Differential Inhibition of Arabidopsis Methionine Adenosyltransferases by Protein S-Nitrosylation
2005; Elsevier BV; Volume: 281; Issue: 7 Linguagem: Inglês
10.1074/jbc.m511635200
ISSN1083-351X
AutoresChristian Lindermayr, Gerhard Saalbach, Günther Bahnweg, Jörg Durner,
Tópico(s)Redox biology and oxidative stress
ResumoIn animals, protein S-nitrosylation, the covalent attachment of NO to the thiol group of cysteine residues, is an intensively investigated posttranslational modification, which regulates many different processes. A growing body of evidence suggests that this type of redox-based regulation mechanism plays a pivotal role in plants, too. Here we report the molecular mechanism for S-nitrosylation of methionine adenosyltransferase (MAT) of Arabidopsis thaliana, thereby presenting the first detailed characterization of S-nitrosylation in plants. We cloned three MAT isoforms of Arabidopsis and tested the effect of NO on the activity of the purified, recombinant proteins. Our data showed that incubation with GSNO resulted in blunt, reversible inhibition of MAT1, whereas MAT2 and MAT3 were not significantly affected. Cys-114 of MAT1 was identified as the most promising target of NO-induced inhibition of MAT1, because this residue is absent in MAT2 and MAT3. Structural analysis of MAT1 revealed that Cys-114 is located nearby the putative substrate binding site of this enzyme. Furthermore, Cys-114 is flanked by S-nitrosylation-promoting amino acids. The inhibitory effect of GSNO was drastically reduced when Cys-114 of MAT1 was replaced by arginine, and mass spectrometric analyses of Cys-114-containing peptides obtained after chymotryptic digestion demonstrated that Cys-114 of MAT1 is indeed S-nitrosylated. Because MAT catalyzes the synthesis of the ethylene precursor S-adenosylmethionine and NO is known to influence ethylene production in plants, this enzyme probably mediates the cross-talk between ethylene and NO signaling. In animals, protein S-nitrosylation, the covalent attachment of NO to the thiol group of cysteine residues, is an intensively investigated posttranslational modification, which regulates many different processes. A growing body of evidence suggests that this type of redox-based regulation mechanism plays a pivotal role in plants, too. Here we report the molecular mechanism for S-nitrosylation of methionine adenosyltransferase (MAT) of Arabidopsis thaliana, thereby presenting the first detailed characterization of S-nitrosylation in plants. We cloned three MAT isoforms of Arabidopsis and tested the effect of NO on the activity of the purified, recombinant proteins. Our data showed that incubation with GSNO resulted in blunt, reversible inhibition of MAT1, whereas MAT2 and MAT3 were not significantly affected. Cys-114 of MAT1 was identified as the most promising target of NO-induced inhibition of MAT1, because this residue is absent in MAT2 and MAT3. Structural analysis of MAT1 revealed that Cys-114 is located nearby the putative substrate binding site of this enzyme. Furthermore, Cys-114 is flanked by S-nitrosylation-promoting amino acids. The inhibitory effect of GSNO was drastically reduced when Cys-114 of MAT1 was replaced by arginine, and mass spectrometric analyses of Cys-114-containing peptides obtained after chymotryptic digestion demonstrated that Cys-114 of MAT1 is indeed S-nitrosylated. Because MAT catalyzes the synthesis of the ethylene precursor S-adenosylmethionine and NO is known to influence ethylene production in plants, this enzyme probably mediates the cross-talk between ethylene and NO signaling. NO is a lipophilic, highly reactive gaseous molecule that plays important roles in regulation of stomatal closure (1.Neill S.J. Desikan R. Clarke A. Hancock J.T. Plant Physiol. 2002; 128: 13-16Crossref PubMed Scopus (445) Google Scholar), programmed cell death (2.Beligni M.V. Fath A. Bethke P.C. Lamattina L. Jones R.L. Plant Physiol. 2002; 129: 1642-1650Crossref PubMed Scopus (304) Google Scholar), abiotic stress (3.Garcia-Mata C. Lamattina L. 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Okamoto M. Crawford M.J. Science. 2003; 302: 100-103Crossref PubMed Scopus (707) Google Scholar, 9.Zeidler D. Zähringer U. Gerber I. Dubery I. Hartung T. Bors W. Hutzler P. Durner J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15811-15816Crossref PubMed Scopus (479) Google Scholar). Other sources of endogenous NO are nitrate reductase (10.Yamasaki H. Sakihama Y. FEBS Lett. 2000; 468: 89-92Crossref PubMed Scopus (419) Google Scholar, 11.Rockel P. Strube F. Rockel A. Wildt J. Kaiser W.M. J. Exp. Bot. 2002; 53: 103-110Crossref PubMed Google Scholar) and nitrite:NO reductase (12.Stöhr C. Strube F. Marx G. Ullrich W.R. Rockel P. Planta. 2001; 212: 835-841Crossref PubMed Scopus (234) Google Scholar). Next to this enzymatic sources, non-enzymatic production of NO via reduction of apoplastic nitrite is also described (13.Bethke P.C. Badger M.R. Jones R.L. Plant Cell. 2004; 16: 332-341Crossref PubMed Scopus (387) Google Scholar).Despite the multiplex importance of NO, less is known about the targets of this redox molecule in plant metabolism. Many of the biological functions of NO arise as a direct consequence of chemical reactions between proteins and NO or NO oxides generated as NO/O2 or NO/superoxide reaction products. Different types of NO-dependent protein modification are described. NO is a precursor of the reactive nitrating species, peroxynitrite and nitrogen dioxide, which modify proteins to generate 3-nitrotyrosine as it is shown for the tyrosine residues 161 and 357 of α-tubulin (14.Tedeschi G. Cappelletti G. Negri A. Pagliato L. Maggioni M.G. Maci R. Ronchi S. Proteomics. 2005; 5: 2422-2432Crossref PubMed Scopus (45) Google Scholar). Protein nitration is an irreversible reaction, which is of importance for pathophysiological but probably not for signaling. Furthermore, NO can bind to metal ions of heme groups as it is reported for the activation of guanylate cyclase (15.Russwurm M. Koesling D. EMBO J. 2004; 23: 4443-4450Crossref PubMed Scopus (165) Google Scholar, 16.Brandish P.E. Buechler W. Marletta M.A. Biochemistry. 1998; 37: 16898-16907Crossref PubMed Scopus (86) Google Scholar) or it can form dinitrosyl complexes together with iron ions and low molecular weight thiols. Latter reactions seem to be important for iron uptake, trafficking, storage, and delivery in plant mesophyll cells (17.Graziano M. Beligni M.V. Lamattina L. Plant Physiol. 2002; 130: 1852-1859Crossref PubMed Scopus (209) Google Scholar, 18.Graziano M. Lamattina L. Trends Plant Sci. 2005; 10: 4-8Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Finally, NO can react with the thiol group of cysteine residues to form S-nitrosothiols (S-nitrosylation) as it is described e.g. for mammalian methionine adenosyltransferase (19.Perez-Mato I. Castro C. Ruiz F.A. Corrales F.J. Mato J.M. J. Biol. Chem. 1999; 274: 17075-17079Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and ryanodine receptor/Ca2+ channel (20.Eu J.P. Sun J. Xu L. Stamler J.S. Meissner G. Cell. 2000; 102: 499-509Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). Because of their reactivity with intracellular reducing agents like ascorbic acid or glutathione, and with reduced metal ions, especially Cu+, the half-lives of S-nitrosothiols are very short (from seconds to a few minutes). This lability makes protein S-nitrosylation a very sensitive regulation mechanism, and there are many reports about the importance of this cysteine-dependent redox-based mechanism for controlling different cellular processes in animals (21.Mannick J.B. Schonhoff C.M. Free Radic. Res. 2004; 38: 1-7Crossref PubMed Scopus (41) Google Scholar, 22.Martinez-Ruiz A. Lamas S. Cardiovasc. Res. 2004; 62: 43-52Crossref PubMed Scopus (216) Google Scholar, 23.Hess D.T. Matsumoto A. Kim S.O. Marshall H.E. Stamler J.S. Nat. Rev. Mol. Cell. Biol. 2005; 6: 150-166Crossref PubMed Scopus (1706) Google Scholar, 24.Gaston B.M. Carver J. Doctor A. Palmer L.A. Mol. Interv. 2003; 3: 253-263Crossref PubMed Scopus (167) Google Scholar).Because most proteins possess cysteine residues, substrate specificity is a very important feature of endogenous protein S-nitrosylation. This includes structural factors that influence the susceptibility to S-nitrosylation like surrounding acidic or basic amino acids and the presence of a hydrophobic environment that enables the formation of S-nitrosylating species via the reaction between oxygen and NO (25.Stamler J.S. Toone E.J. Lipton S.A. Sucher N.J. Neuron. 1997; 18: 691-696Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar). Additionally, trans-S-nitrosylation from the in vivo NO donor nitrosoglutathione (GSNO) 2The abbreviations used are: GSNO, nitrosoglutathione; GST, glutathione S-transferase; MS, mass spectrometry; MAT, methionine adenosyltransferase; NEM, N-ethylmaleimide; DTNB, 5,5′-dithiobis-2-nitrobenzoic acid; DTT, dithiothreitol; AdoMet, S-adenosylmethionine; ACC, 1-aminocyclopropane-1-carboxlic acid.2The abbreviations used are: GSNO, nitrosoglutathione; GST, glutathione S-transferase; MS, mass spectrometry; MAT, methionine adenosyltransferase; NEM, N-ethylmaleimide; DTNB, 5,5′-dithiobis-2-nitrobenzoic acid; DTT, dithiothreitol; AdoMet, S-adenosylmethionine; ACC, 1-aminocyclopropane-1-carboxlic acid. to proteins seemed to be promoted by acidic, basic, and hydrophobic side chains neighboring the target cysteine residue (23.Hess D.T. Matsumoto A. Kim S.O. Marshall H.E. Stamler J.S. Nat. Rev. Mol. Cell. Biol. 2005; 6: 150-166Crossref PubMed Scopus (1706) Google Scholar).Until now more than 100 proteins were reported as targets for protein S-nitrosylation in animals. In plants there is experimental evidence for only three plant proteins to be regulated by S-nitrosylation. Arabidopsis hemoglobin 1 can scavenge NO through the formation of S-nitrosohemoglobin and in vitro experiments suggested that glyceraldehyde 3-phosphate dehydrogenase and the K+-channel in guard cells are regulated by NO via S-nitrosylation (26.Perazzolli M. Dominici P. Romero-Puertas M.C. Zago E. Zeier J. Sonoda M. Lamb C. Delledonne M. Plant Cell. 2004; 16: 2785-2794Crossref PubMed Scopus (279) Google Scholar, 27.Sokolovski S. Blatt M.R. Plant Physiol. 2004; 136: 4275-4284Crossref PubMed Scopus (107) Google Scholar, 28.Lindermayr C. Saalbach G. Durner J. Plant Physiol. 2005; 137: 921-930Crossref PubMed Scopus (588) Google Scholar). Recently, we identified 63 proteins from Arabidopsis cell cultures and 52 proteins from Arabidopsis leaves representing candidates for protein S-nitrosylation including stress-related, redox-related, signaling/regulating, cytoskeleton, and metabolic proteins (28.Lindermayr C. Saalbach G. Durner J. Plant Physiol. 2005; 137: 921-930Crossref PubMed Scopus (588) Google Scholar). The latter group contains enzymes of the methylmethionine cycle: cobalamin-independent methionine synthase, S-adenosylhomocysteinase and S-methionine adenosyltransferase (MAT) (see Fig. 1). MAT catalyzes the biosynthesis of S-adenosylmethionine (AdoMet), which is the most important methyl donor in transmethylation reactions and a substrate for the biosynthesis of polyamines and the plant hormone ethylene. Because of this important function of AdoMet it is not surprising that most species studied today have more than one MAT isoform (29.Sanchez-Perez G.F. Bautista J.M. Pajares M.A. J. Mol. Biol. 2004; 335: 693-706Crossref PubMed Scopus (46) Google Scholar). In mammals, two genes (MAT1A and MAT2A) encode different MAT isoenzymes that show an organ-specific expression pattern. MAT1A is expressed in the liver only, whereas MAT2A is expressed in all other tissues (30.Mato J.M. Alvarez L. Ortiz P. Mingorance J. Duran C. Pajares M.A. Adv. Exp. Med. Biol. 1994; 368: 113-117Crossref PubMed Scopus (23) Google Scholar). However, the most interesting difference between these two isoenzymes is that MAT1A is reversibly inactivated by NO, whereas MAT2A is not. Responsible for the inactivation is the S-nitrosylation of the cysteine residue 121 of MAT1A, which is located within a flexible loop that can gate access to the active center (19.Perez-Mato I. Castro C. Ruiz F.A. Corrales F.J. Mato J.M. J. Biol. Chem. 1999; 274: 17075-17079Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 31.Avila M.A. Mingorance J. Martinez-Chantar M.L. Casado M. Martin-Sanz P. Bosca L. Mato J.M. Hepatology. 1997; 25: 391-396PubMed Google Scholar, 32.Corrales F.J. Perez-Mato I. Sanchez Del Pino M.M. Ruiz F. Castro C. Garcia-Trevijano E.R. Latasa U. Martinez-Chantar M.L. Martinez-Cruz A. Avila M.A. Mato J.M. J. Nutr. 2002; 132: 2377-2381Crossref PubMed Google Scholar, 33.Castro C. Ruiz F.A. Perez-Mato I. Sanchez del Pino M.M. LeGros L. Geller A.M. Kotb M. Corrales F.J. Mato J.M. FEBS Lett. 1999; 459: 319-322Crossref PubMed Scopus (12) Google Scholar, 34.Takusagawa F. Kamitori S. Markham G.D. Biochemistry. 1996; 35: 2586-2596Crossref PubMed Scopus (104) Google Scholar, 35.Takusagawa F. Kamitori S. Misaki S. Markham G.D. J. Biol. Chem. 1996; 271: 136-147Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 36.Taylor J.C. Markham G.D. Arch. Biochem. Biophys. 2003; 415: 164-171Crossref PubMed Scopus (18) Google Scholar, 37.Taylor J.C. Takusagawa F. Markham G.D. Biochemistry. 2002; 41: 9358-9369Crossref PubMed Scopus (26) Google Scholar). Moreover, it has been shown that the inducible NO synthase controls the activity of liver MAT (31.Avila M.A. Mingorance J. Martinez-Chantar M.L. Casado M. Martin-Sanz P. Bosca L. Mato J.M. Hepatology. 1997; 25: 391-396PubMed Google Scholar). Thus, NO regulates the synthesis of AdoMet and as consequence the synthesis of the metabolites AdoMet is necessary for.In this paper, we report the differential inhibition of MAT isoenzymes of Arabidopsis by the NO donor GSNO. We isolated the coding sequences of three MAT isoforms and produced the corresponding enzymes as GST-fusion proteins in Escherichia coli. Activity assays with recombinant GST-MAT fusion proteins pretreated with GSNO demonstrated that the activity of isoenzyme MAT1 is reduced to 30%, whereas the other two isoforms are only weakly affected by this treatment. Site-directed mutagenesis and mass spectrometric analysis showed that S-nitrosylation of the cysteine residue 114 of MAT1 is responsible for the inhibition of the enzymatic activity. Thus, MAT1 represents the first metabolic plant enzyme that is regulated by S-nitrosylation of a redox-sensitive cysteine residue. Furthermore, our results led us to speculate about the involvement of MAT in the regulation of ethylene biosynthesis.EXPERIMENTAL PROCEDURESStructural Analysis—Amino acid sequences were aligned and modeled using SWISS-Model (www.expasy.ch). The crystal structure of E. coli methionine adenosyltransferase (Protein Data Bank code 1RG9, chains A and B) was used as template for the prediction of the putative conformations of MAT1, MAT2, and MAT3.Isolation of Coding Sequences of MAT Isoforms—For cloning the cDNAs of the different MAT isoforms the λ phage-based site-specific recombination (Stratagene) was used (38.Landy A. Annu. Rev. Biochem. 1989; 58: 913-949Crossref PubMed Google Scholar). Briefly, RNA from Arabidopsis leaves and stems was used for reverse transcription-PCR using PfuTurbo DNA polymerase, gene-specific primers, and the following PCR conditions: 2 min at 94 °C, 35 cycles consisting of 30 s at 94 °C, 30 s at 57 °C for amplification of MAT1 and MAT3 cDNA, and 30 s at 54 °C for amplification of MAT2 cDNA, and 2.5 min at 72 °C, followed by a final extension step of 10 min at 72 °C. The introduction of the DNA recombination sequence (att) at the 5′- and 3′-end of the coding sequence of each isoform was achieved by PCR using the isoform-specific att-primers and the amplified cDNAs as template. The resulting PCR products were introduced into pDONR221 by recombination using BP Clonase enzyme mixture according to the instructions of the manufacturer. After verifying the sequences of the different MAT isoforms by sequencing they were transferred into the expression vector pDEST15 by recombination using LP Clonase enzyme mixture.Expression in E. coli and Purification of Recombinant Proteins—E. coli strain BL21 DE3 pLysS harboring the plasmids pDEST15-MAT1, pDEST15-MAT2, pDEST15-MAT3, or pDEST15-MAT1/C114R were grown in Luria-Bertani medium until A600 ∼0.5 was reached. Production of recombinant GST fusion proteins was induced with 1 mm isopropyl-β-d-thiogalactopyranoside. After incubation for 4 h at 37°C bacterial cells were harvested by centrifugation. For protein isolation the cells were resuspended in an appropriate volume of buffer (100 mm Tris-HCl, pH 7.5, 2 mm EDTA, 20% (v/v) glycerol, 20 mm β-mercaptoethanol, 1 mm dithiothreitol (DTT)) and disrupted by sonication. Cellular debris was removed by centrifugation (20,000 × g, 20 min, 4°C). The recombinant GST fusion proteins were purified by affinity chromatography using glutathione-Sepharose 4B (Amersham Biosciences). Adsorbed proteins were eluted from the matrix with 100 mm Tris-HCl, pH 8.0, containing 20 mm glutathione. After adding 20% (v/v) glycerol to the eluates they were frozen in liquid nitrogen and stored at -20 °C until analysis.MAT Activity Assay—Purified enzymes were activated with 10 mm DTT (20 min, 4 °C), and residual DTT was removed using Micro Biospin 6 columns (Bio-Rad). The enzyme activity was assayed as described by (39.Shen B. Li C. Tarczynski M.C. Plant J. 2002; 29: 371-380Crossref PubMed Scopus (143) Google Scholar) with minor modifications. Briefly, 50 μl of purified enzyme (∼15 μg of protein) were added to 150 ml of a reaction mixture containing 100 mm Tris-HCl, pH 8.0, 30 mm MgSO4, 10 mm KCl, 10 mm ATP, and 5 mm [35S]Met (15 μCi). Control reactions contained all agents except for ATP. Reactions were incubated for 1–2 h at 35 °C and were terminated by placement on ice-water. Fifty microliters of the reaction mixture were then spotted onto chromatography paper P81 (2 cm × 2 cm) in duplicate or triplicate. The papers were air-dried and washed two times with ice-water for 5 min. The washed chromatography papers were transferred to scintillation vials containing 1 ml of 1.5 mm ammonium hydroxide. After 5 min, scintillation liquid was added, and the samples were counted by scintillation spectrometry.Site-directed Mutagenesis—The modification of single nucleotide residues was performed as previously described (40.Lindermayr C. Fliegmann J. Ebel J. J. Biol. Chem. 2003; 278: 2781-2786Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Briefly, for mutation, a pair of oligonucleotides was synthesized harboring the desired alterations. The size of the primers was adjusted to yield a melting temperature of 68 °C by using the following formula: Tm = 81.5 + 0.41 × GC (%) - 675/number of bases - sequence deviation (%). For amplification, 20 ng of plasmid DNA was used in a total volume of 15 μl, including 1 μm each primer, 200 μm dNTPs, and 1 unit of PfuTurbo DNA polymerase. After denaturation (2 min at 94 °C) 18 cycles were conducted, consisting of 45 s at 94 °C, 30 s at 55 °C, and 15 min at 72 °C, followed by a final extension step at 72 °C for 10 min. Subsequently, the parental and hemi-parental template DNA was digested with DpnI, and the amplified plasmids were transformed into E. coli DH5α. The mutation was verified by sequencing.Mass Spectrometric Analyses—Purified recombinant MAT1 protein was dissolved in 0.1 m NH4HCO3 at 0.5 μg/μl. Aliquots of 10–20 μg of were treated with 500 ng of chymotrypsin at room temperature for 3 h. The digest was then diluted 1:8 into 50% (v/v) methanol/5% (v/v) formic acid. For analysis by mass spectrometry, this solution was applied to a Q-TOF Ultima™ Global (Micromass, Manchester, UK) by electrospray ionization using the nanospray kit coupled to a 100-μl Hamilton syringe. The mass spectrometer was calibrated using MS/MS fragments of GluFib (Sigma). Full mass scans of 1 s were recorded at a collision energy of 10 V, loss of the NO group was achieved at 20–25 V, and MS/MS fragmentation was achieved at 30 V. A number of scans recorded over time were combined and smoothed (Savitzky-Golay, 3/2).Determination of Ethylene—Seven-day-old Arabidopsis cell cultures were portioned in 10-ml fractions in 25-ml Erlenmeyer flasks under sterile conditions. The following day the cell cultures were treated with water (control) or different concentrations of GSNO and incubated in dark at 26 °C (126 rpm). To measure the ethylene production of the cells the flasks were air-tight and after 30 min 1 ml of gas was collected from the gas phase of the cultures using a syringe. The produced ethylene was measured with a PerkinElmer Autosystem XL gas chromatograph equipped with a Porapak Q column (Supelco) and a flame ionization detector as described previously (41.Tuomainen J. Betz C. Kangasjarvi J. Ernst D. Yin Z.H. Langebartels C. Sandermann H. Plant J. 1997; 12: 1151-1162Crossref Scopus (111) Google Scholar).RESULTSIn a recent publication (28.Lindermayr C. Saalbach G. Durner J. Plant Physiol. 2005; 137: 921-930Crossref PubMed Scopus (588) Google Scholar), we identified more than 100 proteins as S-nitrosylation targets, among them MAT, which is responsible for supplying AdoMet, e.g. for ethylene biosynthesis. Interestingly, NO inhibits ethylene production in plants (42.Leshem Y. Haramaty E. J. Plant Physiol. 1996; 148: 258-263Crossref Scopus (289) Google Scholar, 43.Leshem Y.Y. Wills R.B.H. Ku V.V. Plant Physiol. Biochem. 1998; 36: 825-826Crossref Scopus (365) Google Scholar). We treated Arabidopsis cell cultures with different concentrations of the NO donor GSNO and determined their ethylene emission (Fig. 2). With 0.5 mm GSNO we detected a maximal inhibition of the ethylene production of 20% after a 3-h incubation. Longer incubation times reduced the inhibition of ethylene emission to 10% (6 and 24 h). Treatment with 1 mm GSNO decreased ethylene production to 55% after 6 h of treatment, and the production is restored with longer incubation times (24 h).FIGURE 2Ethylene emission of Arabidopsis cell cultures after treatment with GSNO. Arabidopsis cell cultures were treated with 0.5 mm (light gray) and 1 mm GSNO (dark gray). The cell cultures were air-tight and after the indicated times the produced ethylene was collected. After 30 min the amount of produced ethylene was determined as described previously (41.Tuomainen J. Betz C. Kangasjarvi J. Ernst D. Yin Z.H. Langebartels C. Sandermann H. Plant J. 1997; 12: 1151-1162Crossref Scopus (111) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)A possible mechanism for NO-dependent decrease of ethylene production could be the S-nitrosylation of MAT. Searching the NCBI nucleotide data base we found four different MAT-encoding sequences in Arabidopsis. The deduced amino acid sequences of the different isoenzymes show high homology among each other (89.3–96.4% amino acid sequence identity) and possess seven highly conserved cysteines (Fig. 3). MAT1 and MAT3 have one additional cysteine at positions 114 and 302, respectively.FIGURE 3Alignment of the amino acid sequences of Arabidopsis MAT isoforms. GenBank™ accession numbers are as follows: MAT1 (NP_849577), MAT2 (AAA32869), MAT3 (AAD31573), MAT4 (AAO11581). Highly conserved cysteine residues are bordered, and Cys-114 of MAT1 is marked with an arrow. Amino acids forming the active site loop acids are highlighted with gray. Dots mark amino acids identical to the sequence of MAT1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To investigate whether the activity of the Arabidopsis MAT isoenzymes is regulated by NO we first had to isolate the coding sequence of the Arabidopsis proteins. Because the localization of the cysteines of MAT2 and MAT4 are identical we concentrated our investigations on the isoforms 1, 2, and 3. The isolation of the cDNAs of these three MAT isoforms was achieved by reverse transcription-PCR using gene-specific oligonucleotides, and the amplified coding sequences were expressed in E. coli as inducible fusion proteins containing N-terminal glutathione S-transferase. After affinity chromatography on glutathione-Sepharose 4B, the three fusion proteins showed the expected relative molecular masses of 68 kDa in SDS-polyacrylamide gels.The effect of NO on the activity of the different MAT isoforms was tested by incubating the purified enzymes with 1 mm GSNO and determining their activities afterward. The activity of MAT1 was reduced to ∼30%, whereas neither the activity of MAT2 nor of MAT3 was significantly affected (Fig. 4A). The activity of the inhibited MAT1 could completely be restored by adding 10 mm DTT. Incubation of MAT1 with different doses of GSNO showed that a concentration of 10 μm already reduced the enzyme activity to 50% (Fig. 4B).FIGURE 4Differential inhibition of Arabidopsis MAT isoforms by GSNO. A, recombinant MAT isoenzymes were produced in E. coli and purified as described under "Experimental Procedures." Purified enzymes were incubated in the absence (black) or presence (light gray) of 1 mm GSNO for 20 min at room temperature before enzyme activity was measured. For restoring MAT activity, 10 mm DTT was added to extracts with inhibited enzymes (dark gray). The activity of untreated enzymes was set to 100%. Values are the mean ± S.D. of at least four different measurements. B, dose-dependent inhibition of Arabidopsis MAT1 was analyzed by incubating purified, recombinant MAT1 with different concentrations of GSNO for 20 min at room temperature before measuring the enzyme activity. Values are the mean of at least three different experiments. C, SH-modifying agents DTNB and NEM reduce activity of Arabidopsis MAT isoforms. Recombinant enzymes purified from E. coli were incubated with 250 mm DTNB (light gray) or 500 mm NEM (dark gray) for 20 min at room temperature. Afterward, the activity of the enzymes were measured. Control treatment was done with water (black). The activity of the controls was set to 100%. Values represent means of two independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further characterize the mechanism of GSNO-mediated MAT inhibition, we tested the influence of the cysteine residue-modifying agents N-ethylmaleimide (NEM) and 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) on the activity of the different MAT isoforms. NEM is a highly reactive agent that covalently and irreversibly alkylates free cysteine thiol groups. DTNB is an oxidizing reagent that acts by formation of a disulfide bond between itself and free cysteine thiol groups resulting in a dithiobenzoate complex with proteins. As shown in Fig. 4C exposure to 250 μm DTNB led to complete inactivation of MAT1, whereas MAT2 and MAT3 activity was reduced to 40 and 35%, respectively. We observed a similar result after treatment with 500 μm NEM. Although MAT1 was totally inhibited, MAT2 and MAT3 showed a residual activity of 40 and 20%, respectively (Fig. 4C).E. coli MAT has been crystallized in a ternary complex with the S-adenosylmethionine and imidotriphosphate (44.Komoto J. Yamada T. Takata Y. Markham G.D. Takusagawa F. Biochemistry. 2004; 43: 1821-1831Crossref PubMed Scopus (71) Google Scholar). Because this enzyme shares 54.4–55.4% identical amino acid residues with the Arabidopsis MAT isoforms, it was possible to use the structure of E. coli MAT as a template to model the hypothetical three-dimensional conformation of the Arabidopsis MAT isoenzymes. Actually, MAT is a tetramer of two asymmetric dimers. Each dimer has two-substrate binding site, which are located between the interface of the monomers and are gated by an flexible loop (34.Takusagawa F. Kamitori S. Markham G.D. Biochemistry. 1996; 35: 2586-2596Crossref PubMed Scopus (104) Google Scholar, 35.Takusagawa F. Kamitori S. Misaki S. Markham G.D. J. Biol. Chem. 1996; 271: 136-147Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 36.Taylor J.C. Markham G.D. Arch. Biochem. Biophys. 2003; 415: 164-171Crossref PubMed Scopus (18) Google Scholar, 37.Taylor J.C. Takusagawa F. Markham G.D. Biochemistry. 2002; 41: 9358-9369Crossref PubMed Scopus (26) Google Scholar). The structural models gave us insight into the spatial disposition of the cysteine residues of the MAT isoforms to identify cysteine residues representing possible candidates for S-nitrosylation. All of the seven highly conserved cysteine residues are present as free amino acids, and therefore all are possible targets for NO. More interestingly, the additional cysteine residue of MAT1 (Cys-114, Fig. 3) is located directly next to the putative catalytic center as part of the active
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