Functional and Structural Aspects of Poplar Cytosolic and Plastidial Type A Methionine Sulfoxide Reductases
2006; Elsevier BV; Volume: 282; Issue: 5 Linguagem: Inglês
10.1074/jbc.m605007200
ISSN1083-351X
AutoresNicolas Rouhier, Brice Kauffmann, F. Favier, Pasquale Palladino, Pierre Gans, Guy Branlant, Jean‐Pierre Jacquot, Sandrine Boschi‐Müller,
Tópico(s)Sulfur Compounds in Biology
ResumoThe genome of Populus trichocarpa contains five methionine sulfoxide reductase A genes. Here, both cytosolic (cMsrA) and plastidial (pMsrA) poplar MsrAs were analyzed. The two recombinant enzymes are active in the reduction of methionine sulfoxide with either dithiothreitol or poplar thioredoxin as a reductant. In both enzymes, five cysteines, at positions 46, 81, 100, 196, and 202, are conserved. Biochemical and enzymatic analyses of the cysteine-mutated MsrAs support a catalytic mechanism involving three cysteines at positions 46, 196, and 202. Cys46 is the catalytic cysteine, and the two C-terminal cysteines, Cys196 and Cys202, are implicated in the thioredoxin-dependent recycling mechanism. Inspection of the pMsrA x-ray three-dimensional structure, which has been determined in this study, strongly suggests that contrary to bacterial and Bos taurus MsrAs, which also contain three essential Cys, the last C-terminal Cys202, but not Cys196, is the first recycling cysteine that forms a disulfide bond with the catalytic Cys46. Then Cys202 forms a disulfide bond with the second recycling cysteine Cys196 that is preferentially reduced by thioredoxin. In agreement with this assumption, Cys202 is located closer to Cys46 compared with Cys196 and is included in a 202CYG204 signature specific for most plant MsrAs. The tyrosine residue corresponds to the one described to be involved in substrate binding in bacterial and B. taurus MsrAs. In these MsrAs, the tyrosine residue belongs to a similar signature as found in plant MsrAs but with the first C-terminal cysteine instead of the last C-terminal cysteine. The genome of Populus trichocarpa contains five methionine sulfoxide reductase A genes. Here, both cytosolic (cMsrA) and plastidial (pMsrA) poplar MsrAs were analyzed. The two recombinant enzymes are active in the reduction of methionine sulfoxide with either dithiothreitol or poplar thioredoxin as a reductant. In both enzymes, five cysteines, at positions 46, 81, 100, 196, and 202, are conserved. Biochemical and enzymatic analyses of the cysteine-mutated MsrAs support a catalytic mechanism involving three cysteines at positions 46, 196, and 202. Cys46 is the catalytic cysteine, and the two C-terminal cysteines, Cys196 and Cys202, are implicated in the thioredoxin-dependent recycling mechanism. Inspection of the pMsrA x-ray three-dimensional structure, which has been determined in this study, strongly suggests that contrary to bacterial and Bos taurus MsrAs, which also contain three essential Cys, the last C-terminal Cys202, but not Cys196, is the first recycling cysteine that forms a disulfide bond with the catalytic Cys46. Then Cys202 forms a disulfide bond with the second recycling cysteine Cys196 that is preferentially reduced by thioredoxin. In agreement with this assumption, Cys202 is located closer to Cys46 compared with Cys196 and is included in a 202CYG204 signature specific for most plant MsrAs. The tyrosine residue corresponds to the one described to be involved in substrate binding in bacterial and B. taurus MsrAs. In these MsrAs, the tyrosine residue belongs to a similar signature as found in plant MsrAs but with the first C-terminal cysteine instead of the last C-terminal cysteine. The production and accumulation of reactive oxygen and nitrogen intermediates, inherent to metabolic processes such as respiration or photosynthesis or to stress conditions, initiate oxidative reactions that affect the biochemical constituents of the cells (1Imlay J.A. Linn S. Science. 1986; 240: 1302-1309Crossref Scopus (1670) Google Scholar, 2Berlett B. Stadman E. J. Biol. Chem. 1997; 272: 20313-20316Abstract Full Text Full Text PDF PubMed Scopus (2809) Google Scholar). Living organisms use different strategies to prevent oxidative damage and lethal effects that would result from these compounds. Reactive species are trapped and degraded, or modifications that occur anyway are reversed by repair systems, and finally nonrepaired macromolecules can be degraded and removed. Methionine residues of proteins were shown to be one of the preferred targets of oxidation with the formation of methionine sulfoxide (MetSO) 3The abbreviations used are: MetSO, methionine sulfoxide; Msr, methionine sulfoxide reductase; DTT, dithiothreitol; Trx, thioredoxin; TNB-, thionitrobenzoate. (3Vogt W. Free Radic. Biol. Med. 1995; 18: 93-105Crossref PubMed Scopus (793) Google Scholar). Enzymes named methionine sulfoxide reductases were found to catalyze the reduction of MetSO back to methionine residues (4Brot N. Weissbach L. Werth J. Weissbach H. Biochemistry. 1981; 78: 2155-2158Google Scholar, 5Grimaud R. Ezraty B. Mitchell J.K. Lafitte D. Briand C. Derrick P.J. Barras F. J. Biol. Chem. 2001; 276: 48915-48920Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). The consequences of this side-chain modification are variable and can be partial to protein unfolding (6Gao J. Yin D.H. Yao Y. Sun H. Qin Z. Schoneich C. Williams T.D. Squier T.C. Biophys. J. 1998; 74: 1115-1134Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 7Harndahl U. Kokke B.P. Gustavsson N. Linse S. Berggren K. Tjerneld F. Boelens W.C. Sundby C. Biochim. Biophys. Acta. 2001; 1545: 227-237Crossref PubMed Scopus (37) Google Scholar) and modification of biological functions (8Taggart C. Cervantes-Laurean D. Kim G. McElvaney N.G. Wehr N. Moss J. Levine R.L. J. Biol. Chem. 2000; 275: 27258-27265Abstract Full Text Full Text PDF PubMed Google Scholar, 9Khor H.K. Fisher M. Schöneich C. J. Biol. Chem. 2004; 279: 19486-19493Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 10Sun H. Gao J. Ferrington D.A. Biesiada H. Williams T.D. Squier T.C. Biochemistry. 1999; 38: 105-112Crossref PubMed Scopus (148) Google Scholar). Sometimes surface methionine residues can undergo oxidation without much impact on the protein properties, and this modification can be seen as a mechanism to scavenge oxidative species in a detoxification process based on methionine sulfoxide reductase activity (11Levine R.L. Mosoni L. Berlett B.S. Stadman E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15036-15040Crossref PubMed Scopus (893) Google Scholar). Because of its asymmetric sulfur atom, MetSO exists as two stereoisomeric forms, Met-(S)-SO and Met-(R)-SO. Their reduction back to methionine is catalyzed by two structurally unrelated classes of Msr, MsrAs are specific for Met-(S)-SO, whereas Met-(R)-SO is the substrate of MsrBs. MsrAs and MsrBs display no significant sequence identity and have different three-dimensional structures. Only three MsrA x-ray structures from Escherichia coli, Bos taurus, and Mycobacterium tuberculosis and two MsrB structures from Neisseria species have been described so far (12Tete-Favier F. Cobessi D. Boschi-Muller S. Azza S. Branlant G. Aubry A. Structure. 2000; 8: 1167-1178Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 13Lowther W.T. Brot N. Weissbach H. Matthews B.W. Biochemistry. 2000; 39: 13307-13312Crossref PubMed Scopus (125) Google Scholar, 14Taylor A.B. Benglis Jr., D.M. Dhandayuthapani S. Hart P.J. J. Bacteriol. 2003; 185: 4119-4126Crossref PubMed Scopus (68) Google Scholar, 15Lowther W.T. Weissbach H. Etienne F. Brot N. Matthews B.W. Nat. Struct. Biol. 2002; 9: 348-352PubMed Google Scholar, 16Kauffmann B. Aubry A. Favier F. Biochim. Biophys. Acta. 2005; 1703: 249-260Crossref PubMed Scopus (39) Google Scholar). Both classes of Msrs share, for most of them, a similar three-step chemical mechanism, including the following: 1) a nucleophilic attack of the catalytic CysA residue on the sulfur atom of the sulfoxide substrate leading to the formation of a sulfenic acid intermediate and to the release of 1 mol of Met per mol of enzyme; 2) a formation of an intramonomeric disulfide bond between the catalytic CysA and the recycling CysB with a concomitant release of 1 mol of water; and 3) a reduction of the CysA-CysB methionine sulfoxide reductase disulfide bond by thioredoxin (Trx) (Fig. 1) (17Boschi-Muller S. Olry A. Antoine M. Branlant G. Biochim. Biophys. Acta. 2005; 1703: 231-238Crossref PubMed Scopus (160) Google Scholar, 18Boschi-Muller S. Azza S. Sanglier-Cianferani S. Talfournier F. Van Dorsselear A. Branlant G. J. Biol. Chem. 2000; 275: 35908-35913Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 19Kumar R.A. Koc A. Cerny R.L. Gladyshev V.N. J. Biol. Chem. 2002; 277: 37527-37535Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Nevertheless, for MsrAs, at least three subclasses, based on the number and the position of the recycling Cys residues, have been proposed (20Boschi-Muller S. Azza S. Branlant G. Protein Sci. 2001; 10: 2272-2279Crossref PubMed Scopus (44) Google Scholar). The Neisseria meningitidis and M. tuberculosis MsrA represent the first subclass, characterized by the presence of the recycling CysB in the C-terminal end, and the Bacillus subtilis enzyme represents the second one with the CysB located three amino acids behind CysA. The third subclass, represented by E. coli and B. taurus MsrAs, contains two recycling Cys residues in the C-terminal end and requires the formation not of one but of two successive disulfide bonds. The first one is formed between the catalytic CysA and the recycling CysB. The second one, formed between CysB and the second recycling cysteine CysC, is the one preferentially reduced by Trx in the last step (18Boschi-Muller S. Azza S. Sanglier-Cianferani S. Talfournier F. Van Dorsselear A. Branlant G. J. Biol. Chem. 2000; 275: 35908-35913Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). 4S. Boschi-Muller and G. Branlant, unpublished results. The denomination of the catalytic cysteines as CysA, -B, and -C is based on the primary structure order. Most of the MsrAs studied so far are bacterial or mammalian enzymes. In comparison, little has been done concerning plants. Five MsrA-like genes were identified in Arabidopsis thaliana; one encodes chloroplastic isoforms, and another one is predicted to be targeted to the secretory pathway, and three are cytosolic enzymes (21Sadanandom A. Poghosyan Z. Fairbairn D.J. Murphy D.J. Plant Physiol. 2000; 123: 255-264Crossref PubMed Scopus (71) Google Scholar, 22Romero H.M. Berlett B.S. Jensen P.J. Pell E.J. Tien M. Plant Physiol. 2004; 136: 3784-3794Crossref PubMed Scopus (137) Google Scholar). The expression of the chloroplastic isoform, found mainly in photosynthetic tissues, is strongly induced by illumination of etiolated seedlings and is responsive to various oxidative stress conditions (21Sadanandom A. Poghosyan Z. Fairbairn D.J. Murphy D.J. Plant Physiol. 2000; 123: 255-264Crossref PubMed Scopus (71) Google Scholar, 22Romero H.M. Berlett B.S. Jensen P.J. Pell E.J. Tien M. Plant Physiol. 2004; 136: 3784-3794Crossref PubMed Scopus (137) Google Scholar, 23Vieira Dos Santos C. Cuine S. Rouhier N. Rey P. Plant Physiol. 2005; 138: 909-922Crossref PubMed Scopus (133) Google Scholar). Moreover, this plastidial MsrA was also shown to maintain chaperonin activity of a small heat-shock protein Hsp21 by preventing its denaturation and consequently inactivation after methionine oxidation (24Gustavsson N. Kokke B.P. Harndahl U. Silow M. Bechtold U. Poghosyan Z. Murphy D. Boelens W.C. Sundby C. Plant J. 2002; 29: 545-553Crossref PubMed Scopus (89) Google Scholar). Finally, the expression of cytosolic MsrAs was also shown to respond to various changing conditions as follows: (i) in the dark period of A. thaliana plants growing in short-day conditions (25Bechtold U. Murphy D.J. Mullineaux P.M. Plant Cell. 2004; 16: 908-919Crossref PubMed Scopus (109) Google Scholar), (ii) during a pathogen infection by the cauliflower mosaic virus (21Sadanandom A. Poghosyan Z. Fairbairn D.J. Murphy D.J. Plant Physiol. 2000; 123: 255-264Crossref PubMed Scopus (71) Google Scholar), or (iii) during softening of cold-hardened leaves (26In O. Berberich T. Romdhane S. Feierabend J. Planta. 2005; 220: 941-950Crossref PubMed Scopus (16) Google Scholar). The previous reports about the plant MsrAs have focused essentially on their expression patterns, but the catalytic mechanism, in particular that related to Trx-dependent recycling process, and the three-dimensional structure of a plant MsrA have not yet been addressed. One of the first methionine sulfoxide reductase activities that was evidenced for a plant enzyme was established for a chloroplast-targeted MsrA from Brassica napus (28Jacquot J.P. Stein M. Suzuki A. Liottet S. Sandoz G. Miginiac-Maslow M. FEBS Lett. 1997; 400: 293-296Crossref PubMed Scopus (47) Google Scholar). In this study, the biochemical and catalytic properties of poplar MsrA are presented, in particular those related to the Trx-dependent recycling process. The crystal structure of a poplar MsrA in complex with a mercaptoethanol molecule bound to the catalytic CysA is also reported. Altogether, the data support a Trx-recycling process with formation of a disulfide bond first between the catalytic Cys46 (CysA) and Cys202 (CysC) and then between Cys202 and Cys196 (CysB). This latter disulfide bond was reduced by Trx. The open reading frame sequences encoding a cytosolic MsrA (cMsrA) and a plastidial MsrA (pMsrA) (respective GenBank™ accession numbers AAS46231 and AAS46232) were cloned by PCR into the expression plasmid pET-3d using as templates a root cDNA library of Populus × interamericana (clone Beaupré) and a leaf cDNA library of Populus tremula × tremuloides, respectively. Both reactions also contained Pfu DNA polymerase (Promega) and the forward and reverse MsrA oligonucleotides described in Table 1. In the pMsrA cloning, a codon for alanine was inserted downstream from the methionine closest to the putative cleavage site and the corresponding N-terminal amino acid sequence starts thus with MANIL. The five cysteines of cMsrA were substituted into serine one by one using either two complementary mutagenic primers per mutation (C46S, C81S, C100S, C196S cMsrA and C46S, C196S pMsrA) (Table 1), using a two-step procedure described previously (28Jacquot J.P. Stein M. Suzuki A. Liottet S. Sandoz G. Miginiac-Maslow M. FEBS Lett. 1997; 400: 293-296Crossref PubMed Scopus (47) Google Scholar), or a one-step procedure when the mutation is directly inserted in the reverse primers (C202S cMsrA and C202S pMsrA). In addition, various combinations of cysteine substitutions by serine were also introduced in cMsrA (C81S/C100S; C81S/C100S/C196S; C81S/C100S/C202S; and C81S/C100S/C196S/C202S cMsrAs). The introduction of the mutation in the cDNA sequence was verified by DNA sequencing.TABLE 1Cloning and mutagenic oligonucleotidescMsrA forward5′-CCCCCCATGGCAACCAGCACCACCAAT-3′cMsrA reverse5′-CCCCGGATCCTTAACCATAGCATCTAATAGG-3′cMsrA C46S forward5′-GCTCAATTCGGAGCTGGAAGTTTCTGGGGGGTT-3′cMsrA C46S reverse5′-AACCCCCCAGAAACTTCCAGCTCCGAATTGAGC-3′cMsrA C81S forward5′-ACTTACAAGCTGGTATCCACCAACACCACCAAC-3′cMsrA C81S reverse5′-GTTGGTGGTGTTGGTGGATACCAGCTTGTAAGT-3′cMsrA C100S forward5′-TTTGACCCGGAAGTTTCCCCATATACCAACCTC-3′cMsrA C100S reverse5′-GAGGTTGGTATATGGGGAAACTTCCGGGTCAAA-3′cMsrA C196S forward5′-TCTGCTGAAAAAGGTTCCAATGACCCTATTAGA-3′cMsrA C196S reverse5′-TCTAATAGGGTCATTGGAACCTTTTTCAGCAGA-3′cMsrA C202S reverse5′-CCCCGGATCCTTAACCATAGCTTCTAATAGGGTCATTGCA-3′pMsrA forward5′-CCCCCCATGGCTAACATCCTTAGCAAACTAGGC-3′pMsrA reverse5′-CCCCGGATCCTTAGCCATAGCATCGGATTGGATC-3′pMsrA C46S forward5′-TTTGGAGCTGGTTCTTTTTGGGGTGTT-3′pMsrA C46S reverse5′-AACACCCCAAAAAGAACCAGCTCCAAA-3′pMsrA C196S forward5′-GCTGAGAAAGGATCCAATGATCCAATC-3′pMsrA C196S reverse5′-GATTGGATCATTGGATCCTTTCTCAGC-3′pMsrA C202S reverse5′-CCCCGGATCCTTAGCCATAGGATCGGATTGG-3′ Open table in a new tab The recombinant plasmids were used to transform the BL21(DE3) E. coli strain, which also contains the helper plasmid pSBET (29Schenk P.M. Baumann S. Mattes R. Steinbiss H.H. Bio-Techniques. 1995; 19: 196-200Google Scholar). Cultures of 5 liters of a kanamycin-resistant (50 μg/ml) and ampicillin-resistant (50 μg/ml) colony were grown at 37 °C and induced by 100 μm isopropyl 1-thio-β-d-galactopyranoside in the exponential phase. Bacteria were harvested by centrifugation, resuspended in buffer A (30 mm Tris-HCl, 1 mm EDTA, 200 mm NaCl) containing 20 mm DTT, and lysed by sonication. The soluble and insoluble fractions were separated by centrifugation (16,000 × g, 30 min). The recombinant wild-type pMsrA was in the soluble fraction and precipitated between 0 and 50% of ammonium sulfate. All the other recombinant proteins were produced essentially as inclusion bodies with only a small soluble part when cultures were grown at 30 °C without induction. When needed, the insoluble fraction was thus resuspended in buffer A in the presence of 20 mm DTT and 8 m urea, centrifuged, and then dialyzed against 1 liter of buffer A containing 500 mm urea for at least 5 h at 5°C (all subsequent steps were realized at that temperature). The extract was centrifuged, and the soluble fraction was dialyzed against 1 liter of buffer A for 5 h and finally centrifuged again. The resulting soluble fraction was purified by exclusion size chromatography onto an ACA 44 column equilibrated in buffer A. The fractions of interest were pooled, dialyzed to remove salts, and separated by DEAE-Sephacel chromatography. The recombinant proteins were eluted around 100 mm NaCl using a linear gradient from 0 to 400 mm NaCl. The purity of the proteins was assessed using 15% SDS-PAGE. The protein concentrations were estimated spectrophotometrically using a molar extinction coefficient of 25,700 m-1 cm-1 for cMsrA and pMsrA. The proteins were stored at -30 °C in buffer A either in the presence of 14 mm β-mercaptoethanol or 25 mm DTT. Crystallization of pMsrA was achieved using the hangingdrop vapor-diffusion method in Linbro multiwell tissue culture plates at room temperature. Many crystallization conditions resulted in very thin needles that were not usable for data collection and were impossible to improve. Only one condition described here gave suitable crystals for x-ray crystallography. The purified enzyme was concentrated to 40 mg/ml in a solution containing 30 mm Tris-HCl, pH 7.0, 14 mm β-mercaptoethanol, and 1 mm EDTA. The crystals were grown from 4-μl droplets composed of equal volumes of the protein solution and of the precipitant solution (10% w/v polyethyleneglycol 6000, 2 m NaCl) and equilibrated against 700-μl reservoirs. Long needles (1 mm) with a thin triangular cross-section (0.03 mm) appeared after 6 weeks. Crystals were briefly soaked in a cryoprotectant solution (10% v/v methylpentanediol mixed with the precipitant solution) and flash-frozen by fast immersion in nitrogen gas stream at 100 K, maintained during the x-ray diffraction experiments performed on beamline BM30A (FIP) at the ESRF. Crystals belong to space group P31 with unit cell parameters a = b = 68.6 Å, c = 40.7 Å and contain one monomer per asymmetric unit. Using a wavelength of 1.009 Å, one native data set was collected up to a resolution of 1.7 Å and processed using DENZO (30Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). Further details are given in Table 2.TABLE 2Statistics of X-ray diffraction data collection for the pMsrA crystalsWavelength1.009 Å (ESRF, BM30)Temperature100 KResolution25.0-1.7 (1.74-1.70) ÅNo. of measured reflections114,471No. of independent reflections23,121Completeness98.2 (83.4)%Rsym4.8 (21.9)% / 8.6 (2.5) Open table in a new tab The structure was solved using the molecular replacement method implemented in Molrep (31Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4175) Google Scholar) of the CCP4 program suite. The initial model used in Molrep consisted of the core (41Gly-Pro194) of the E. coli MsrA structure (Protein Data Bank entry 1FF3). The molecular replacement solution was submitted to the Molrep mode and then to the warpNtrace mode of the Arp/wArp5.1 automatic model building and refinement program (32Perrakis A. Morris J.R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2565) Google Scholar). It produced a model that contained four polypeptide chains representing 164 amino acids, with R and Rfree factors of 20.6 and 25.8%, respectively. Manual corrections (in particular, building of the missing residues) and automatic CNS refinement (33Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunsteleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) of the model were then performed in an iterative procedure, until the model fulfilled satisfactory criteria. The final structure corresponds to 183 amino acids among 204 (residues 22Pro-Gly204), 183 water molecules, with R = 19.5%, Rfree = 20.1%. Further details are given in Table 3.TABLE 3Refinement and model statisticsResolution rangeaValues in parentheses correspond to statistics in the outer resolution shell. (Å)25.0 to 1.7 (1.76 to 1.70)No. of reflections used for R calculationsaValues in parentheses correspond to statistics in the outer resolution shell.21,710 (1812)No. of reflections used for Rfree calculationsaValues in parentheses correspond to statistics in the outer resolution shell.1103 (84)Data cutoff F/s(F)0.0R valueaValues in parentheses correspond to statistics in the outer resolution shell. (%)19.5 (23.4)Rfree valueaValues in parentheses correspond to statistics in the outer resolution shell. (%)20.1 (23.4)No. of non-hydrogen protein atoms1478No. of water molecules183Mean B-factor, protein main-chain atoms (Å2)23.6Mean B-factor, protein side-chain atoms (Å2)25.2Mean B-factor, solvent atoms (Å2)28.5B-factor from the Wilson plot (Å2)27.6Ramachandran plotResidues in most favored regions (%)92.7Residues in additionally allowed regions (%)7.3Residues in generously allowed regions (%)0Residues in disallowed regions (%)0Root mean square deviation from ideal geometryBond length (Å)0.006Bond angle (°)1.31Root mean square deviation for isotropic thermal factor restraints (Å2)Main-chain bond0.98Main-chain angle1.53Side-chain bond1.82Side-chain angle2.65a Values in parentheses correspond to statistics in the outer resolution shell. Open table in a new tab Known concentrations (generally around 25 μm) of recombinant proteins were reduced with 50 mm DTT, extensively dialyzed, and then treated or not with 100 mm l-MetSO for 1 h at room temperature. The proteins were then precipitated on ice by addition of 1 volume of 20% trichloroacetic acid for 30 min. The proteins were pelleted by centrifugation and washed twice with 2% trichloroacetic acid. The pellets were resuspended in 30 mm Tris-HCl, pH 8.0, 1 mm EDTA, and 2% SDS. The concentrations of the proteins were determined spectrophotometrically at this stage, and then 5,5′-dithiobis(nitrobenzoic acid) was added to a final concentration of 100 μm, and the absorbance was read at 412 nm 1 h later. The thiol content was determined using a molar extinction coefficient of 13,600 m -1 cm-1 for thionitrobenzoate (TNB-). The sulfenic acid intermediate was characterized spectrophotometrically by using TNB- under nondenaturing conditions, as described previously (18Boschi-Muller S. Azza S. Sanglier-Cianferani S. Talfournier F. Van Dorsselear A. Branlant G. J. Biol. Chem. 2000; 275: 35908-35913Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Briefly, progress curves of TNB- disappearance were recorded at 412 nm in 50 mm Tris-HCl, pH 8.0, 1 mm EDTA buffer. Enzyme concentrations were 7.35 and 14.7 μm, and the TNB- concentration was 60 μm. The amount of TNB- consumed was calculated using an extinction coefficient at 412 nm of 13,600 m -1 cm-1. NMR Determination of Activity in the Presence of DTT—The catalytic activity was determined by monitoring the reduction of MetSO to Met using DTT as the reducing agent. The concentrations of the substrate and product of the reaction were obtained from the intensity of the resonance signals at 2.65 and 2.15 ppm corresponding to the MetSO methyl resonance and the Met methyl resonance, respectively. The assay conditions were 100 mm phosphate buffer, 50 mm DTT, 20 mm l-Met-(RS)-SO at pH 8.5 in 90/10% H2O/D2O. l-Alanine (10 mm) was used for internal concentration calibration. The enzyme was added directly in the NMR cell, and careful homogenization of the sample was performed just before recording. NMR spectra were recorded with eight scans at 27 °C every 79 s on Varian Inova 400 MHz spectrometer equipped with a triple resonance (1H, 13C, and 15N) probe including shielded z-gradients. Data were processed using FELIX 97 (Accelrys). Thioredoxin-dependent Methionine Sulfoxide Reductase Activity—The activity of cMsrA and pMsrA was also measured by following the NADPH oxidation at 340 nm in the presence of Trx and NADPH Trx reductase system. A 500-μl cuvette contained 30 mm Tris-HCl, pH 8.0, 1 mm EDTA, 200 μm NADPH, 2 μm A. thaliana NADPH thioredoxin reductase (purified as in Ref. 34Jacquot J.P. Rivera-Madrid R. Marinho P. Kollarova M. Le Marechal P. Miginiac-Maslow M. Meyer Y. J. Mol. Biol. 1994; 235: 1357-1363Crossref PubMed Scopus (129) Google Scholar), various concentrations of a cytosolic poplar Trx h1, and 100 mm l-Met-(RS)-SO. After 1 min of incubation, MsrA was added to the reaction mixture. Poplar Trx h1 was purified as described previously (35Behm M. Jacquot J.P. Plant Physiol. Biochem. 2000; 38: 363-369Crossref Scopus (31) Google Scholar). The reaction was carried out at 30 °C with a Cary 50 spectrophotometer. The catalytic parameters for Trx and MetSO were determined at saturating concentrations of the other substrate and adjusted using GraFit. Stoichiometry of Methionine Formation in the Absence of Reductants—The different proteins were reduced by 50 mm DTT and dialyzed twice against 1 liter of 30 mm Tris-HCl, pH 8.0, 1 mm EDTA. A typical 200-μl reaction mixture containing 100-400 μm of recombinant proteins and 100 mm l-Met-(RS)-SO was incubated at room temperature for 10 min. After adding 2% trifluoroacetic acid to stop the reaction, 100 μl were injected onto a Sephasil C18 column to quantify the concentration of Met formed as described previously (18Boschi-Muller S. Azza S. Sanglier-Cianferani S. Talfournier F. Van Dorsselear A. Branlant G. J. Biol. Chem. 2000; 275: 35908-35913Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Genome and Sequence Analysis—Among the five isoforms found in the released genome of Populus trichocarpa, two very close genes (86% identity) are predicted to be located in plastids and two other (93% identity) to be cytosolic. Except for the presence of an N-terminal targeting sequence, the four genes are very similar. It is likely that these genes have been actually duplicated two by two. The fifth isoform (EST accession number DT503157) is quite divergent (28Jacquot J.P. Stein M. Suzuki A. Liottet S. Sandoz G. Miginiac-Maslow M. FEBS Lett. 1997; 400: 293-296Crossref PubMed Scopus (47) Google Scholar, 29Schenk P.M. Baumann S. Mattes R. Steinbiss H.H. Bio-Techniques. 1995; 19: 196-200Google Scholar, 30Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar, 31Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4175) Google Scholar, 32Perrakis A. Morris J.R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2565) Google Scholar% identity) compared with the four other sequences, although it displays the canonical GCFW active site sequence that allows us to classify it as an MsrA, but it does not possess the two C-terminal cysteines (see below). The cDNA sequences of a chloroplastic and a cytosolic isoform, which we call here conveniently pMsrA (plastidial MsrA) and cMsrA (cytosolic MsrA), were isolated by PCR from poplar leaf and root cDNA libraries, respectively. Based on transit peptide prediction programs and amino acid comparisons with homologous proteins from A. thaliana, pMsrA (260 amino acids for the precursor) is predicted to present a 57-amino acid-long N-terminal chloroplastic transit peptide. The size of the mature recombinant pMsrA devoid of the transit peptide produced here (see "Materials and Methods") is 204 amino acids (including the initial methionine and an alanine added for cloning facility). The cmsra open reading frame encodes a protein of 190 amino acids. The additional 14 amino acids of the plastidial form are all located in the N-terminal part of the sequence. The two mature enzymes possess 62% strict identity at the amino acid level. Fig. 2 displays an amino acid sequence comparison of various plant MsrAs with enzymes from other kingdoms with known catalytic mechanisms or structures. For the comprehensive analysis of this work, we used the numbering of the recombinant pMsrA both for pMsrA and cMsrA cysteines, although they are not exactly at the same position because of the N-terminal extension in pMsrA. Only the first cysteine at position 46, the catalytic CysA, is conserved among all the sequences presented here. In plants, there are three other strictly conserved cysteines at positions 81, 196, and 202, whereas a fourth at position 100 is present in all sequences but B. napus. The Cys at position 81 is equivalent to Cys86 of E. coli MsrA, which has been shown to play no role in the catalytic mechanism (18Boschi-Muller S. Azza S. Sanglier-Cianferani S. Talfournier F. Van Dorsselear A. Branlant G. J. Biol. Chem. 2000; 275: 35908-35913Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The C-terminal part of plant MsrAs also contains two cysteines, located in a consensus sequen
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