The Gene glvA of Bacillus subtilis 168 Encodes a Metal-requiring, NAD(H)-dependent 6-Phospho-α-glucosidase
1998; Elsevier BV; Volume: 273; Issue: 42 Linguagem: Inglês
10.1074/jbc.273.42.27347
ISSN1083-351X
AutoresJohn F. Thompson, Andreas Pikis, Sergei B. Ruvinov, Bernard Henrissat, Hiroki Yamamoto, Junichi Sekiguchi,
Tópico(s)Biochemical and Molecular Research
ResumoThe gene glvA (formerlyglv-1) from Bacillus subtilis has been cloned and expressed in Escherichia coli. The purified protein GlvA (449 residues, M r = 50,513) is a unique 6-phosphoryl-O-α-d-glucopyranosyl:phosphoglucohydrolase (6-phospho-α-glucosidase) that requires both NAD(H) and divalent metal (Mn2+, Fe2+, Co2+, or Ni2+) for activity. 6-Phospho-α-glucosidase (EC3.2.1.122) from B. subtilis cross-reacts with polyclonal antibody to maltose 6-phosphate hydrolase from Fusobacterium mortiferum, and the two proteins exhibit amino acid sequence identity of 73%. Estimates for the M r of GlvA determined by SDS-polyacrylamide gel electrophoresis (51,000) and electrospray-mass spectroscopy (50,510) were in excellent agreement with the molecular weight of 50,513 deduced from the amino acid sequence. The sequence of the first 37 residues from the N terminus determined by automated analysis agreed precisely with that predicted by translation of glvA. The chromogenic and fluorogenic substrates, p-nitrophenyl-α-d-glucopyranoside 6-phosphate and 4-methylumbelliferyl-α-d-glucopyranoside 6-phosphate were used for the discontinuous assay and in situ detection of enzyme activity, respectively. Site-directed mutagenesis shows that three acidic residues, Asp41, Glu111, and Glu359, are required for GlvA activity. Asp41 is located at the C terminus of a βαβ fold that may constitute the dinucleotide binding domain of the protein. Glu111 and Glu359 may function as the catalytic acid (proton donor) and nucleophile (base), respectively, during hydrolysis of 6-phospho-α-glucoside substrates including maltose 6-phosphate and trehalose 6-phosphate. In metal-free buffer, GlvA exists as an inactive dimer, but in the presence of Mn2+ ion, these species associate to form the NAD(H)-dependent catalytically active tetramer. By comparative sequence alignment with its homologs, the novel 6-phospho-α-glucosidase from B. subtilis can be assigned to the nine-member family 4 of the glycosylhydrolase superfamily. The gene glvA (formerlyglv-1) from Bacillus subtilis has been cloned and expressed in Escherichia coli. The purified protein GlvA (449 residues, M r = 50,513) is a unique 6-phosphoryl-O-α-d-glucopyranosyl:phosphoglucohydrolase (6-phospho-α-glucosidase) that requires both NAD(H) and divalent metal (Mn2+, Fe2+, Co2+, or Ni2+) for activity. 6-Phospho-α-glucosidase (EC3.2.1.122) from B. subtilis cross-reacts with polyclonal antibody to maltose 6-phosphate hydrolase from Fusobacterium mortiferum, and the two proteins exhibit amino acid sequence identity of 73%. Estimates for the M r of GlvA determined by SDS-polyacrylamide gel electrophoresis (51,000) and electrospray-mass spectroscopy (50,510) were in excellent agreement with the molecular weight of 50,513 deduced from the amino acid sequence. The sequence of the first 37 residues from the N terminus determined by automated analysis agreed precisely with that predicted by translation of glvA. The chromogenic and fluorogenic substrates, p-nitrophenyl-α-d-glucopyranoside 6-phosphate and 4-methylumbelliferyl-α-d-glucopyranoside 6-phosphate were used for the discontinuous assay and in situ detection of enzyme activity, respectively. Site-directed mutagenesis shows that three acidic residues, Asp41, Glu111, and Glu359, are required for GlvA activity. Asp41 is located at the C terminus of a βαβ fold that may constitute the dinucleotide binding domain of the protein. Glu111 and Glu359 may function as the catalytic acid (proton donor) and nucleophile (base), respectively, during hydrolysis of 6-phospho-α-glucoside substrates including maltose 6-phosphate and trehalose 6-phosphate. In metal-free buffer, GlvA exists as an inactive dimer, but in the presence of Mn2+ ion, these species associate to form the NAD(H)-dependent catalytically active tetramer. By comparative sequence alignment with its homologs, the novel 6-phospho-α-glucosidase from B. subtilis can be assigned to the nine-member family 4 of the glycosylhydrolase superfamily. phosphoenol pyruvate-dependent sugar phosphotransferase system polyacrylamide gel electrophoresis 6-phospho-α-glucosidase maltose 6-phosphate hydrolase p-nitrophenyl-α-d-glucopyranoside 6-phosphate 4-methylumbelliferyl-α-d-glucopyranoside 6-phosphate kilobase(s) polymerase chain reaction 2[N-morpholino]ethane sulfonic acid β-mercaptoethanol. The serendipitous discovery in 1964 (1Kundig W. Ghosh S. Roseman S. Proc. Natl. Acad. Sci. U. S. A. 1964; 52: 1067-1074Crossref PubMed Scopus (322) Google Scholar, 2Roseman S. FEMS Microbiol. Rev. 1989; 63: 3-12Google Scholar) of the bacterial phosphoenol pyruvate-dependent sugar phosphotransferase system (PEP-PTS)1 by Roseman and colleagues represents a landmark in our understanding of carbohydrate transport by microorganisms (3Saier Jr., M.H. Reizer J. Mol. Microbiol. 1994; 13: 755-764Crossref PubMed Scopus (171) Google Scholar, 4Saier Jr., M.H. Res. Microbiol. 1996; 147: 435-594Google Scholar). Since the initial description in Escherichia coli, this phosphoryl group-transfer system (5Danchin A. FEMS Microbiol. Rev. 1989; 63: 1-200Crossref Scopus (6) Google Scholar, 6Erni B. Int. Rev. Cytol. 1992; 137: 127-148Crossref PubMed Scopus (22) Google Scholar) has been established as the primary mechanism for the accumulation of sugars by bacteria from both Gram-negative (7Meadow N.D. Fox D.K. Roseman S. Annu. Rev. Biochem. 1990; 59: 497-542Crossref PubMed Scopus (302) Google Scholar, 8Postma P.W. Lengeler J.W. Jacobson G.R. Microbiol. 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London. 1990; 326: 489-504Crossref PubMed Scopus (62) Google Scholar) comprises both membrane-localized and cytoplasmic proteins that in concert catalyze the simultaneous phosphorylation and vectorial translocation of sugar across the cytoplasmic membrane. Catalytically, each PEP-PTS requires two general components (Enzyme I and HPr) that, allied with sugar-specific proteins (IIA, -B, and -C; for discussion, see Ref. 14Saier Jr., M.H. Reizer J. J. Bacteriol. 1992; 174: 1433-1438Crossref PubMed Google Scholar), promote the sequential transfer of the high energy, phosphoryl moiety from PEP to the incoming sugar. Prior to catabolism via energy-yielding pathways, the intracellular disaccharide phosphates must first be hydrolyzed to their constituent hexose 6-phosphate and aglycone moieties. Several phosphoglycosylhydrolases (whose genes are frequently encoded within PTS operons) have been purified, cloned, and sequenced. Particularly well characterized are the 6-phospho-β-galactosidases (EC 3.2.1.85; Refs. 15Hengstenberg W. Penberthy W.K. Morse M.L. Eur. J. Biochem. 1970; 14: 27-32Crossref PubMed Scopus (20) Google Scholar, 16Johnson K.G. McDonald I.J. J. Bacteriol. 1974; 117: 667-674Crossref PubMed Google Scholar, 17Calmes R. Brown A.T. Infect. Immun. 1979; 23: 68-79Crossref PubMed Google Scholar, 18Breidt Jr., F. Stewart G.C. Appl. Environ. Microbiol. 1987; 53: 969-973Crossref PubMed Google Scholar, 19Boizet B. Villeval D. Slos P. Novel M. Novel G. Mercenier A. Gene (Amst .). 1988; 62: 249-261Crossref PubMed Scopus (33) Google Scholar, 20Porter E.V. Chassy B.M. Gene (Amst.). 1988; 62: 263-276Crossref PubMed Scopus (50) Google Scholar, 21Wiesmann C. Beste G. Hengstenberg W. Schulz G.E. Structure. 1995; 3: 961-968Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and 6-phospho-β-glucosidases (EC 3.2.1.86; Refs.22Schaefler S. Schenkein I. Proc. Natl. Acad. Sci. U. S. 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J. 1991; 280: 309-316Crossref PubMed Scopus (2574) Google Scholar, 32Henrissat B. Bairoch A. Biochem. J. 1993; 293: 781-788Crossref PubMed Scopus (1753) Google Scholar). In 1996, as participants in the Bacillus genome project (33Kunst F. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3088) Google Scholar), Sekiguchi and co-workers determined the nucleotide sequence of a 12.4-kb fragment of DNA near the 76° region of the Bacillus subtilis chromosome (34Yamamoto H. Uchiyama S. Fajar A.N. Ogasawara N. Sekiguchi J. Microbiology. 1996; 142: 1417-1421Crossref PubMed Scopus (20) Google Scholar). Following translation of the ten open reading frames within this fragment, a search of the protein data bases revealed that the deduced amino acid sequences of open reading framesglv-1 and glv-2 exhibited similarity to 6-phospho-β-glucosidase and the IIC domain (GlvC) of the arbutin (PEP-PTS) of E. coli, respectively. It seemed likely, as suggested by Yamamoto et al. (34Yamamoto H. Uchiyama S. Fajar A.N. Ogasawara N. Sekiguchi J. Microbiology. 1996; 142: 1417-1421Crossref PubMed Scopus (20) Google Scholar), that the products of glv-1 and glv-2 might participate in the transport and dissimilation of β-glucosides in the Gram-positive spore-forming organism. Contemporaneous with the Bacillusstudies in Japan, our program at the National Institutes of Health was directed toward the purification and characterization of a metal-dependent maltose 6-phosphate hydrolase (MalH) from the anaerobic pathogen Fusobacterium mortiferum (35Thompson J. Gentry-Weeks C.R. Nguyen N.Y. Folk J.E. Robrish S.A. J. Bacteriol. 1995; 177: 2505-2512Crossref PubMed Google Scholar). This novel enzyme, together with an inducible maltose PEP-PTS permits growth of F. mortiferum on a wide variety of α-glucosides including maltose, α-methyl glucoside, trehalose, palatinose, and turanose (36Robrish S.A. Fales H.M. Gentry-Weeks C.R. Thompson J. J. Bacteriol. 1994; 176: 3250-3256Crossref PubMed Google Scholar). The 6-phospho-α-glucosidase gene (malH) has recently been cloned, sequenced, and expressed in E. coli(37Bouma C.L. Reizer J. Reizer A. Robrish S.A. Thompson J. J. Bacteriol. 1997; 179: 4129-4137Crossref PubMed Google Scholar). Remarkably, the deduced amino acid sequence of MalH exhibited 73% identity (88% similarity) to that deduced from the nucleotide sequence of glv-1 in B. subtilis. These findings raised doubts concerning the initial classification and catalytic activity of the polypeptide encoded by glv-1 (34Yamamoto H. Uchiyama S. Fajar A.N. Ogasawara N. Sekiguchi J. Microbiology. 1996; 142: 1417-1421Crossref PubMed Scopus (20) Google Scholar). In a collaborative program, we have addressed and resolved these issues. This communication describes the cloning, expression, and site-directed mutagenesis of glv-1 (now designatedglvA (33Kunst F. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3088) Google Scholar)) from B. subtilis. Purification and characterization of the novel 6-phospho-α-glucosidase (GlvA) encoded by glvA was facilitated by the availability of the natural substrate for the enzyme (maltose 6-phosphate) and by chemical synthesis of the chromogenic and fluorogenic analogs pNPαGlc6P and 4MUαGlc6P, respectively. In contrast to other phosphoglycosylhydrolases, GlvA from B. subtilis exhibits specific requirements for both divalent metal (Mn2+, Fe2+, Co2+, or Ni2+) and NAD(H) for activity. Furthermore, by sequence similarity and conservation of functionally important acidic residues, GlvA can be assigned to the nine-member family 4 of the glycosylhydrolase superfamily (31Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2574) Google Scholar, 37Bouma C.L. Reizer J. Reizer A. Robrish S.A. Thompson J. J. Bacteriol. 1997; 179: 4129-4137Crossref PubMed Google Scholar). PD-10 gel filtration columns, isoelectric focusing standards, Ampholine PAG plates (pH 3.5–9.0), DEAE-Sephacel, and phenyl-Sepharose CL- 4B were purchased from Amersham Pharmacia Biotech. Ultrogel AcA 44 and TrisAcryl DEAE-M were supplied by Sepracor. Enzymes, nucleotides, cofactors, and trehalose 6-phosphate were obtained from Sigma. Trimethylphosphate, phosphorus oxychloride, and cyclohexylamine were obtained from Aldrich. Pressure concentration cells and Diaflo PM-10 ultrafiltration membranes were from Amicon Corp. [U-14C]Maltose 6-phosphate was prepared enzymatically by PEP-dependent phosphorylation of the disaccharide by the maltose PEP-PTS in permeabilized cells of F. mortiferum. The radiolabeled disaccharide phosphate was purified by ion exchange chromatography, Ba2+ and ethanol precipitation, and finally by paper chromatography (36Robrish S.A. Fales H.M. Gentry-Weeks C.R. Thompson J. J. Bacteriol. 1994; 176: 3250-3256Crossref PubMed Google Scholar). Chemical syntheses of pNPαGlc6P, pNPαMan6P, pNPαGal6P and 4MUαGlc6P were initiated with the commercially available non-phosphorylated glycosides (Sigma) using the procedure of Wilson and Fox (24Wilson G. Fox C.F. J. Biol. Chem. 1974; 249: 5586-5598Abstract Full Text PDF PubMed Google Scholar). Selective phosphorylation at the C-6 hydroxyl group of the nonreducing glucopyranose was achieved by use of a mixture of phosphorus oxychloride in trimethylphosphate containing small amounts of water. The phosphorylated derivatives were obtained as white, crystalline cyclohexylamine salts in 25–30% yield. B. subtilis 168 and E. coli strains JM109 and XL1-Blue were grown in Luria-Bertani (LB) medium at 37 °C as described previously (38Ishikawa S. Yamane K. Sekiguchi J. J. Bacteriol. 1998; 180: 1375-1380Crossref PubMed Google Scholar). When required, ampicillin was included in the medium at a final concentration of 50 μg/ml. E. coliplasmids pUC119 (Takara Shuzo Co., Kyoto) and a high expression vector pKP1500 (39Miki T. Yasukochi T. Nagatani H. Furuno M. Orita T. Yamada H. Imoto T. Horiuchi T. Protein Eng. 1987; 1: 327-332Crossref PubMed Scopus (107) Google Scholar) were used to construct a plasmid (pKPglv-1) containingglvA. Site-directed mutagenesis was carried out by thePfu polymerase method (QuickChange site-directed mutagenesis kit, Stratagene). The desired mutations and the primers used to effect these changes are described in the text (Table IV).Table IVOligonucleotides used for site-directed mutagenesis of glvA from B. subtilis 168 and resultant activities of the mutant 6-phospho-α-glucosidaseMutationPrimer sequence4-aBase substitutions are shown underlined.Activity4-bActivity expressed as nmol of pNPαGlc6P hydrolyzed/min/mg protein. Each value is an average of duplicate determinations.None492D41GG CTG AAG CTG TAT GGT AAT GAT AAG GAG AGA CAG G4D41EG CTG AAG CTG TAT GAG AAT GAT AAG GAG AGA CAG G3E111GGGA GTT GTC GGC CAG GGG ACG TGC GGG CCG1E111DGGA GTT GTC GGC CAG GAT ACG TGC GGG CCG3E359GC CCG ACT GCG ATG GTT GGG GTG CCA TGC ATC GTC GGC5E359DCCG ACT GCG ATG GTT GAT GTG CCA TGC ATC GTC GGC54-a Base substitutions are shown underlined.4-b Activity expressed as nmol of pNPαGlc6P hydrolyzed/min/mg protein. Each value is an average of duplicate determinations. Open table in a new tab For the amplification of the gene glvA (alsoglv-1 (33Kunst F. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3088) Google Scholar, 34Yamamoto H. Uchiyama S. Fajar A.N. Ogasawara N. Sekiguchi J. Microbiology. 1996; 142: 1417-1421Crossref PubMed Scopus (20) Google Scholar)), two primers were synthesized: forward primer G1PEF, 5′-GCCGGAATTC ATGAAGAAAAAATCATTCTCAA-3′ (theglvA sequence is italicized and the EcoRI site is underlined) and reverse primer G1PBR, 5′-GCGCGGATCC CTGATTGATCAGTTCTTCG-3′ (the sequence complimentary to the downstream region of glvA is italicized and the BamHI site is underlined). PCR amplification was performed with the GeneAmp PCR Core kit (Perkin Elmer) using 0.1 μg of B. subtilis 168 genomic DNA as template, 10 μl of 10 × reaction buffer, 8 μl of 25 mm MgCl2, 2 μl each of 10 mmdNTP, 30 pmol of each primer, and 0.5 unit of Taq polymerase in a total volume of 100 μl. The annealing temperature was 56 °C. The PCR product was analyzed by agarose gel electrophoresis and subsequently digested with EcoRI and BamHI. The 0.5-kb EcoRI and 0.95-kb EcoRI-BamHI restriction fragments corresponding to the 5′ and 3′ regions of glvA, respectively, were purified with GeneClean II kit (BIO 101). The fragments were ligated into the corresponding sites of pUC119 and used for transformation of E. coli JM109. Nucleotide sequences of the fragments inserted into the recombinant plasmids (designated pUC119EE and pUC119EB, respectively) were determined by the following procedure. Transformed cells were boiled in water, and the sample was transferred to the PCR reaction mixture containing universal forward and reverse primers of pUC plasmids (M13 primers M4 and RV; Takara), Taq polymerase, and dNTP. After amplification, the primers were removed by Microcon (Amicon). Sequences were determined using a Taq Dye Primer Cycle Sequencing kit (Perkin Elmer) and a 373A DNA sequencer from Applied Biosystems. After confirmation of the sequences, the 0.95-kb EcoRI-BamHI fragment from pUC119EB was purified and ligated to the EcoRI and BamHI sites of pKP1500 to form pKPGEB. Transformants of E. coli JM109 containing the recombinant plasmid were boiled in water and transferred to the PCR reaction mixture containing primers G1PEF and G1PBR, Taq polymerase, and dNTP. After amplification and transformation, those colonies that contained a 1.45-kb DNA restriction fragment were considered to harbor a plasmid (pKPglv-1) containing the insert in the correct orientation. For confirmation, pKPglv-1 plasmid DNA was isolated from the transformant, digested with EcoRI or HindIII, and subjected to agarose gel electrophoresis. The size of the restriction fragments corresponded to those expected from the sequence of glvA, thereby confirming the presence of the complete gene in pKPglv-1. E. coli JM109 (pKPglv-1) was grown at 37 °C in LB medium containing ampicillin (100 μg/ml). Cells were harvested by centrifugation (13,000 × g for 10 min at 5 °C) and washed by resuspension and centrifugation from 25 mmTris-HCl buffer (pH 7.5) containing 1 mm MnSO4(TM buffer). The washed cell pellet (∼80 g) was resuspended in 120 ml of TM buffer, and the cells were disrupted (at 0 °C) by 2 × 1.5-min periods of sonic oscillation with a Branson model 350 sonifier operating at ∼75% of maximum power. The enzyme was purified by conventional low pressure chromatography, and all procedures were performed at 4 °C in a cold room. Column flow rates were maintained by a P-1 peristaltic pump interfaced with a Frac-100 collector. Protein in column eluents was monitored at 280 nm by a UV-1 optical control unit connected to a single channel chart recorder (all instrumentation from Pharmacia Biotech). The sonicated preparation was centrifuged (25,000 ×g for 30 min at 5 °C) to remove intact cells and cell debris. The supernatant was collected and centrifuged at 180,000 × g for 2 h at 5 °C. The clarified HSS was transferred to dialysis sacs (molecular weight cut-off, 6000–8000) and dialyzed overnight against 4 liters of TM buffer. Dialyzed HSS (∼120 ml) was transferred (0.8 ml/min) to a column (2.6 × 16 cm) of DEAE-TrisAcryl-M previously equilibrated with TM buffer. The column was washed to remove nonadsorbed material, and 6-phospho-α-glucosidase was eluted with 800 ml of a linear, increasing concentration gradient of NaCl (0–300 mm) in TM buffer. Fractions of 10 ml were collected, and 10 μl of each fraction was tested for enzyme activity by the formation of a yellow color in microtiter wells containing 100 μl of the standard pNPαGlc6P reaction mixture. Fractions 22–27 (inclusive) were pooled and concentrated to 25 ml by pressure filtration (Amicon PM-10 membrane, 35 psi). Ammonium sulfate was then added slowly, and with gentle stirring, to a final concentration of 0.75m. The solution from step 2 was transferred (0.4 ml/min) to a 2.6 × 16-cm column of phenyl-Sepharose CL-4B equilibrated with TM buffer containing 0.75 m(NH4)2SO4. The column was washed with equilibration buffer to remove material that did not bind, and then 600 ml of a decreasing, linear gradient of (NH4)2SO4 (300–0 mm) in TM buffer was passed through the column. Fractions of 10 ml were collected, and enzyme was recovered in a broad protein peak comprising fractions 25–45. These fractions were pooled and concentrated to 7.5 ml. Approximately 2.5 ml of the preparation from step 3 was applied at a flow rate of 0.15 ml/min to a column (1.6 × 94 cm) of Ultrogel AcA-44 previously equilibrated with TM buffer containing 0.1 m NaCI. Fractions of 2.15 ml were collected, and maximum levels of enzyme activity were found in fractions 49–53, inclusive. These fractions were concentrated to 2 ml, and aliquots were either frozen directly in dry ice or glycerol was added to a final concentration of 10% prior to storage of the enzyme at −20 °C. The chromogenic analog pNPαGlc6P was used as substrate in the discontinuous assay for 6-phospho-α-glucosidase activity. The 2-ml reaction mixture (at 37 °C) contained, when required, 50 mm Tris-HCl buffer (pH 7.5), 1 mm pNPαGlc6P, 0.5 mm MnS04, and 0.1 mm NAD(H). After addition of the enzyme preparation, samples of 0.25 ml were removed at intervals of 0.5, 1, 1.5, 2, 2.5, and 3 min and immediately injected into 0.75 ml of 0.5 mNa2CO3 solution. TheA 400 nm of the yellow solution was measured, and the amount of pNP was calculated by assuming a molar extinction coefficient for the p-nitrophenoxide anion ε = 18,300m−1 cm−1. One unit of 6-phospho-α-glucosidase activity is the amount of enzyme that catalyzes the formation of 1 μmol of pNP per min at 37 °C. Native gel electrophoresis and SDS-PAGE were carried out in the Novex XCell Mini-Cell system according to manufacturer's instructions. Electrophoresis of proteins under nonreducing (native) conditions was performed in Tris-glycine (4–20%) gels, from Novex, with Tris-glycine (pH 8.3) running buffer. For SDS-PAGE experiments, Novex NuPage (4–12%) Bis-Tris gels and MES-SDS running buffer (pH 7.3) were used together with Novex Mark 12 wide range or Bio-Rad low range molecular weight protein standards. In Western blots, proteins were transferred to nitrocellulose membranes using NuPage transfer buffer (pH 7.2) and SeeBlue prestained standards. Immunodetection of 6-phospho-α-glucosidase with polyclonal antibody to maltose 6-phosphate hydrolase was as described previously (35Thompson J. Gentry-Weeks C.R. Nguyen N.Y. Folk J.E. Robrish S.A. J. Bacteriol. 1995; 177: 2505-2512Crossref PubMed Google Scholar). The concentrations of glucose and Glc6P were determined enzymatically in an NADP+-coupled assay that contained (in 1 ml) 0.1 m potassium phosphate (pH 7) buffer, 1 mm MgCl2, 5 mm ATP, 1 mm NADP+, and 2 units each of Glc6P dehydrogenase (EC 1.1.1.49) and hexokinase (EC 2.7.1.1). Formation of NADPH was followed in a Beckman DU 70 recording spectrophotometer, and a molar extinction coefficient ε = 6,220 m−1cm−1 was assumed for calculation of NADPH produced (i.e., equivalent to glucose or Glc6P formed). The Pharmacia Biotech Multiphor flat-bed electrophoresis unit and precast Ampholine PAG plates (pH range 3.5–9.5) were used for electrofocusing experiments as described previously (40Thompson J. J. Biol. Chem. 1989; 264: 9592-9601Abstract Full Text PDF PubMed Google Scholar). Protein concentrations were routinely determined by the BCA protein assay kit (Pierce). Chromatographic procedures are described in a previous report (35Thompson J. Gentry-Weeks C.R. Nguyen N.Y. Folk J.E. Robrish S.A. J. Bacteriol. 1995; 177: 2505-2512Crossref PubMed Google Scholar). The N-terminal amino acid sequence of 6-phospho-α-glucosidase was determined by automated Edman degradation in a 494A Procise sequenator (Applied Biosystems, Inc.) using pulse liquid chemistry. PTH derivatives were identified by on-line high pressure liquid chromatography. The mass of purified 6-phospho-α-glucosidase was determined by electrospray in an HP1100 mass spectrometer. The enzyme sample was dissolved in 0.05% trifluoroacetic acid, injected onto a Zorbax 300SB-C3 narrow bore (2.1 × 150 mm) high pressure liquid chromatography column, and eluted with a gradient of 5% acetic acid to 100% acetonitrile. A mass range from m/z 700 to 2000 was scanned every 4 s, and the protein mass (50,510) was obtained by deconvolution from the only peak that eluted from the column. Analytical ultracentrifugation experiments were conducted at 20 °C in a Beckman Optima model XL-I instrument equipped with a four-place An-Ti rotor. Specific absorption coefficients were determined by a combination of absorbance and interference optics. Absorbance readings on protein samples were measured in a Perkin Elmer model 320 double-beam spectrophotometer at 20 °C. Protein concentrations (mg/ml) were determined on the same solutions using the analytical ultracentrifuge in an interference mode as a differential refractometer (41Maurizi M.R. Pinkofsky H.B. McFarland P.J. Ginsburg A. Arch. Biochem. Biophys. 1986; 246: 494-500Crossref PubMed Scopus (21) Google Scholar) and an experimentally determined value of 3.191 ± 0.005 fringes (mg/ml)−1 for the same instrument. 2M. Zolkiewski and A. Ginsburg, unpublished data. A 12-mm cell housing equipped with a double-sector capillary synthetic boundary centerpiece and sapphire windows was used with initial volumes of 130 μl of protein solution and 410 μl of dialysate buffer. After temperature equilibration at 20.0 °C at 3000 rpm, the rotor speed was increased to 10,000 rpm to initiate boundary formation and to 20,000 rpm (in 2000-rpm steps) until protein and solvent-side menisci were matched (total time ∼10 min) and then decelerated to 3000 rpm; 10 interference scans were then recorded at 2-min intervals (42Nosworthy N.J. Peterkofsky A. Konig S. Seok Y.-J. Szczepanowski R.H. Ginsburg A. Biochemistry. 1998; 37: 6718-6726Crossref PubMed Scopus (41) Google Scholar). For 10 scans, the difference between the fringes in the plateau and solvent sides of the boundary (100 data points in both radial positions) was constant and within 0.1% accuracy during the period of data collection. The specific absorption coefficient for 6-phospho-α-glucosidase was determined to beA 280 nm1 cm = 1.25 ± 0.01 ml/mg (average of three independent measurements). This value is similar to that calculated from the amino acid composition of the protein (1.20 ml/mg). Sedimentation velocity experiments were conducted using charcoal-filled double-sector Epon 12-mm centerpieces. Enzyme solution (A 280 nm ∼1.0) was loaded on the right (330 μl per channel) with the reference buffer on the left (340 μl per channel). After thermal equilibration at 3000 rpm, the rotor was accelerated to 40,000 rpm, and radial scans were collected at 280 nm (0.003-cm step size, 4-min intervals) with triple averaging in a continuous scan mode. For sedimentation equilibrium experiments, a 12-mm cell equipped with a carbon-filled 6-channel centerpiece and plane quartz windows was used. Enzyme solutions (A 280 nm from 0.17 to 0.35) were loaded on the right (100 μl per channel) with the reference buffer on the left (110 μl per channel). Radial scans at 10,000 rpm with 13 averages were made at 280 nm in 0.001-cm steps (step mode) after 18 and 20 h (equilibrium was reached by 16 h). Analysis of ultracentrifugation data was performed as described previously (43Maurizi M.R. Singh S.K. Thompson M.W. Kessel M. Ginsburg A. Biochemistry. 1998; 37: 7778-7786Crossref PubMed Scopus (82) Google Scholar) with software from Beckman, Inc. and A. P. Minton (NIDDK, National Institutes of Health). The densities of dialysate buffers (20.0 °C) were determined using an Anton Paar model DMA 58 densitometer. The partial specific volume for the protein (υ = 0.720 ml/g) was calculated from the amino acid composition (44Zamyatnin A. Annu. Rev. Biophys. Bioeng. 1984; 13: 145-165Crossref PubMed Scopus (268) Google Scholar). The nucleotide sequence of the gene glvA (previously called glv-1 (34Yamamoto H. Uchiyama S. Fajar A.N. Ogasawara N. Sekiguchi J. Microbiology. 1996; 142: 1417-1421Crossref PubMed Scopus (20) Google Scholar)) is presented in Fig. 1. The 1347-base pair open reading frame begins with an ATG initiation codon at nucleotide position 15 and terminates with a TAA stop codon at position 1362. A putative ribosome binding site AAGGAGGT precedes the start codon. Translation of the codon sequence of glvA predicts a polypeptide of 449 residues of calculated M r = 50,513 and theoretical pI = 4.77. The 48.1 mol% (G+C) base composition of glv
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