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• W1969751337 abstract "ATP-dependent β-glucoside kinase (BglK) has been purified from cellobiose-grown cells of Klebsiella pneumoniae. In solution, the enzyme (EC2.7.1.85) exists as a homotetramer composed of non-covalently linked subunits of Mr ∼33,000. Determination of the first 28 residues from the N terminus of the protein allowed the identification and cloning of bglK from genomic DNA ofK. pneumoniae. The open reading frame (ORF) ofbglK encodes a 297-residue polypeptide of calculatedMr 32,697. A motif of 7 amino acids (AFD7IG9GT) near the N terminus may comprise the ATP-binding site, and residue changes D7G and G9A yielded catalytically inactive proteins. BglK was progressively inactivated (t12 ∼ 19 min) by N-ethylmaleimide, but ATP afforded considerable protection against the inhibitor. By the presence of a centrally located signature sequence, BglK can be assigned to the ROK (Repressor, ORF,Kinase) family of proteins. Preparation ofHis6BglK by nickel-nitrilotriacetic acid-agarose chromatography provided high purity enzyme in quantity sufficient for the preparative synthesis (200–500 mg) of ten 6-phospho-β-d-glucosides, including cellobiose-6′-P, gentiobiose-6′-P, cellobiitol-6-P, salicin-6-P, and arbutin-6-P. These (and other) derivatives are substrates for phospho-β-glucosidase(s) belonging to Families 1 and 4 of the glycosylhydrolase superfamily. The structures, physicochemical properties, and phosphorylation site(s) of the 6-phospho-β-d-glucosides have been determined by fast atom bombardment-negative ion spectrometry, thin-layer chromatography, and 1H and 13C NMR spectroscopy. The recently sequenced genomes of two Listeria species, L. monocytogenes EGD-e and L. innocua CLIP 11262, contain homologous genes (lmo2764 and lin2907, respectively) that encode a 294-residue polypeptide (Mr ∼ 32,200) that exhibits ∼58% amino acid identity with BglK. The protein encoded by the two genes exhibits β-glucoside kinase activity and cross-reacts with polyclonal antibody to His6BglK from K. pneumoniae. The location oflmo2764 and lin2907 within a β-glucoside (cellobiose):phosphotransferase system operon, may presage both enzymatic (kinase) and regulatory functions for the BglK homolog inListeria species. ATP-dependent β-glucoside kinase (BglK) has been purified from cellobiose-grown cells of Klebsiella pneumoniae. In solution, the enzyme (EC2.7.1.85) exists as a homotetramer composed of non-covalently linked subunits of Mr ∼33,000. Determination of the first 28 residues from the N terminus of the protein allowed the identification and cloning of bglK from genomic DNA ofK. pneumoniae. The open reading frame (ORF) ofbglK encodes a 297-residue polypeptide of calculatedMr 32,697. A motif of 7 amino acids (AFD7IG9GT) near the N terminus may comprise the ATP-binding site, and residue changes D7G and G9A yielded catalytically inactive proteins. BglK was progressively inactivated (t12 ∼ 19 min) by N-ethylmaleimide, but ATP afforded considerable protection against the inhibitor. By the presence of a centrally located signature sequence, BglK can be assigned to the ROK (Repressor, ORF,Kinase) family of proteins. Preparation ofHis6BglK by nickel-nitrilotriacetic acid-agarose chromatography provided high purity enzyme in quantity sufficient for the preparative synthesis (200–500 mg) of ten 6-phospho-β-d-glucosides, including cellobiose-6′-P, gentiobiose-6′-P, cellobiitol-6-P, salicin-6-P, and arbutin-6-P. These (and other) derivatives are substrates for phospho-β-glucosidase(s) belonging to Families 1 and 4 of the glycosylhydrolase superfamily. The structures, physicochemical properties, and phosphorylation site(s) of the 6-phospho-β-d-glucosides have been determined by fast atom bombardment-negative ion spectrometry, thin-layer chromatography, and 1H and 13C NMR spectroscopy. The recently sequenced genomes of two Listeria species, L. monocytogenes EGD-e and L. innocua CLIP 11262, contain homologous genes (lmo2764 and lin2907, respectively) that encode a 294-residue polypeptide (Mr ∼ 32,200) that exhibits ∼58% amino acid identity with BglK. The protein encoded by the two genes exhibits β-glucoside kinase activity and cross-reacts with polyclonal antibody to His6BglK from K. pneumoniae. The location oflmo2764 and lin2907 within a β-glucoside (cellobiose):phosphotransferase system operon, may presage both enzymatic (kinase) and regulatory functions for the BglK homolog inListeria species. phosphoenolpyruvate phosphotransferase system β-glucoside kinase 6-phospho-β-d-glucoside nickel-nitrilotriacetic acid 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol open reading frame glucose-6-phosphate dehydrogenase pyruvate kinase l-lactate dehydrogenase 4-morpholineethanesulfonic acid fast atom bombardment glucose 6-phosphate phospho-β-glucosidase high speed supernatant fluid cellobiose-6′-phosphate hydrolase repressor, ORF, kinase N-ethylmaleimide glucokinase The phosphoenolpyruvate-dependent sugar:phosphotransferase system (P-enolpyruvate:PTS),1 was first described by Roseman and colleagues in Escherichia coli in 1964 (1Kundig W. Ghosh S. Roseman S. Proc. Natl. Acad. Sci. U. S. A. 1964; 52: 1067-1074Crossref PubMed Scopus (326) Google Scholar). This multicomponent group translocation system is now recognized as the primary route for entry, and simultaneous phosphorylation, of carbohydrates by many bacterial species from both Gram-negative (2Meadow N.D. Fox D.K. Roseman S. Annu. Rev. Biochem. 1990; 59: 497-542Crossref PubMed Scopus (302) Google Scholar, 3Postma P.W. Lengeler J.W. Jacobson G.R. Microbiol. Rev. 1993; 57: 543-594Crossref PubMed Google Scholar) and Gram-positive genera (4Reizer J. Saier M.H., Jr. Deutscher J. Grenier F. Thompson J. Hengstenberg W. Crit. Rev. Microbiol. 1988; 15: 297-338Crossref PubMed Scopus (130) Google Scholar, 5Thompson J. Reizer J. Peterkofsky A. Sugar Transport and Metabolism in Gram-positive Bacteria. Ellis Horwood, Chichester, Great Britain1987: 13-38Google Scholar). Hexose monophosphates may immediately enter the central energy-generating pathways, but prior to their dissimilation, accumulated disaccharide phosphates must first be hydrolyzed by sugar-specific disaccharide-phosphate hydrolases. Of the latter inducible enzymes, phospho-β-galactosidase (EC 3.2.1.85) and phospho-β-glucosidase (EC3.2.1.86) have received considerable attention (6Witt E. Frank R. Hengstenberg W. Protein Eng. 1993; 6: 913-920Crossref PubMed Scopus (35) Google Scholar, 7Wiesmann C. Hengstenberg W. Schultz G.E. J. Mol. Biol. 1997; 269: 851-860Crossref PubMed Scopus (53) Google Scholar). By sequence-based alignment these enzymes are assigned to Family 1 of the 87-member superfamily of glycosylhydrolases (8Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2616) Google Scholar). 2P. M. Coutinho and B, Henrissat (1999) Carbohydrate-Active Enzymes, available at afmb.cnrs-mrs.fr/∼cazy/CAZY/index.html.2P. M. Coutinho and B, Henrissat (1999) Carbohydrate-Active Enzymes, available at afmb.cnrs-mrs.fr/∼cazy/CAZY/index.html. Until recently, microbial phosphoglycosylhydrolases were found only in Family 1 of glycosylhydrolases. However, studies begun in our laboratory (9Thompson J. Gentry-Weeks C.R. Nguyen N.Y. Folk J.E. Robrish S.A. J. Bacteriol. 1995; 177: 2505-2512Crossref PubMed Google Scholar) and, subsequently, in other institutions, have identified a novel group of phosphoglucosylhydrolases in an increasing number of bacterial species, including Bacillus subtilis(10Varrot A. Yamamoto H. Sekiguchi J. Thompson J. Davies G.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1212-1214Crossref PubMed Scopus (10) Google Scholar, 11Thompson J. Pikis A. Ruvinov S.B. Henrissat B. Yamamoto H. Sekiguchi J. J. Biol. Chem. 1998; 273: 27347-27356Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), Fusobacterium mortiferum (12Bouma C.L. Reizer J. Reizer A. Robrish S.A. Thompson J. J. Bacteriol. 1997; 179: 4129-4137Crossref PubMed Google Scholar), E. coli (13Nagao Y. Nakada T. Imoto M. Shimamoto T. Sakai S. Tsuda M. Tsuchiya T. Biochem. Biophys. Res. Commun. 1988; 151: 236-241Crossref PubMed Scopus (30) Google Scholar, 14Thompson J. Ruvinov S.B. Freedberg D.I. Hall B.G. J. Bacteriol. 1999; 181: 7339-7345Crossref PubMed Google Scholar), Klebsiella pneumoniae (15Thompson J. Robrish S.A. Immel S. Lichtenthaler F.W. Hall B.G. Pikis A. J. Biol. Chem. 2001; 276: 37415-37425Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), andThermotoga maritima (16Raasch C. Streit W. Schanzer J. Bibel M. Gosslar U. Liebl W. Extremophiles. 2000; 4: 189-200Crossref PubMed Scopus (47) Google Scholar). By their inherent instability, oligomeric structure, sequence homology, and conserved signature pattern, these enzymes are assigned to Family 4 of glycosylhydrolases.2 Remarkably, and in contrast to other glycosylhydrolases described thus far, members of Family 4 require both a dinucleotide (NAD+) and a divalent metal ion (Mn2+, Fe2+, Co2+, or Ni2+) for activity. The one or more functions of these unique cofactors are presently unknown, and the catalytic mechanism for cleavage of the O-glycosyl linkage in their substrates has yet to be established. Significantly, the crystal structure has not been solved for any member of Family 4. In large measure, these deficiencies are directly attributable to the lack of the appropriate P-enolpyruvate:PTS-derived substrates for the enzymes. In the absence of the natural disaccharide-6′-Ps, chromogenic analogs such asp-nitrophenyl-α- andp-nitrophenyl-β-d-glucopyranoside 6-phosphates, respectively, have been used extensively for the assay and partial characterization of phosphoglycosylhydrolase activities. The former chromogenic substrates (and structurally similar alkyl and aryl glycoside phosphates) have usually been prepared by phosphorylation of the parent glycosides at the single primary -OH group with 2-cyanoethyl phosphate or phosphorous oxychloride (17Hengstenberg W. Morse M.L. Carbohydr. Res. 1968; 7: 180-183Crossref Scopus (11) Google Scholar, 18Hengstenberg W. Morse M.L. Carbohydr. Res. 1969; 10: 463-465Crossref Scopus (18) Google Scholar, 19Wilson G. Fox C.F. J. Biol. Chem. 1974; 249: 5586-5596Abstract Full Text PDF PubMed Google Scholar). Unfortunately, these non-selective phosphorylating agents cannot be used for synthesis of the disaccharide-6′-P products of the P-enolpyruvate:PTS, because the presence of two primary -OH groups yields a tripartite mixture of the -6-P, -6′-P, and -6,6′-P2 derivatives. Regiospecific chemical syntheses of disaccharide 6′-phosphates would entail the addition and subsequent removal of protecting groups, together with potentially laborious methods for purification of the derivatives. It is perhaps in light of these difficulties that compounds such as cellobiose-6′-phosphate and maltose-6′-phosphate are not commercially available, and it was for these reasons that we sought enzymatic routes for the biosynthesis of the α- and β-conformers of disaccharide monophosphates. Fortuitously, during studies of PTS functions in permeabilized cells of K. pneumoniae, we discovered that, although low pH caused inactivation of intracellular phospho-α-glucosidase, the α-glucoside-specific PTS remained operative under the same conditions. This serendipitous finding permitted the synthesis and facile isolation of a wide variety of phospho-α-glucoside products of the PTS, including maltose-6′-P and the 6-phospho- derivatives of sucrose and its five linkage isomers (20Thompson J. Robrish S.A. Pikis A. Brust A. Lichtenthaler F.W. Carbohydr. Res. 2001; 331: 149-161Crossref PubMed Scopus (42) Google Scholar). As a potential route for the biosynthesis of phospho-β-glucosides, we turned our attention to the report some 30 years ago by Palmer and Anderson (21Palmer R.E. Anderson R.L. J. Biol. Chem. 1972; 247: 3415-3419Abstract Full Text PDF PubMed Google Scholar) of an ATP-dependent β-glucoside kinase present in K. pneumoniae. Although only partially purified, the enzyme preparation of Palmer and Anderson catalyzed the in vitro phosphorylation of several β-glucosides and, indeed, a small quantity of cellobiose-6′-phosphate was prepared by these investigators. In the present communication we describe the purification, substrate specificity, and kinetic parameters of β-glucoside kinase (BglK, EC2.7.1.85) from K. pneumoniae. The chromosomal genebglK has been cloned, and catalytically activeHis6BglK has been purified from a high expression system by Ni2+-NTA-agarose chromatography. The availability ofHis6BglK in quantity (and high purity) has allowed the first preparative synthesis of 10 phospho-β-glucosides. The biosynthesis, method of isolation, and some of the physicochemical properties of these novel phospho-β-glucosylhydrolase substrates are presented herein. By sequence-based alignment we show that BglK ofK. pneumoniae can be assigned to the ROK (Repressor, ORF, Kinase) family of proteins (22Titgemeyer F. Reizer J. Reizer A. Saier M.H., Jr. Microbiology (UK). 1994; 140: 2349-2354Crossref PubMed Scopus (151) Google Scholar) 3Welcome Trust Sanger Institute. ROK Family, available at www.sanger.ac.uk/cgi-bin/Pfam/getacc?PF00480.3Welcome Trust Sanger Institute. ROK Family, available at www.sanger.ac.uk/cgi-bin/Pfam/getacc?PF00480. and that a homolog of BglK is encoded within the cel-PTS operon in two species of Listeria. Salicin and gentiobiose were obtained from Pfanstiehl Laboratories, and thiocellobiose was purchased from Toronto Research Chemicals, Inc. Other carbohydrates and reagents, including dinucleotides, ATP, P-enolpyruvate, DEAE-TrisAcryl M, and ATP-agarose (4% cross-linked, nine spacer atoms) were purchased from Sigma Chemical Co. Other materials and suppliers, included Ni2+-NTA-agarose (Qiagen), Ultrogel-AcA (Sepracor), and Polygram cell 400 thin-layer microcrystalline sheets (Macherey and Nagel). Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and the mixture of pyruvate kinase (PK, EC 2.7.1.40)/l-lactate dehydrogenase (LDH, EC 1.1.1.27) were obtained from Boehringer and Roche Diagnostics, respectively. Polyclonal (rabbit) antibody to BglK was prepared by Covance Research Products using purifiedHis6BglK as immunogen. K. pneumoniae ATCC 23357 was grown in the medium described by Sapico et al. (23Sapico V. Hanson T.E. Walter R.W. Anderson R.L. J. Bacteriol. 1968; 96: 51-54Crossref PubMed Google Scholar), supplemented with 0.4% (w/v) of appropriate sugar. The medium (800 ml) was contained in 1-liter bottles, and cells were grown at 37 °C to the stationary phase. Cells were harvested by centrifugation (13,000 ×g for 10 min at 5 °C) and washed twice by resuspension and centrifugation from 25 mm Tris-HCl buffer (pH 7.5) containing 1 mm MgCl2 (designated TM buffer). The yield of cells was approximately 2 g of wet weight/liter. Cells of E. coli TOP 10 (pTrcHisbglK) were grown with vigorous aeration at 37 °C in LB medium containing ampicillin (150 μg/ml). Isopropyl-1-thio-β-d-galactopyranoside (1 mm) was added to the culture at A600 nm ∼ 0.4, and 4 h later, the cells were harvested and washed with TM buffer. The yield was approximately 3.7 g of wet weight of cells/liter. The Novex X-Cell mini system (Invitrogen) was used for SDS-PAGE procedures. Denatured samples of cell extracts and Novex Mark 12 protein standards were electrophoresed in Novex NuPage (4–12%) BisTris gels with MES-SDS (pH 7.3) as the running buffer. Proteins were visualized by staining with Coomassie Brilliant Blue R-250. The pI of BglK was determined by analytical electrofocusing (at 10 °C) in anAmersham Biosciences Multiphor flat-bed electrophoresis unit, using precast Ampholine PAG layers (pH range, 3.5–9.5) and broad range pI standards. Cell extracts and SeeBlue (Invitrogen)-prestained markers were electrophoresed by SDS-PAGE, and proteins were electrophoretically transferred to nitrocellulose membranes in NuPage transfer buffer. Immunodetection of native and mutated forms of BglK was achieved by sequential incubation of the membranes with polyclonal antibody toHis6BglK and goat anti-rabbit horseradish peroxidase-conjugated antibody, as described previously (9Thompson J. Gentry-Weeks C.R. Nguyen N.Y. Folk J.E. Robrish S.A. J. Bacteriol. 1995; 177: 2505-2512Crossref PubMed Google Scholar). Negative-ion FAB spectra of the P-β-d-glucosides were obtained on a JEOL SX102 mass spectrometer. The compounds were desorbed from a glycerol matrix with 6 KeV xenon atoms, and mass measurements [M-H]−1 in FAB mode were performed at 10,000 times resolution using electric field scans and matrix ions as reference material. Low resolution analysis provided integer-mass information, and high resolving power was used for determination (and confirmation) of molecular formulae. Thin-layer chromatographic analyses were performed using 0.1-mm-thick layers of microcrystalline cellulose and a solvent containing n-butanol/acetic acid/water (5:2:3, v/v). The phosphate-containing derivatives were visualized by sequential dipping of the air-dried layers in solutions containing: (i) 50 mg of ferric chloride and 1 ml of 1 nHCl, dissolved in 100-ml of acetone, and (ii) 1.25 g of sulfosalicylic acid in 100 ml of acetone (24Wade H.E. Morgan D.M. Nature. 1953; 171: 529-530Crossref PubMed Scopus (63) Google Scholar). 1H and13C NMR spectra of the phosphates and their parent sugars were recorded on a Bruker AVANCE 500 spectrometer. Signal assignments were confirmed by COSY, heteronuclear correlation, and total correlation spectroscopy experiments. Chemical shifts (listed below in Tables IV and V) are reported in D2O relative to sodium 2,2,3,3-tetradeutero-3-trimethylsilyl propionate as internal standard.Table IV1H NMR data (500 MHz in D2O, 25 °C, ppm from internal TSP4-aTSP, sodium 2,2,3,3-tetradeutero-3-trimethylsilyl-propionate. of β-d-glucosyl-glucoses, cellobiitol, and β-d-glucosides compared with their monophosphates, uniformly carrying the phosphate group at the terminal primary OHCompoundβ-d-Glucopyranoside residueReducing glucose residueH-1H-2H-3H-4H-56-H2H-1H-2H-3H-4H-56-H2β-p-Cellobiose4.513.333.513.433.493.74, 3.924.673.293.633.633.633.81, 3.96β-p-Cellobiose-6′-P4.523.353.533.573.574.02, 4.074.673.293.633.633.633.82, 3.96β-p-Thiocellobiose4.653.363.523.433.473.71, 3.894.643.293.582.893.693.93, 4.09β-p-Thiocellobiose-6′-P4.673.373.543.593.544.014.673.293.612.873.723.92, 4.11β-p-Gentiobiose4.523.333.513.403.473.73, 3.934.663.263.483.473.633.85, 4.21β-p-Gentiobiose-6′-P5.473.353.513.603.494.034.673.273.493.443.653.85, 4.21d-Glucitol residueCellobiitol4.583.343.513.443.443.76, 3.913.60, 3.783.973.853.883.943.75, 3.88Cellobiitol-6-P4.573.373.533.583.514.033.63, 3.823.983.843.893.953.74, 3.89Methyl β-d-glucoside4.373.273.493.383.463.73, 3.93Methyl β-d-glucoside-6-P4.393.293.523.573.524.05Phenyl β-d-glucoside5.143.583.633.513.633.76, 3.94Phenyl β-d-glucoside-6-P5.143.633.663.713.664.04, 4.10iPropyl 1-thio-β-d-glucoside4.613.293.513.413.483.70, 3.90iPropyl 1-thio-β-d-glucoside-6-P4.623.303.523.623.524.02Salicin5.123.653.623.523.623.77, 3.93Salicin-6-P5.143.693.693.693.694.08Arbutin4.953.593.643.553.553.80, 3.95Arbutin-6-P4.973.573.623.713.624.064-Methylumbelliferyl β-d-glucoside5.203.653.653.563.713.81, 3.984-Methylumbelliferyl β-d-glucoside-6-P5.173.693.743.773.744.10, 4.184-a TSP, sodium 2,2,3,3-tetradeutero-3-trimethylsilyl-propionate. Open table in a new tab Table V13C chemical shifts (125 MHz in D2O, 25 °C, ppm from internal TSP5-aTSP, sodium 2,2,3,3-tetradcutero-3-trimethylsilyIpropionate.) of β-d-glucosyl-glucoses, cellobiitol, and β-d-glucosides compared with their monophosphates, uniformly carrying the phosphate group at the terminal primary OHCompoundβ-d-Glucopyranoside residueReducing glucose residueC-1C-2C-3C-4C-5C-6C-1C-2C-3C-4C-5C-6β-p-Cellobiose105.375.978.372.378.863.498.576.776.977.681.562.9β-p-Cellobiose-6′-P105.776.278.171.978.265.998.676.777.277.682.263.1β-p-Thiocellobiose86.775.579.972.282.663.698.578.275.850.079.364.3β-p-Thiocellobiose-6′-P86.875.479.571.782.066.098.478.375.850.479.064.3β-p-Gentiobiose105.575.978.572.578.963.699.076.978.572.477.771.7β-p-Gentiobiose-6′-P105.876.278.071.878.265.598.876.978.572.577.772.0d-Glucitol residueCellobiitol105.476.278.472.278.763.365.575.172.382.174.064.9Cellobiitol-6-P105.576.478.072.078.165.865.374.972.682.474.265.0Methyl β-d-glucoside106.075.978.672.578.763.6Methyl β-d-glucoside-6-P106.175.977.971.677.865.5Phenyl β-d-glucoside103.075.878.572.379.063.4Phenyl β-d-glucoside-6-P102.775.577.471.177.865.0iPropyl 1-thio-β-d-glucoside87.375.380.172.482.663.8iPropyl 1-thio-β-d-glucoside-6-P87.775.479.671.781.965.8Salicin103.475.878.572.378.963.4Salicin-6-P103.576.078.071.678.365.6Arbutin104.275.878.572.378.863.0Arbutin-6-P104.476.077.971.678.365.44-Methylumbelliferyl β-d-glucoside102.675.778.472.379.163.44-Methylumbelliferyl β-d-glucoside-6-P102.875.977.771.578.565.55-a TSP, sodium 2,2,3,3-tetradcutero-3-trimethylsilyIpropionate. Open table in a new tab The procedure outlined for cellobiose-6′-P (with adjustment for the limited quantity of some substrates) was used for the preparation of other phosphorylated compounds. Cellobiose (2 mmol) was dissolved in 10 ml of 25 mm HEPES buffer (pH 7.5) containing 2 mm MgSO4 and immediately added to 10 ml of water containing 1 mmol of ATP (adjusted to pH 7.5 with ∼0.6 ml of 3m NH4OH). His6BglK was added (∼40 units), and, throughout a 2-h incubation period at room temperature, the pH of the reaction mixture was maintained at 7.5 by addition of a 3m NH4OH solution. Thereafter, the pH was adjusted to 8.2 with NH4OH, and 4 ml of barium acetate solution (3 mmol) was added with stirring. The heavy white precipitate of the barium salts of ADP and residual ATP was removed by centrifugation, and the supernatant fluid was clarified by filtration through a Millex (0.22-μm pore size) membrane. The filtrate (approximately 22 ml) was chilled on ice, 4 volumes of absolute ethanol (0 °C) was added, and the mixture was transferred to a cold room overnight. The flocculent precipitate of the Ba2+ salt of cellobiose-6′-P (together with trace amounts of nucleotide salts) was collected by centrifugation. The white pellet was dried at 37 °C for about 30 min, and Ba2+ ions were exchanged for H+ by addition of 2–3 ml of an aqueous suspension of Bio-Rad AG 50Wx2 (H+ form) resin. Resin beads were removed by filtration, and the filtrate was adjusted to pH 7.2 by NH4OH addition. The solution was frozen and lyophilized, to yield 300–500 mg of the white, crystalline ammonium salt of cellobiose-6′-P. Except for thiocellobiose-6′-P, all P-β-glucosides were quantitatively determined by enzymatic assay of Glc6P released by acid hydrolysis (1 n HCl for 2 h at 100 °C). Prior to mass spectrometry and NMR spectroscopy, trace contaminants of ADP and ATP were removed from ∼50 mg of each derivative by paper chromatography (20Thompson J. Robrish S.A. Pikis A. Brust A. Lichtenthaler F.W. Carbohydr. Res. 2001; 331: 149-161Crossref PubMed Scopus (42) Google Scholar). Using sequence information from the unfinished genome project ofK. pneumoniae strain MGH 78578 (Washington University Genome sequencing Center, St. Louis, MO), two primers, KPC808 (5′-TTGCCCCTGCGGAAAAATAC-3′) and KPC2116 (5′-TACAGTCTGGTGCTTGCCCTCTACG-3′), were designed to amplify the DNA fragment encoding bglK and a portion of a putative phospho-β-glucosidase (pbgA) gene of K. pneumoniae ATCC 23357. PCR amplification was carried out in a thermal cycler (PerkinElmer Life Sciences Model 9600) in a reaction mixture (100 μl) containing 100 ng of K. pneumoniae ATCC 23357 chromosomal DNA, 10 μl of 10× reaction buffer, 20 mm each of the four DNTPs, 250 ng of each primer, 5 units of Pfu DNA polymerase (Stratagene, La Jolla, CA), and 1% (v/v) Me2SO. After an initial 2-min denaturation at 95 °C, the mixture was subjected to 30 cycles of amplification. Each cycle consisted of 1 min of denaturation at 95 °C, 1 min of annealing at 50 °C, and 2 min and 36 s of extension at 72 °C. These were followed by a 10-min runoff at 72 °C. The PCR product was purified (QIAquick PCR purification kit, Qiagen) and ligated into the pCR-Blunt vector (Invitrogen, Carlsbad, CA). The recombinant plasmid was transformed into E. coli TOP 10-competent cells, and colonies were selected on Luria-Bertani agar plates containing 50 μg/ml kanamycin. Sequencing was accomplished by the dideoxynucleotide chain-termination method using the Sequenase 7-deaza-dGTP sequencing kit (U.S. Biochemicals) and [α-35S]dATP for labeling. Both strands of the DNA insert were sequenced. Sequences were assembled, edited, and analyzed with MacVector sequence analysis package (version 7.0, Genetics Computer Group, Madison, WI). For the amplification of bglK, two primers were synthesized from the sequence data presented in Fig. 2. Forward primer F1, 5′-CCCCGGATCCCATGAAGATTGCGGCATTTGATATCGG-3′ (the bglK sequence is in boldface, and the BamHI site is underlined); reverse primer R1, 5′-GGGGGTAAGCTTCTACTAATGTCGATCGTCGTCTGGCG-3′ (the sequence complimentary to the downstream region of bglKis in boldface, and the HindIII site is underlined). After amplification with high fidelity Pfu DNA polymerase, the DNA fragment was digested with restriction endonucleases (BamHI and HindIII), electrophoresed (1% agarose), and purified (QIAquick gel extraction kit). The purified 0.9-kb DNA fragment was ligated into the similarly digested and purified high expression vector pTrcHisB. The recombinant plasmid (pTrcHisBbglK) was transformed into competent cells ofE. coli TOP 10, and transformants were selected in LB agar plates containing 150 μg/ml ampicillin. A high level of expression of the histidine-tagged enzyme His6BglK was initiated by addition of 1 mmisopropyl-1-thio-β-d-galactopyranoside to logarithmic phase cultures (A600 nm ∼ 0.3) ofE. coli TOP 10 (pTrcHisBbglK). From the complete genome sequence(s) of L. monocytogenes EGD-e and L. innocua CLIP 11262 (25Glaser P. Frangeul L. Buchrieser C. Rusniok C. Amend A. Baquero F. Berche P. Bloecker H. Brandt P. Chakraborty T. Charbit A. Chetouani F. Couveá E. de Daruvar A. Dehoux P. Domann E. Dominguez-Bernal G. Duchaud E. Durant L. Dussurget O. Entian K.-D. Fsihi H. Garcia-Del Portillo F. Garrido P. Gautier L. Goebel W. Goámez-Loápez N. Hain T. Hauf J. Jackson D. Jones L.-M. Kaerst U. Kreft J. Kuhn M. Kunst F. Kurapkat G. Madueño E. Maitournam A. Mata Vicente J., Ng, E. Nedjari H. Nordsiek G. Novella S. de Pablos B. Peárez-Diaz J.-C. Purcell R. Remmel B. Rose M. Schlueter T. Simoes N. Tierrez A. Vázquez-Boland J.-A. Voss H. Wehland J. Cossart P. Science. 2001; 294: 849-852PubMed Google Scholar), the following pairs of primers were designed for amplification of geneslmo2764 and lin2907, respectively. For amplification of lmo2764, the forward primer EGD-eF1 was 5′-CCCCGGATCCCATGAAAATTGCAGCTTTTGATATCGG-3′ (the lmo2764 sequence is in boldface, and theBamHI site is underlined) and reverse primer EGD-eR1 was 5′-GGGGGTAAGCTTCATCATTCATGTCTATTTTCCTCC-3′ (the sequence complimentary to the downstream region of lmo2764is in boldface, and the HindIII site is underlined). For amplification of lin2907, the forward primer CLIP-F1 was 5′-CCCCGGATCCCATGAAAATTGCAGCATTTGATATTGG-3′ (thelin2907 sequence is in boldface, and the BamHI site is underlined), and reverse primer CLIP-R1 was identical to EGD-eR1. After amplification, digestion, and purification, the two ∼0.9-kb DNA fragments were ligated (as described previously) into the high expression vector pTrcHis2B to yield plasmids pTrcHisBlmo2764 and pTrcHisBlin2907, respectively. The two plasmids were transformed into E. coliTOP 10-competent cells for expression of the Listeria gene products. (Note: incorporation of the two stop codons in our reverse primers prevents expression and incorporation of the C-terminal His6 fusion peptides normally generated by use of pTrcHis2 vectors.) The method required the use of PfuTurbo DNA polymerase, a temperature cycler, and reagents that were obtained as a kit (QuikChange site-directed mutagenesis kit, Stratagene). E. coli TOP 10-competent cells were transformed with plasmids of pTrcHisBbglK containing the desired mutation. Base changes effected by site-directed mutagenesis were confirmed by sequence analysis. Washed cells (∼16 g of wet wt) grown previously on cellobiose as the energy source were resuspended in 24 ml of TM buffer. Cells were disrupted at 0 °C, by 2× 1.5-min periods of sonic oscillation in a Branson (Model 350) instrument operated at ∼75% of maximum power. BglK was purified in four stages by low pressure chromatography. Column flow rates were maintained by a P-1 peristaltic pump interfaced with a Frac-100 collector. Proteins present in column eluents were monitored at 280 nm by a UV-1 optical control unit connected to a single-channel chart recorder. (All instruments were from Amersham Biosciences.) The sonicated extract was clarified by ultracentrifugation (180,000 × g for 2 h at 5 °C). The HSS was transferred to sacs and dialyzed overnight against 4 liters of TM buffer (at 4 °C) The dialyzed HSS (∼35 ml) was transferred at a flow rate of 0.8 ml/min to a column of DEAE-TrisAcryl M (2.6 × 10 cm) previously equilibrated with TM buffer. Non-adsorbed materials were removed by washing with TM buffer, and BglK was eluted with 400 ml of a linear, increasing concentration gradient of NaCl (0–300 mm) in TM buffer. Fractions of 6 ml were collected, and samples (30 μl) were assayed for enzymatic activity. Fractions (33Tarelli E. Wheeler S.F. Carbohydr. Res. 1994; 261: 25-36Crossref Scopus (16) Google Scholar, 34Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55635) Google Scholar, 35Davies G. Henrissat B. Structure. 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1609) Google Scholar, 36Henrissat B. Davies G. Curr. Op" @default.
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