matamoros MA, Rubio MC (2000) Reactive oxygen species and antioxidants in legume nodules. Physiol Plant 109: 372-381 Dalton DA, Russell SA, Hanus FJ, Pascoe GA, Evans HJ (1986) Enzymatic reactions of ascorbate and glutathione that prevent peroxide damage in soybean root nodules. Proc Natl Acad Sci USA 83: 3811-3815 Fahey RC, Newton GL (1987) Determination of low-molecular-weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography. Methods Enzymol 143: 85-96 Frendo P, Gallesi D, Turnbull R, Van de Sype G, Hérouart D, Puppo A (1999) Localisation of glutathione and homoglutathione in Medicago truncatula is correlated to a differential expression of genes involved in their synthesis. 13 Plant J 17: 215-219 Frendo P, Hernández MJ, Mathieu C, Duret L, Gallesi D, Van de Sype G, Hérouart D, Puppo A (2001) A Medicago truncatula homoglutathione synthetase is derived from glutathione synthetase by gene duplication. Plant Physiol 126: 1706-1715 Gogorcena Y, Gordon AJ, Escuredo PR, Minchin FR, Witty JF, Moran JF, Becana M (1997) N2 fixation, carbon metabolism, and oxidative damage in nodules of dark-stressed common bean plants. Plant Physiol 113: 11931201 Iturbe-Ormaetxe I, Matamoros MA, Rubio MC, Dalton DA, Becana M (2001) The antioxidants of legume nodule mitochondria. Mol Plant-Microb Interact 14: 1189-1196 Klapheck S (1988) Homoglutathione: isolation, quantification, and occurrence in legumes. Physiol Plant 74: 727-732 Klapheck S, Zopes H, Levels HG, Bergmann L (1988) Properties and localization of the homoglutathione synthetase from Phaseolus coccineus leaves. Physiol Plant 74: 733-739 Klapheck S, Chrost B, Starke J, Zimmermann H (1992) γ−Glutamylcysteinylserine, a new homologue of glutathione in plants of the family Poaceae. Bot Acta 105: 174-179 Macnicol PK (1987) Homoglutathione and glutathione synthetases of legume seedlings: partial purification and substrate specificity. Plant Sci 53: 229-235 Matamoros MA, Moran JF, Iturbe-Ormaetxe I, Rubio MC, Becana M (1999) Glutathione and homoglutathione synthesis in legume root nodules. Plant Physiol 121: 879-888 Meuwly P, Thibault P, Schwan AL, Rauser WE (1995) Three families of thiol peptides are induced by cadmium in maize. Plant J 7: 391-400 Moran JF, Iturbe-Ormaetxe I, Matamoros MA, Rubio MC, Clemente MR, Brewin NJ, Becana M (2000) Glutathione and homoglutathione synthetases of 14 legume nodules. Cloning, expression, and subcellular localization. Plant Physiol 124: 1381-1392 Rennenberg H (1997) Molecular approaches to glutathione biosynthesis. In: Cram WJ, DeKok LJ, Stulem I, Brunold C, Rennenberg H (eds) Sulphur Metabolism in Higher Plants. Backhuys Publishers, Leiden, The Netherlands, pp 59-70 Riccillo PM, Muglia CI, de Bruijn FJ, Roe AJ, Booth IR, Aguilar OM (2000) Glutathione is involved in environmental stress responses in Rhizobium tropici, including acid tolerance. J Bacteriol 182: 1748-1753 Edited by J. I. Sprent 15 Legends for Figures Fig. 1. Proposed pathway for the synthesis of thiol tripeptides in plants. The synthesis of GSH proceeds through two steps catalyzed by γECS and GSHS. The synthesis of hGSH, a GSH homolog found exclusively in legumes, is thought to proceed through the same γECS enzyme and then by either a specific hGSHS or by a GSHS isozyme with broad substrate specificity. Hydroxymethyl-glutathione, found in cereals such as wheat and rice, could be synthesized by addition of a Ser residue to the C-terminus of γEC or by hydroxymethylation of the C-terminal Gly of GSH (KIapheck et al. 1992), and γGlu-Cys-Glu, detected in maize seedlings exposed to Cd, is thought to be synthesized from γEC (Meuwly et al. 1995). Fig. 2. Overproduction in insect cells and purification of pea nodule GSHS2. (A) Red Pounceau-stained SDS-gel of proteins from control (uninfected) and infected cells after 24, 48, 72, and 96 h. (B) Western blot of the same gel using 6xHis monoclonal antibody. (C) Coomassie-stained SDS-gel of proteins from cell free extracts prior to loading on the metal-affinity column (fraction 0) or subsequently eluted with 1 ml of imidazole elution buffer per fraction (fractions 1 and 2). (D) Western blot of a gel similar to (C) using the same antibody and conditions as in (B). Fig. 3. Southern blot analysis of GSHS2 in pea and bean. Genomic DNA was extracted from leaves, digested with restriction enzymes, electrophoresed (10 µg of pea DNA per lane or 5 µg of bean DNA per lane), blotted onto Hybond N+ membranes, and hybridized with 32P-labeled probes. Fig. 1 + Ala Glu-Cys-Ala (hGSH) hGSHS + Gly Glu-Cys-Gly (GSH) GSHS L-Glu + L-Cys ECS Glu-Cys (EC) ? + Ser ? + Glu ? Glu-Cys-Ser (hydroxymethylglutathione) Glu-Cys-Glu Fig. 2