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• W1973587531 abstract "The biosynthetic reaction scheme for the compatible solute mannosylglycerate in Rhodothermus marinusis proposed based on measurements of the relevant enzymatic activities in cell-free extracts and in vivo 13C labeling experiments. The synthesis of mannosylglycerate proceeded via two alternative pathways; in one of them, GDP mannose was condensed withd-glycerate to produce mannosylglycerate in a single reaction catalyzed by mannosylglycerate synthase, in the other pathway, a mannosyl-3-phosphoglycerate synthase catalyzed the conversion of GDP mannose and d-3-phosphoglycerate into a phosphorylated intermediate, which was subsequently converted to mannosylglycerate by the action of a phosphatase. The enzyme activities committed to the synthesis of mannosylglycerate were not influenced by the NaCl concentration in the growth medium. However, the combined mannosyl-3-phosphoglycerate synthase/phosphatase system required the addition of NaCl or KCl to the assay mixture for optimal activity. The mannosylglycerate synthase enzyme was purified and characterized. Based on partial sequence information, the corresponding mgs gene was identified from a genomic library of R. marinus. In addition, the mgs gene was overexpressed inEscherichia coli with a high yield. The enzyme had a molecular mass of 46,125 Da, and was specific for GDP mannose andd-glycerate. This is the first report of the characterization of a mannosylglycerate synthase. The biosynthetic reaction scheme for the compatible solute mannosylglycerate in Rhodothermus marinusis proposed based on measurements of the relevant enzymatic activities in cell-free extracts and in vivo 13C labeling experiments. The synthesis of mannosylglycerate proceeded via two alternative pathways; in one of them, GDP mannose was condensed withd-glycerate to produce mannosylglycerate in a single reaction catalyzed by mannosylglycerate synthase, in the other pathway, a mannosyl-3-phosphoglycerate synthase catalyzed the conversion of GDP mannose and d-3-phosphoglycerate into a phosphorylated intermediate, which was subsequently converted to mannosylglycerate by the action of a phosphatase. The enzyme activities committed to the synthesis of mannosylglycerate were not influenced by the NaCl concentration in the growth medium. However, the combined mannosyl-3-phosphoglycerate synthase/phosphatase system required the addition of NaCl or KCl to the assay mixture for optimal activity. The mannosylglycerate synthase enzyme was purified and characterized. Based on partial sequence information, the corresponding mgs gene was identified from a genomic library of R. marinus. In addition, the mgs gene was overexpressed inEscherichia coli with a high yield. The enzyme had a molecular mass of 46,125 Da, and was specific for GDP mannose andd-glycerate. This is the first report of the characterization of a mannosylglycerate synthase. isopropyl-β-d-thiogalactopyranoside d-3-phosphoglycerate mannosylglycerate polyacrylamide gel electrophoresis polymerase chain reaction 2-(N-morpholino)ethanesulfonic acid kilobase pair(s) 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol Most microorganisms capable of osmotic adaptation accumulate compatible solutes in response to increases in the levels of salts or sugars in the environment. Some compatible solutes, such as glutamate, betaine, and trehalose are widespread in mesophilic organisms, but compatible solutes unique to thermophiles and hyperthermophiles, that appear to be associated with thermal adaptation, have also been identified in recent years. Newly discovered solutes from thermophilic and hyperthermophilic organisms include cyclic-2,3-bisphosphoglycerate (1Tolman C.J. Kanodia S. Roberts M.F. Daniels L. Biochim. Biophys. Acta. 1986; 886: 345-352Crossref PubMed Scopus (19) Google Scholar), two isomers of di-myo-inositolphosphate (2Scholz S. Sonnenbichler J. Schäfer W. Hensel R. FEBS Lett. 1992; 306: 239-242Crossref PubMed Scopus (134) Google Scholar, 3Martins L.O. Santos H. Appl. Environ. Microbiol. 1995; 61: 3299-3303Crossref PubMed Google Scholar, 4Martins L.O. Carreto L.S. Da Costa M.S. Santos H. J. Bacteriol. 1996; 178: 5644-5651Crossref PubMed Scopus (76) Google Scholar), mannosylglycerate and mannosylglyceramide (3Martins L.O. Santos H. Appl. Environ. Microbiol. 1995; 61: 3299-3303Crossref PubMed Google Scholar, 5Nunes O.C. Manaia C.M. Da Costa M.S. Santos H. Appl. Environ. Microbiol. 1995; 61: 2351-2357Crossref PubMed Google Scholar, 6Silva Z. Borges N. Martins L.O. Wait R. Da Costa M.S. Santos H. Extremophiles. 1999; 3: 163-172Crossref PubMed Scopus (70) Google Scholar), di-mannosyl-di-myo-inositolphosphate (4Martins L.O. Carreto L.S. Da Costa M.S. Santos H. J. Bacteriol. 1996; 178: 5644-5651Crossref PubMed Scopus (76) Google Scholar), diglycerolphosphate (7Martins L.O. Huber R. Huber H. Stetter K.O. Da Costa M.S. Santos H. Appl. Environ. Microbiol. 1997; 63: 896-902Crossref PubMed Google Scholar), and galactosyl-5-hydroxylysine (8Lamosa P. Martins L.O. Da Costa M.S. Santos H. Appl. Environ. Microbiol. 1998; 64: 3591-3598Crossref PubMed Google Scholar). Many of these solutes have only been identified in marine thermophilic and hyperthermophilic organisms. The observation that some of these solutes accumulate at supraoptimal growth temperatures, combined to their effectiveness in protecting enzymes in vitro supports the hypothesis that they play a role in thermoprotection of living cells (2Scholz S. Sonnenbichler J. Schäfer W. Hensel R. FEBS Lett. 1992; 306: 239-242Crossref PubMed Scopus (134) Google Scholar, 3Martins L.O. Santos H. Appl. Environ. Microbiol. 1995; 61: 3299-3303Crossref PubMed Google Scholar, 4Martins L.O. Carreto L.S. Da Costa M.S. Santos H. J. Bacteriol. 1996; 178: 5644-5651Crossref PubMed Scopus (76) Google Scholar, 6Silva Z. Borges N. Martins L.O. Wait R. Da Costa M.S. Santos H. Extremophiles. 1999; 3: 163-172Crossref PubMed Scopus (70) Google Scholar, 7Martins L.O. Huber R. Huber H. Stetter K.O. Da Costa M.S. Santos H. Appl. Environ. Microbiol. 1997; 63: 896-902Crossref PubMed Google Scholar, 8Lamosa P. Martins L.O. Da Costa M.S. Santos H. Appl. Environ. Microbiol. 1998; 64: 3591-3598Crossref PubMed Google Scholar, 9Da Costa M.S. Santos H. Galinski E.A. Adv. Biochem. Eng. Biotechnol. 1998; 61: 117-153PubMed Google Scholar, 10Hensel R. König H. FEMS Microbiol. Lett. 1988; 49: 75-79Crossref Scopus (160) Google Scholar, 11Ciulla R.A. Burggraf S. Stetter K.O. Roberts M.F. Appl. Environ. Microbiol. 1994; 60: 3660-3664Crossref PubMed Google Scholar, 12Ramos A. Raven N.D.H. Sharp R. Bartolucci S. Rossi M. Cannio R. Lebbink J. Van der Oost J. De Vos W. Santos H. Appl. Environ. Microbiol. 1997; 63: 4020-4025Crossref PubMed Google Scholar). Our knowledge of the biosynthetic pathways for compatible solutes in prokaryotes has increased significantly in recent years to include the synthesis of trehalose (13Strøm A.R. Kaasen I. Mol. Microbiol. 1993; 8: 205-210Crossref PubMed Scopus (274) Google Scholar), ectoine (14Peters P. Galinski E.A. Trüper H.G. FEMS Microbiol. Lett. 1990; 71: 157-162Crossref Google Scholar, 15Louis P. Galinski E. Microbiology. 1997; 143: 1141-1149Crossref PubMed Scopus (144) Google Scholar), glucosylglycerol (16Hagemann M. Erdmann N. Microbiolology. 1994; 140: 1427-1431Crossref Scopus (94) Google Scholar,17Hagemann M. Schoor A. Erdmann N. J. Plant Physiol. 1996; 149: 746-752Crossref Scopus (21) Google Scholar), galactosylglycerol (18Kauss H. Schobert B. FEBS Lett. 1971; 19: 131-135Crossref PubMed Scopus (11) Google Scholar), cyclic-2,3-bisphosphoglycerate (19Evans J.N.S. Tolman C.J. Kanodia S. Roberts M.F. Biochemistry. 1985; 24: 5693-5698Crossref PubMed Scopus (35) Google Scholar, 20Lechmacher A. Vogt A.-B. Hensel R. FEBS Lett. 1990; 272: 94-98Crossref PubMed Scopus (25) Google Scholar), and di-myo-inositolphosphate (21Chen L. Spiliotis E.T. Roberts M.F. J. Bacteriol. 1998; 180: 3785-3792Crossref PubMed Google Scholar, 22Scholz S. Wolff S. Hensel R. FEMS Microbiol. Lett. 1998; 168: 37-42Google Scholar). Mannosylglycerate, a solute initially identified in a few red algae (23Karsten U. Barrow K.D. Mostaert A.S. King R.J. West J.A. Plant Physiol. Biochem. 1994; 32: 669-676Google Scholar), was recently identified in thermophilic bacteria of the generaRhodothermus, Thermus (5Nunes O.C. Manaia C.M. Da Costa M.S. Santos H. Appl. Environ. Microbiol. 1995; 61: 2351-2357Crossref PubMed Google Scholar), Petrotoga(4Martins L.O. Carreto L.S. Da Costa M.S. Santos H. J. Bacteriol. 1996; 178: 5644-5651Crossref PubMed Scopus (76) Google Scholar), and hyperthermophilic archaea of the genera Pyrococcus(3Martins L.O. Santos H. Appl. Environ. Microbiol. 1995; 61: 3299-3303Crossref PubMed Google Scholar), Thermococcus (8Lamosa P. Martins L.O. Da Costa M.S. Santos H. Appl. Environ. Microbiol. 1998; 64: 3591-3598Crossref PubMed Google Scholar), and Methanothermus(7Martins L.O. Huber R. Huber H. Stetter K.O. Da Costa M.S. Santos H. Appl. Environ. Microbiol. 1997; 63: 896-902Crossref PubMed Google Scholar). Mannosylglycerate appears to be one of the most prevalent compatible solutes in thermophilic and hyperthermophilic bacteria and archaea, and is believed to be, along with di-myo-inositolphosphate, an archetypal osmolyte of organisms living near or at the highest growth temperatures for life. In Rhodothermus marinus, mannosylglycerate accumulates in response to growth at supraoptimal temperature and salinity while the amide form, mannosylglyceramide, accumulates exclusively in response to salt stress (5Nunes O.C. Manaia C.M. Da Costa M.S. Santos H. Appl. Environ. Microbiol. 1995; 61: 2351-2357Crossref PubMed Google Scholar, 6Silva Z. Borges N. Martins L.O. Wait R. Da Costa M.S. Santos H. Extremophiles. 1999; 3: 163-172Crossref PubMed Scopus (70) Google Scholar). Furthermore, mannosylglycerate was found to protect several enzymes against inactivation by temperature and freeze-drying (12Ramos A. Raven N.D.H. Sharp R. Bartolucci S. Rossi M. Cannio R. Lebbink J. Van der Oost J. De Vos W. Santos H. Appl. Environ. Microbiol. 1997; 63: 4020-4025Crossref PubMed Google Scholar). The elucidation of metabolic pathways for the synthesis of compatible solutes is essential to understand the regulatory mechanisms involved in the adaptation of many thermophilic and hyperthermophilic organisms to fluctuations in salt or temperature. Moreover, the biochemical and genetic characterization of key enzymes is also a prerequisite for achieving overproduction of these compounds in suitable hosts. In this study we elucidated the biosynthetic pathways of mannosylglycerate in R. marinus, and characterized the enzyme mannosylglycerate synthase, catalyzing a final step in the synthesis of mannosylglycerate. In addition, we report the cloning and overexpression of the respective gene in Escherichia coli. The type strain of R. marinus DSM 4252 (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) was grown on Degryse medium containing 2.5 g of tryptone and 2.5 g of yeast extract/liter (24Nunes O.C. Donato M.M. Da Costa M.S. Syst. Appl. Microbiol. 1992; 15: 92-97Crossref Scopus (43) Google Scholar), and supplemented with 1, 4, and 6% NaCl (w/v). Cultures were grown in a 5-liter fermentor at 65 °C, with continuous gassing with air and stirred at 150 rpm. Cell growth was monitored by measuring the turbidity at 600 nm. Escherichia coli TG1 (supE thi hsdΔ5 Δ(lac-proAB) [F′traD36 proAB lacI q ZΔM15]) and E. coliXL1-Blue (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac[F′proAB lacI q ZΔM15 Tn10 (Tetr)]) were used as hosts for the cloning vectors pUC18, pGEM-T Easy (Promega), and pKK223–3 (Amersham Pharmacia Biotech). E. coli was grown in YT-medium containing 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl/liter. Ampicillin was added at a final concentration of 100 μg/ml for selection of plasmids. 5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside and IPTG1 were obtained from Roche Molecular Biochemicals (Germany) and added at a final concentration of 80 μg/ml and 0.5 mm, respectively. Cells were harvested by centrifugation (8,000 ×g, 15 min, 20 °C) during the late exponential phase of growth and frozen at −80 °C. The cell sediment was suspended in Tris-HCl (20 mm, pH 7.6) containing DNase I (10 μg/ml extract) and MgCl2 (5 mm), and a mixture of protease inhibitors: phenylmethylsulfonyl fluoride (0.08 mg/ml), leupeptine (0.02 mg/ml), and antipain (0.02 mg/ml). Cells were disrupted in a French pressure cell, followed by centrifugation (20,000 × g, 1 h, 10 °C) to remove cell debris. Cell extracts were applied to a Sephadex G-25 column (Amersham Pharmacia Biotech), equilibrated with 20 mm Tris-HCl (pH 7.6) to remove mannosylglycerate (MG) and other low molecular weight compounds prior to measuring enzyme activities. The protein content was determined by the Bradford assay (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211906) Google Scholar) using bovine serum albumin as standard. The specific activities of phosphoglucose isomerase (EC5.3.1.9), phosphomannose isomerase (EC 5.3.1.8), and phosphomannomutase (EC 5.4.2.8) were determined by spectrophotometry at 55 °C using coupled reaction assays. Phosphoglucose isomerase and phosphomannose isomerase assays were based on the method described by Slein (26Slein M.W. Methods Enzymol. 1955; 1: 299-306Crossref Scopus (68) Google Scholar). Phosphomannomutase activity was assayed by the method described by Pindar and Bucke (27Pindar D.F. Bucke C. Biochem. J. 1975; 152: 617-622Crossref PubMed Scopus (105) Google Scholar). Mannose-1-phosphate guanylyltransferase (EC2.7.7.13), mannosylglycerate synthase, and the combined mannosyl-3-phosphoglycerate synthase/phosphatase activities were assayed by 1H NMR to detect and quantify substrate consumption and product formation. The assay mixture for measurement of mannose-1-phosphate guanylyltransferase activity was based on that suggested by Munch-Peterson (28Munch-Peterson A. Acta Chem. Scand. 1956; 10: 928-934Crossref Google Scholar) and contained 10 mmMgCl2, 4 mm EDTA, 4 mmguanosine-5′-triphosphate (Sigma), 4 mm mannose 1-phosphate (Sigma), and 2 units of inorganic pyrophosphatase in Tris-HCl (20 mm, pH 7.6). For determination of the combined activity of mannosyl-3-phosphoglycerate synthase/phosphatase the reaction mixture contained 10 mm MgCl2, 4 mm EDTA, 4 mm GDP mannose (Sigma), 4 mmd-3-P-glycerate (sodium salt, Sigma), and 0.3 mKCl in 20 mm Tris-HCl (pH 7.6). Mannosylglycerate synthase was measured in an assay mixture containing 10 mmMgCl2, 4 mm EDTA, 4 mm GDP mannose, and 4 mmd-glycerate (hemicalcium salt, Sigma) in the same buffer. Discontinuous or continuous methods were used to measure the activities of these enzymes at 65 °C. In the discontinuous method, cell extracts (generally, 100 μl) were incubated with the substrates, in a total reaction volume of 0.6 ml, for various periods of time, and the reactions were stopped by cooling and acidification with 25 mm HCl. After centrifugation, the pH of the supernatants was adjusted to 7.0 before freeze-drying. The residues were dissolved in 2H2O and analyzed by NMR. In the continuous method, the substrates were added to a 5-mm NMR tube and the reactions started by addition of aliquots of the enzyme preparation. The time course of product formation was followed by sequential acquisition of spectra. The reactions were stopped by rapid cooling followed by acidification. After centrifugation, the supernatant was neutralized and MG quantified by 1H NMR. Control assays lacking the cell extracts or the substrates were also carried out. All specific activities are the mean values of six assays in crude extracts prepared from cells derived from three independent growths. NMR spectra were acquired on Bruker DRX500 or AMX300 spectrometers. 1H NMR spectra were acquired with a 5-mm broad band inverse probe head with presaturation of the water signal. For quantification spectra were acquired with a repetition delay of 35 s and acetate was used as an internal concentration standard. Proton chemical shifts are relative to 3-(trimethylsilyl)propanesulfonic acid (sodium salt). Chromatograms were run on silica gel plates (Silica 60; Merck) with a solvent system composed of chloroform, methanol, 25% ammonia (6:10:5, v/v). The sugar and sugar derivatives were visualized by spraying with α-naphtol-sulfuric acid solution followed by charring at 120 °C (29Jacin H. Mishkin A.R. J. Chromatogr. 1965; 18: 170-173Crossref PubMed Google Scholar). Authentic standards of mannose, mannose 1-phosphate, GDP mannose, GDP, GMP, guanosine, and mannosylglycerate were used for comparative purposes. The enzyme, catalyzing the synthesis of MG from GDP mannose andd-glycerate, was purified by fast protein liquid chromatography (Amersham Pharmacia Biotech) at room temperature fromR. marinus cells grown in medium containing 4% NaCl. Fractions were examined for the presence of the enzyme by the mannosylglycerate synthase assay method described above and visualization of MG formation by TLC. Cell-free extract was applied to a column (XK50/30; bed volume, 250 ml) packed with DEAE-Sepharose fast flow (Amersham Pharmacia Biotech) equilibrated with Tris-HCl (20 mm, pH 7.6). Elution was carried out with a two-step linear NaCl gradient (0–0.2 m and 0.2–0.4 m) in the same buffer. Mannosylglycerate synthase activity was found in the fractions eluting at approximately 0.3 m NaCl. Ammonium sulfate was added to the pooled fractions from the previous step to a final concentration of 0.5 m. This sample was applied to a column (XK16/20; bed volume, 90 ml) packed with phenyl-Sepharose (Amersham Pharmacia Biotech) equilibrated with 50 mm potassium phosphate (pH 7.0), containing 0.5 m ammonium sulfate. Elution was carried out with a decreasing linear gradient of ammonium sulfate (0.5–0.0 m) in potassium phosphate buffer. Mannosylglycerate synthase eluted toward the end of the gradient. The active fractions were pooled and concentrated by ultrafiltration (30-kDa cutoff). Fractions (1 ml) were applied to a gel filtration column (XK16/60; bed volume, 120 ml) packed with Superdex-200 (Amersham Pharmacia Biotech) equilibrated with 0.15 m NaCl in 50 mm Tris-HCl (pH 7.6), and eluted with the same buffer. Active fractions were pooled, concentrated, and equilibrated to 20 mmTris-HCl (pH 6.7). The resulting sample was applied to a 6-ml Resource Q column. Elution was carried out with a two-step linear NaCl gradient (0–0.2 m and 0.2–1 m). The fractions eluting between 0.22 and 0.25 m NaCl contained mannosylglycerate synthase. Electrophoresis of native proteins was performed on 7.5% polyacrylamide (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205511) Google Scholar). Four protein bands were located by cutting two thin longitudinal strips from the sides of the gel slab and staining with Coomassie Blue. The four bands were recovered by electrophoretic elution from the gel using a BIOTRAP BT 1000 (Schleider & Schuell) at 200 V for 4 h, and each protein was assayed for mannosylglycerate synthase activity. Of these bands, only one had this activity, originating a single band by SDS-PAGE. The N-terminal amino acid sequence of mannosylglycerate synthase was determined by the method of Edman and Begg (31Edman P. Begg G. Eur. J. Biochem. 1967; 1: 80-91Crossref PubMed Scopus (2410) Google Scholar) using an Applied Biosystem Model 477A protein sequencer. The internal sequences were determined by Edman degradation after digestion with trypsin and separation of the peptides by micro-HPLC at the Microchemical Facility, Emory University School of Medicine, GA. Most DNA manipulations followed standard molecular techniques and procedures (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Total DNA from R. marinus was purified by the method of Marmur (33Marmur J. J. Mol. Biol. 1961; 3: 208-218Crossref Scopus (8944) Google Scholar). For sequencing purposes, recombinant plasmid DNAs were prepared using plasmid kits (QIAGEN). Southern blots of restriction endonuclease-cleaved genomic or plasmid DNAs, and colony blots were hybridized with DNA probes labeled with a digoxigenin DNA labeling and detection kit (Roche Molecular Biochemicals). From the N-terminal amino acid sequence of the purified mannosylglycerate synthase, FPFKHEHPEV (amino acids 6–15), the degenerate sense primer 5′-TTCCC(G/C)TTCAAGCA-CGAGCACCC(G/C)GAGGT-3′ was designed. From the partial amino acid sequences of three internal peptides, HFYDADIT (amino acids 97–104), QVELLELFT (amino acids 286–294), and GYDYAQQ (amino acids 359–366), the degenerate antisense primers 5′-GTGATGTC(G/C)GCGTCGTAGAAGTG-3′, 5′-GTGAA(G/C)AGCTC(G/C)AG(G/C)AGCTC(G/C)ACCTG-3′, and 5′-TACTGCTG(G/C)GCGTAGTCGTA(G/C)CC-3′, respectively, were designed. PCR amplifications were carried out in a Perkin-Elmer GeneAmp PCR System 2400 in reaction mixtures (50 μl) containing 200 ng of genomic R. marinus DNA, 100 ng of each primer, 10 mm Tris-HCl (pH 9.0), 1.5 mmMgCl2, 50 mm KCl, 0.5 units of TaqDNA polymerase, and 0.2 mm of each deoxynucleoside triphosphate (Amersham Pharmacia Biotech). The mixture was preincubated for 5 min at 95 °C and then subjected to 20 cycles of denaturation at 95 °C for 1 min. Annealing was performed at gradually decreasing temperatures (59 to 49 °C) for 1 min, and primer extension was at 72 °C for 1 min, followed by 10 cycles in which the annealing temperature remained constant at 49 °C. The extension reaction in the last cycle was prolonged for 5 min. Amplification products were purified from agarose gels for use as hybridization probes, and ligated to the pGEM-T Easy vector (Promega). To obtain a genomic library from R. marinus, total DNA was partially digested with restriction enzyme Sau3A. Fragments ranging from 1 to 5 kbp were purified from agarose gels, and ligated into the BamHI site of dephosphorylated pUC18. The ligation mixture was used to transform E. coli TG1 cells. Transformed cells were plated on YT-broth supplemented with ampicillin, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside, and IPTG. Approximately, 2 × 104 transformants were obtained after 18 h of growth at 37 °C. Similarly, a partial genomic library from R. marinus, containing DNA fragments selected by Southern analysis of genomic DNA preparations with themgs probe, was obtained by complete digestion of total DNA with restriction enzyme PstI, followed by ligation of purified fragments of about 2.1 kbp in size into the PstI site of pUC18, and subsequent transformation of E. coliXL1-Blue cells with the ligation mixture. Positive clones were detected by colony hybridization with digoxigenin-labeled probes as described above. Nucleotide sequences were determined by MWG-BIOTECH (Ebersberg, Germany) using the LI-COR 4200 automated sequencing system. Inserts of positive pUC18 clones (pMG721, pMG7, and pMG161), pGEM-T Easy-clones, and plasmid clone pMG37 were sequenced in both orientations using vector- and insert-specific oligodeoxynucleotide primers. Nucleic acid and protein sequence analyses were conducted with programs in the Wisconsin Genetics Computer Group (GCG) software package (34Devereux J. Haeberli P. Smithies O. Nucleic Acids Res. 1984; 12: 287-395Crossref PubMed Scopus (25) Google Scholar). The European Bioinformatics Institute data bases, and functionally annotated genomes in the Kyoto Encyclopedia of Genes and Genomes, as well as the archaeal genome sequence data base were screened for homologies using the (T)FASTA (35Pearson W.R. Methods Enzymol. 1990; 183: 63-98Crossref PubMed Scopus (1639) Google Scholar, 36Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9328) Google Scholar) and (T)BLAST (37Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (68333) Google Scholar) algorithms. Overexpression of the mgs Gene and Purification of the Enzyme—The mgs gene was amplified by PCR using the forward primer 5′-GCGGAATTCATGAGCCTGGTCGTTTTTCCC-3′ with an additional EcoRI recognition sequence (underlined) immediately upstream of the ATG start codon, and the reversed primer 5′-GCGCTGCAGGGATCCTCAGGCGGTCGACAGTGCC-3′ with additionalPstI and BamHI recognition sequences (underlined) directly behind the TGA stop codon. The PCR product was purified after digestion with EcoRI and PstI, and ligated into the corresponding sites in the pKK223-3 expression vector to obtain plasmid pMG37. E. coli XL1-Blue cells containing pMG37 were grown to the midexponential growth phase (OD600 = 0.4), induced with IPTG, and growth continued for a further 3 to 4 h. Cells were harvested and treated as described above for the preparation of cell-free extracts. The resulting extract was incubated for 30 min at 75 °C and centrifuged. After dialysis against 20 mmTris-HCl (pH 7.6), the sample was loaded onto a Mono Q (Amersham Pharmacia Biotech) column that was eluted by a linear gradient of NaCl (0–500 mm). Fractions with activity were pooled, concentrated, and applied to a Superdex-200 (Amersham Pharmacia Biotech) column. The temperature profile for the activity of mannosylglycerate synthase was determined between 35 and 95 °C, by using the discontinuous method described above. The effect of pH on mannosylglycerate synthase activity was determined at 90 °C in 50 mm MES (pH 5.5–6.5) and 50 mm BisTris-propane buffer (pH 6.5–9.5). All pH values were measured at room temperature; pH values at 90 °C were calculated by using ΔpK a /ΔT °C = −0.011 and −0.015 for MES and BisTris-propane, respectively. Enzyme thermostability was determined at 65 and 90 °C by incubating an enzyme solution in 20 mm Tris-HCl (pH 7.6). At appropriate times, samples were withdrawn and immediately examined for residual mannosylglycerate synthase activity using the discontinuous assay at 90 °C. Kinetic parameters were determined at 90 °C. Reaction mixtures contained GDP mannose (0.165–10.0 mm) plus 10 mmd-glycerate, or d-glycerate (0.188–10.0 mm) plus 10 mm GDP mannose. Samples were preheated for 3 min, the reaction was initiated by the addition of the enzyme preparation, and MG was quantified by 1H NMR. Experiments were performed in duplicate. Values forV max and K m were determined from Hanes plots. The molecular mass of native mannosylglycerate synthase was determined on a Superose 12 column (Amersham Pharmacia Biotech) equilibrated with 50 mm sodium phosphate buffer (pH 7.6) containing 0.15m NaCl. Cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), albumin (66 kDa), aldolase (158 kDa), and ferritin (440 kDa) were used as standards. The isoelectric point of native mannosylglycerate synthase was determined by isoelectric focusing (model 111 mini-IEF cell, Bio-Rad), according to the manufacturer. A pH 3–10 isoelectric focusing gel and standards in a range of pI 4.5–9.6 were used. The nucleotide sequence of mannosylglycerate synthase has been deposited in GenBank under accession number AF173987. By analogy with the known biosynthetic pathways of other sugar derivatives, such as glucosylglycerol (16Hagemann M. Erdmann N. Microbiolology. 1994; 140: 1427-1431Crossref Scopus (94) Google Scholar) and trehalose (38Giæver H.M. Styrvold O.B. Kaasen I. Strøm A.R. J. Bacteriol. 1988; 170: 2841-2849Crossref PubMed Google Scholar), the terminal reaction in the synthesis of MG should involve a sugar nucleotide as donor of the glycosyl moiety, and a phosphorylated acceptor (3-P-glycerate). From an array of experiments with GDP mannose, UDP mannose, and ADP mannose as donors, andd-3-P-glycerate as acceptor, we were unable to detect the formation of MG in cell extracts prepared from R. marinusgrown in medium containing 4% NaCl. Activity for the synthesis of MG, however low, was detected upon the addition of NaCl or KCl (150, 300, and 450 mm), to the enzyme assay, when GDP mannose was used as a glycosyl donor. Optimal activity was observed at 300 mm NaCl or KCl. Unexpectedly, the activity for the formation of MG in cell extracts was significantly higher whend-glycerate, instead of d-3-P-glycerate, was used in addition to GDP mannose (Table I, compare the values for mannosylglycerate synthase and mannosyl 3-P-glycerate synthase/mannosyl 3-P-glycerate phosphatase). Furthermore, this activity did not depend on the salt concentration in the assay mixture (0–450 mm NaCl or KCl). 1H NMR was used to detect and monitor the time course for the formation of MG in crude cell extracts at 65 °C. In the anomeric region of the spectra clear resonances due to the anomeric protons of MG and the ribose and mannose moieties in GDP mannose were detected, allowing to monitor on line the consumption of the substrate and build up of the product (Fig. 1).Table IEnzymatic activities involved in the biosynthesis of mannosylglycerate in cell-free extracts of Rhodothermus marinusEnzymesSpecific activitiesaAll specific activities are the mean values of six assays in crude extracts prepared from cells obtained from three independent growths./NaCl in the growth media1%4%6%nmol/min·mg proteinPhosphoglucose isomerase621 ± 14551 ± 19556 ± 17Phosphomannose isomerase657 ± 28528 ± 8470 ± 12Phosphomannose mutase242 ± 3174 ± 7102 ± 6Mannose-1-P guanylyltransferase107 ± 1491 ± 2098 ± 25Mannosylglycerate synthase17 ± 0.112 ± 617 ± 6Mannosyl-3-P-glycerate synthase bActivities measured in the presence of 0.3 mKCl.3.0 ± 1.42.0 ± 0.11.3 ± 0.3(Mannosyl-3-P-glycerate phosphatase)a All specific activities are the mean values of six assays in crude extracts prepared from cells obtained from three independent growths.b Activities measured in the presence of 0.3 mKCl. Open table in a new tab The use of two different substrates, d-glycerate andd-3-P-glycerate, combined with the differences in the" @default.
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• W1973587531 title "Biosynthesis of Mannosylglycerate in the Thermophilic Bacterium Rhodothermus marinus" @default.
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