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• W1587177180 abstract "CobU is a bifunctional enzyme involved in adenosylcobalamin (coenzyme B12) biosynthesis inSalmonella typhimurium LT2. In this bacterium, CobU is the adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase needed to convert cobinamide to adenosylcobinamide-GDP during the late steps of adenosylcobalamin biosynthesis. The guanylyltransferase reaction has been proposed to proceed via a covalently modified CobU-GMP intermediate. Here we show that CobU requires a nucleoside upper ligand on cobinamide for substrate recognition, with the nucleoside base, but not the 2′-OH group of the ribose, being important for this recognition. During the kinase reaction, both the nucleotide base and the 2′-OH group of the ribose are important for γ-phosphate donor recognition, and GTP is the only nucleotide competent for the complete nucleotidyltransferase reaction. Analysis of the ATP:adenosylcobinamide kinase reaction shows CobU becomes less active during this reaction due to the formation of a covalent CobU-AMP complex that holds CobU in an altered conformation. Characterization of the GTP:adenosylcobinamide-phosphate guanylyltransferase reaction shows the covalent CobU-GMP intermediate is on the reaction pathway for the generation of adenosylcobinamide-GDP. Identification of a modified histidine and analysis of cobU mutants indicate that histidine 46 is the site of guanylylation. CobU is a bifunctional enzyme involved in adenosylcobalamin (coenzyme B12) biosynthesis inSalmonella typhimurium LT2. In this bacterium, CobU is the adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase needed to convert cobinamide to adenosylcobinamide-GDP during the late steps of adenosylcobalamin biosynthesis. The guanylyltransferase reaction has been proposed to proceed via a covalently modified CobU-GMP intermediate. Here we show that CobU requires a nucleoside upper ligand on cobinamide for substrate recognition, with the nucleoside base, but not the 2′-OH group of the ribose, being important for this recognition. During the kinase reaction, both the nucleotide base and the 2′-OH group of the ribose are important for γ-phosphate donor recognition, and GTP is the only nucleotide competent for the complete nucleotidyltransferase reaction. Analysis of the ATP:adenosylcobinamide kinase reaction shows CobU becomes less active during this reaction due to the formation of a covalent CobU-AMP complex that holds CobU in an altered conformation. Characterization of the GTP:adenosylcobinamide-phosphate guanylyltransferase reaction shows the covalent CobU-GMP intermediate is on the reaction pathway for the generation of adenosylcobinamide-GDP. Identification of a modified histidine and analysis of cobU mutants indicate that histidine 46 is the site of guanylylation. adenosylcobalamin adenosylcobinamide adenosylcobinamide-phosphate adenosylcobinamide guanosine diphosphate cobinamide dicyanocobinamide dicyanocobinamide phosphate dicyanocobinamide guanosine diphosphate guanosylcobinamide inosylcobinamide cytosylcobinamide 2′,5′-dideoxyadenosylcobinamide R-1-amino-O-2-propanol R-1-amino-propanol-O-2-phosphate dithiothreitol high performance liquid chromatography Tris(2-carboxyethyl)phosphine hydrochloride polymerase chain reaction polyacrylamide gel electrophoresis A schematic of adenosylcobalamin (AdoCbl)1 and the late steps of the biosynthesis of this coenzyme (referred to as nucleotide loop assembly) are shown in Fig. 1. In Salmonella typhimuriumLT2, nucleotide loop assembly has been divided into two activation branches that involve four enzymes, CobU, CobT, CobC, and CobS (1Rondon M.R. Trzebiatowski J.R. Escalante-Semerena J.C. Prog. Nucleic Acids Res. Mol. Biol. 1997; 56: 347-384Crossref PubMed Scopus (29) Google Scholar, 2O'Toole G.A. Rondon M.R. Trzebiatowski J.R. Suh S.-J. Escalante-Semerena J.C. Neidhardt F.C. Curtis III R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 710-720Google Scholar). CobT and CobC are involved in 5,6-dimethylbenzimidazole activation whereby 5,6-dimethylbenzimidazole is converted to its riboside, α-ribazole (3Trzebiatowski J.R. Escalante-Semerena J.C. J. Biol. Chem. 1997; 272: 17662-17667Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 4O'Toole G.A. Trzebiatowski J.R. Escalante-Semerena J.C. J. Biol. Chem. 1994; 269: 26503-26511Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). The second branch of the nucleotide loop assembly pathway is the cobinamide (Cbi) activation branch where adenosylcobinamide (AdoCbi) or adenosylcobinamide-phosphate (AdoCbi-P) is converted to the activated intermediate AdoCbi-GDP by the bifunctional enzyme CobU (5O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1995; 270: 23560-23569Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The final step in AdoCbl biosynthesis is the condensation of AdoCbi-GDP with α-ribazole, which is catalyzed by cobalamin synthase (CobS) to yield AdoCbl (6Maggio-Hall L.A. Escalante-Semerena J.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11798-11803Crossref PubMed Scopus (57) Google Scholar). Work reported in this paper, further analyzes the bifunctional enzyme CobU. This enzyme has kinase and guanylyltransferase activities. However, it appears that both activities are not required at all times. The kinase activity has been proposed to function only when S. typhimurium LT2 is assimilating Cbi from its environment (7Brushaber K.R. O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1998; 273: 2684-2691Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). 2Thomas, M. G., and Escalante-Semerena, J. C. (2000) J. Bacteriol. 182, 4227–4233. 2Thomas, M. G., and Escalante-Semerena, J. C. (2000) J. Bacteriol. 182, 4227–4233. In contrast, the guanylyltransferase activity is required for both the assimilation of exogenous Cbi and for de novo synthesis of AdoCbl (Fig.1) (7Brushaber K.R. O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1998; 273: 2684-2691Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar).2 Biochemical and structural analysis on CobU have given insights into how this enzyme may work (5O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1995; 270: 23560-23569Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 9Thompson T.B. Thomas M.G. Escalante-Semerena J.C. Rayment I. Biochemistry. 1998; 37: 7686-7695Crossref PubMed Scopus (35) Google Scholar, 10Thompson T. Thomas M.G. Escalante-Semerena J.C. Rayment I. Biochemistry. 1999; 38: 12995-13004Crossref PubMed Scopus (24) Google Scholar). Biochemical studies showed the reactions catalyzed by CobU are as follows: Reaction 1 (kinase): AdoCbi + NTP → AdoCbi-P + NDP; Reaction 2 (guanylyltransferase) (i) CobU + GTP → CobU-GMP + PPi and (ii) CobU-GMP + AdoCbi-P → AdoCbi-GDP + CobU. The kinase reaction (Reaction 1) can use either ATP or GTP as a γ-phosphate donor to generate the AdoCbi-P intermediate (5O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1995; 270: 23560-23569Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), as does its orthologue, CobP, in Pseudomonas denitrificans (11Blanche F. Debussche L. Famechon A. Thibaut D. Cameron B. Crouzet J. J. Bacteriol. 1991; 173: 6052-6057Crossref PubMed Google Scholar). The guanylyltransferase reaction is proposed to occur via two half-reactions (Reaction 2). First, the enzyme forms a CobU-GMP covalent phosphoramidate bond, followed by the transfer of the GMP moiety to AdoCbi-P to generate the final product AdoCbi-GDP (5O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1995; 270: 23560-23569Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Structural data showed CobU functions as a homotrimer, forming three prominent clefts with the conserved phosphate-binding loop of each monomer at the base of each cleft (9Thompson T.B. Thomas M.G. Escalante-Semerena J.C. Rayment I. Biochemistry. 1998; 37: 7686-7695Crossref PubMed Scopus (35) Google Scholar, 10Thompson T. Thomas M.G. Escalante-Semerena J.C. Rayment I. Biochemistry. 1999; 38: 12995-13004Crossref PubMed Scopus (24) Google Scholar). Co-crystallization of CobU with GTP determined there is a substantial substrate-induced conformational change associated with the formation of the CobU-GMP intermediate (10Thompson T. Thomas M.G. Escalante-Semerena J.C. Rayment I. Biochemistry. 1999; 38: 12995-13004Crossref PubMed Scopus (24) Google Scholar). This conformational change closes the clefts and, in the process, forms a GMP-specific binding pocket. The structural work also suggested there may be an overlap between the proposed GTP-binding site for guanylylation and the proposed nucleotide-binding site for phosphorylation. This raises the question of how this enzyme coordinates the binding of two different nucleotides for two separate enzymatic activities. Additionally, the finding that CobU functions as a guanylyltransferase or a kinase/guanylyltransferase raises questions as to how the enzyme binds two relatively large substrates, AdoCbi or AdoCbi-P, and discriminates between kinase or guanylyltransferase activities. Interestingly, CobU was found structurally and topologically similar to the central domain of the RecA protein of Escherichia coli(9Thompson T.B. Thomas M.G. Escalante-Semerena J.C. Rayment I. Biochemistry. 1998; 37: 7686-7695Crossref PubMed Scopus (35) Google Scholar). CobU and RecA both contain a phosphate binding loop (Walker A box), a Walker B box, and a non-prolyl cis peptide bond in similar positions in the three-dimensional structures (9Thompson T.B. Thomas M.G. Escalante-Semerena J.C. Rayment I. Biochemistry. 1998; 37: 7686-7695Crossref PubMed Scopus (35) Google Scholar, 10Thompson T. Thomas M.G. Escalante-Semerena J.C. Rayment I. Biochemistry. 1999; 38: 12995-13004Crossref PubMed Scopus (24) Google Scholar, 12Story R.M. Steitz T.A. Nature. 1992; 355: 374-376Crossref PubMed Scopus (559) Google Scholar, 13Story R.M. Weber I.T. Steitz T.A. Nature. 1992; 355: 318-325Crossref PubMed Scopus (679) Google Scholar). Additionally, both proteins bind nucleotides and three-dimensional structures have been solved for both proteins in the presence of nucleotide; GMP for CobU (10Thompson T. Thomas M.G. Escalante-Semerena J.C. Rayment I. Biochemistry. 1999; 38: 12995-13004Crossref PubMed Scopus (24) Google Scholar) and ADP for RecA (12Story R.M. Steitz T.A. Nature. 1992; 355: 374-376Crossref PubMed Scopus (559) Google Scholar). Based on these similarities, CobU and RecA most likely shared a common ancestor, and information obtained from the analysis of either protein may give insights into how both proteins function. The specificity of the CobU enzyme for the corrin ring substrate during the kinase reaction and the specificity of the enzyme for its nucleotide substrate for both the kinase and nucleotidyltransferase reactions was investigated. It is demonstrated that during the ATP:AdoCbi kinase reaction, in the absence of GTP, CobU is altered to a less active conformation due to the formation of a stable, covalent CobU-AMP complex. It is also shown that the CobU-GMP intermediate is a true intermediate of the guanylyltransferase reaction, and that histidine 46 is the site of guanylylation. Strain JE3207 ((hisG-cob)Δ299/pGP1–2rpo + T7 RNA polymerasekan +, pJO52 cobU +) was used for the overexpression of wild-type CobU protein (5O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1995; 270: 23560-23569Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Conditions for the overexpression of wild-type CobU protein were as follows. Strain JE3207 was grown at 30 °C in 2-liter batches of LB medium containing kanamycin (50 μg/ml) and ampicillin (100 μg/ml) in 4-liter Erlenmeyer flasks. Mid-log phase cultures (A 650 = 0.6) were shifted to 42 °C for 1 h followed by expression at 37 °C for 2 h. After 2 h, cells were harvested and resuspended in 25 ml (per 2-liter batch) of 50 mm Tris-HCl, pH 8.0, at 4 °C containing 1.4m glycerol. Cells were frozen at −20 °C until use. Cell paste obtained from four 2-liter cultures of the overexpression strain JE3207 was used for each purification. Frozen cells were thawed and the following reagents were added: 10 mm (final concentration) dithiothreitol (DTT), 200 μm phenylmethanesulfonyl fluoride, 1 mm EDTA. Cells were broken by sonication (550 Sonic Dismembrator, Fisher Scientific, Itasca, IL) at setting 7, 50% duty. Sonication was performed four times for 5 min at 1-s intervals while keeping the sample below 15 °C. Cell debris was cleared from the extracts by centrifugation at 34,000 × g for 30 min in a Beckman J2–21 centrifuge using a KA21.50 rotor (Composite Rotor, Inc., Mountain View, CA). Finely ground UltrapureTM ammonium sulfate (ICN Biomedicals Inc., Cleveland, OH) was added to the cell-free extracts to 30% saturation. The sample was incubated on ice with stirring for 30 min. Protein precipitate was pelleted by centrifugation at 14,000 ×g (Beckman J2–21 centrifuge, KA21.50 rotor) for 20 min. Supernatant was recovered and ammonium sulfate was added to 55% saturation. Sample was incubated on ice with stirring for 30 min. Protein precipitate was pelleted by centrifugation as above. Supernatant was poured off, and the precipitated protein was resuspended in buffer A (50 mm Tris-HCl, pH 8.0, at 4 °C, 10 mm DTT, 200 mmphenylmethanesulfonyl fluoride, 1 mm EDTA). Resuspended protein was dialyzed against buffer A. Dialyzed, ammonium sulfate-precipitated protein was loaded at 0.4 cm/min onto a 50-ml DEAE Sepharose fast-flow (Sigma) column (10 × 2.5 cm) pre-equilibrated with buffer A. After the sample was loaded, the column was washed with 250 ml of buffer A. CobU was eluted using a 500-ml linear gradient of 0–0.5 m NaCl in buffer A. Fractions containing CobU, detected by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and Coomassie Blue staining, were pooled and dialyzed against buffer A. Pooled fractions from the DEAE column were dialyzed against buffer B (50 mm Tris-HCl, pH 8.0, at 4 °C, 10 mm DTT, 200 μmphenylmethanesulfonyl fluoride, 5 mm MgCl2). After dialysis, pooled fractions were loaded at 0.2 cm/min onto a 50-ml Cibacron Blue type 3000 (Sigma) column (10 × 2.5 cm) pre-equilibrated with buffer B. After loading, the column was washed with 100 ml of buffer B. CobU was subsequently eluted with a 500-ml linear gradient of 0–2.0 m NaCl in buffer B. Fractions containing CobU, detected by 12% SDS-PAGE and Coomassie Blue staining, were pooled and dialyzed against buffer B. Fractions from the Cibacron Blue column containing CobU were pooled, concentrated to a final volume of ∼10 ml with Centriprep 10 concentrators (Amicon, Beverly, MA), and dialyzed against buffer C (1.8 mTris-HCl, pH 8.0, at 4 °C, 5 mm DTT, 5 mmEDTA). After dialysis, sample was concentrated to ∼5 ml and was loaded at 0.3 cm/min through a 130-ml Sephacryl S-300-HR (Sigma) column (75 × 1.5 cm) pre-equilibrated with buffer C. Fractions containing CobU, based on 12% SDS-PAGE and Coomassie Blue staining, were pooled and dialyzed extensively against buffer D (50 mm Tris-HCl, pH 8.0, at 4 °C, 5 mm DTT, 0.1m NaCl, 680 mm glycerol). Protein was concentrated to 1.4 mg/ml and stored at −80 °C after drop-freezing in liquid nitrogen. Protein was stable under these conditions for months. During size exclusion chromatography a high Tris-HCl concentration (1.8m) was used as a mild denaturant, as described previously for actin purification (14Schafer D.A. Jennings P.B. Cooper J.A. Cell Motil. Cytoskeleton. 1998; 39: 166-171Crossref PubMed Scopus (32) Google Scholar). This step was included to enhance the release of any components that may be bound in the active site of CobU. The inclusion of the mild denaturant did not have a detectable effect on the specific activity of CobU when compared with the specific activity of CobU obtained through other protocols (data not shown). This denaturant was essential for consistent CobU crystal formation during structural analysis of the enzyme. 3T. B. Thompson and I. Rayment, unpublished data. Protein quantitation was determined by either the commercially available Bio-Rad Protein Assay kit or trichloroacetic acid-turbidimetric assay (15Kunitz M.J. J. Gen. Physiol. 1952; 35: 423-450Crossref PubMed Scopus (167) Google Scholar) both using bovine serum albumin as a standard. Assays gave comparable results and were consistent with protein concentration determined by amino acid analysis (data not shown). Synthesis and purification of AdoCbi and AdoCbi-P were performed as described elsewhere.2 For dAdoCbi, GuoCbi, InoCbi, and CyoCbi synthesis, the reactions mixtures were identical to those described2 except 800 mm ATP was replaced by 800 mm dATP, GTP, ITP, or CTP. Purification and quantitation of Cbi substrates was performed as described elsewhere.2 All reactions mixtures contained 50 mm Tris-HCl, pH 8.5, at 25 °C, 1.25 mm Tris(2-carboxyethyl)-phosphine hydrochloride (TCEP-HCl), 10 mm MgCl2, 680 mm glycerol, 0.1m NaCl in addition to CobU and substrate at concentrations as indicated. Reactions were terminated by the addition of 5 μl of 0.1 m KCN, pH 10.0, and immediately incubated at 70 °C for 10 min. Samples were centrifuged for 30 s at 14,000 ×g and 5 μl of each sample was spotted onto cellulose TLC plates (Macherey-Nagel PolygramTM CEL 400, Düren, Germany). Products and reactants were separated using ascending TLC with a solvent system of isobutyric acid:water:ammonium hydroxide (66:33:1) (5O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1995; 270: 23560-23569Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 16Ronzio R.A. Barker H.A. Biochemistry. 1967; 6: 2344-2354Crossref PubMed Scopus (18) Google Scholar). TLC resolution was allowed to run for 1.5 h, which corresponded to a 10-cm migration of the solvent front. All assays were performed in dim light to minimize the photolysis of the Co-C bond of the Cbi substrate. To quantitate the amount of product synthesized, the TLC plate was dried, then cut in half to remove the unincorporated label. The portion of the TLC plate containing the (CN)2Cbi-P or (CN)2Cbi-GDP product was exposed to a PhosphorImager screen along with standards containing known amounts of radioactivity. A standard curve was generated, and determination of the product synthesized in each reaction mixture was based on this standard curve. The amount of product synthesized was calculated by adjusting for decay of radioactivity, the known ratio of radiolabeled to unlabeled nucleotide at time 0, and the 32P specific activity of the sample determined by liquid scintillation counting. Calculations were periodically verified by cutting product spots from the TLC plate and determining the amount of radioactivity in each sample by liquid scintillation. At all times, the calculated values using the PhosphorImager standard curve were in agreement with the values obtained by scintillation counting. Reaction mixture components, assay conditions, and assay termination were the same as described above. The enzyme and substrate concentrations were as indicated. For the separation of the products and reactants a previously reported HPLC profile (11Blanche F. Debussche L. Famechon A. Thibaut D. Cameron B. Crouzet J. J. Bacteriol. 1991; 173: 6052-6057Crossref PubMed Google Scholar) was used with the following modifications. The chromatograph used was a computer-controlled Waters model 996 with a photodiode array detector and model 600 quaternary delivery system (Waters, Milford, MA). Separations were performed at room temperature on a LUNA 5 μ C18 (2O'Toole G.A. Rondon M.R. Trzebiatowski J.R. Suh S.-J. Escalante-Semerena J.C. Neidhardt F.C. Curtis III R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 710-720Google Scholar) column (150 × 4.6 mm) (Phenomenex, Torrance, CA) at a flow rate of 1 ml/min. The solvents used were as follows: solvent A (0.1 m PO4, pH 6.5, 10 mmKCN) and solvent B (0.1 m PO4, pH 8.0, 100% acetonitrile (1:1), 10 mm KCN). The complete reaction mixture for each sample (20 μl) was loaded onto the column pre-equilibrated with 98% A, 2% B. The following protocol was used for separation. One-min isocratic development with 98% A, 2% B; 5-min linear gradient from 98% A, 2% B to 75% A, 25% B; 15-min linear gradient from 75% A, 25% B to 65% A, 35% B; 4-min linear gradient from 65% A, 35% B to 98% A, 2% B. Cbi products and substrate eluted at times indicated in Fig. 3 B. Product generated was determined by analyzing the ratio of products ((CN)2Cbi-P and (CN)2Cbi-GDP) and substrate ((CN)2Cbi). Quantitation of product generated using either the HPLC or TLC assay were in good agreement. CobU (60 nm) was incubated in the following 20-μl reaction mixtures. Tris-HCl (100 mm), pH 8.5, at 25 °C, 5 mmMgCl2, 30 μm AdoCbi, 100 μmATP, 0.3 μm [α-32P]ATP (800 Ci/mmol) (NEN Life Science Products) in the presence or absence of 100 μm GTP. Reactions were incubated for 20 min at 25 °C. Reactions were terminated by the addition of 20 μl of 2 × loading buffer (17Gallagher S.R. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 2. Wiley Interscience, New York1996: Unit 10.2AGoogle Scholar) and incubated at 70 °C for 10 min. Separation of the unincorporated label and CobU-[α-32P]AMP was achieved by 12% SDS-PAGE. The gel was dried and exposed to a PhosphorImaging plate for visualization. Analysis of the chemical stability of the CobU-[32P]AMP complex was performed as described previously (18Forst S. Delgado J. Inouye M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6052-6056Crossref PubMed Scopus (193) Google Scholar) with the following modifications. A 120-μl reaction mixture containing 50 mm Tris-HCl, pH 8.5, at 25 °C, 10 mm MgCl2, 1.25 mm TCEP-HCl, 680 mm glycerol, 0.1 m NaCl, 0.1 μm[α-32P]ATP (800 Ci/mmol), and 0.86 μmCobU was incubated at 25 °C. After 10 min, the reaction was stopped by the addition of 18 μl of stop buffer (10% SDS, 1.5 mDTT, 1.36 m glycerol) and the sample was heated to 70 °C for 10 min. Twenty-three-μl samples of the reaction mixture were then transferred to fresh tubes containing 5 μl of 1.0 m HCl, 0.16 m HCl, ddH20, 1.8 m NaOH, or 3.3 m NaOH to alter the final pH to ∼1, 3, 8.5, 11, and 13, respectively. Twenty-eight μl of 2 × loading buffer (17Gallagher S.R. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 2. Wiley Interscience, New York1996: Unit 10.2AGoogle Scholar) was then added to each sample and heated to 70 °C for 10 min. Samples were resolved on 12% SDS-PAGE and visualized by PhosphorImaging. For the analysis of the CobU-[32P]GMP chemical stability, the same procedure was followed, however, [α-32P]GTP replaced [α-32P]ATP. Starting reaction mixtures for CobU-[32P]GMP exchange reactions contained the following: Tris-HCl (50 mm), pH 8.5, at 25 °C, 10 mmMgCl2, 1.25 mm TCEP-HCl, 680 mmglycerol, 0.1 m NaCl, 52 nm[α-32P]GTP (800 Ci/mmol), and 0.25 μmCobU (trimer). This concentration of label to enzyme was chosen to minimize the formation of more than one molecule of CobU-[32P]GMP per trimer. Reaction mixtures (180 μl) were incubated for 5 min at 25 °C. At time 0, a 15-μl sample was removed and added to 15 μl of 2 × loading buffer (17Gallagher S.R. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 2. Wiley Interscience, New York1996: Unit 10.2AGoogle Scholar). After removal of the zero time point sample, nonradioactive GTP was added to a final concentration of 2 mm to bring the final volume to 220 μl. Samples were mixed by pipetting, and 20-μl samples were removed at specified time points and added to 20 μl of 2 × loading buffer (17Gallagher S.R. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 2. Wiley Interscience, New York1996: Unit 10.2AGoogle Scholar). After the addition of loading buffer, the reactions were incubated at 70 °C for 10 min. Equal protein concentrations were separated by 15% SDS-PAGE to differentiate between covalently bound and unincorporated label. Identical reactions were run for the ATP nucleotide exchange reactions where [α-32P]ATP replaced [α-32P]GTP and nonradioactive ATP was added to a final concentration of 2 mm instead of GTP. A previously described protocol (19Yang L.S.-L. Frey P.A. Biochemistry. 1979; 18: 2980-2984Crossref PubMed Scopus (35) Google Scholar, 20Cartwright J.L. McLennan A.G. Arch. Biochem. Biophys. 1999; 361: 101-105Crossref PubMed Scopus (12) Google Scholar), with minor modifications, was used to characterize the guanylylated amino acid residue with minor modifications. CobU (1.4 μg) was added to a reaction mixture (100 μl final volume) containing 50 mmTris-HCl, pH 8.5, at 25 °C, 10 mm MgCl2, 1.25 mm TCEP-HCl, 680 mm glycerol, 0.1m NaCl, 10 μCi of [α-32P]GTP (800 Ci/mmol) and incubated for 10 min at 25 °C to allow formation of the CobU- [32P]GMP intermediate. The reaction was terminated by the addition of 200 μl of 15% trichloroacetic acid and incubated on ice 30 min. Precipitated protein was recovered by centrifugation (14,000 × g for 5 min). The pellet was washed once with 200 μl of 10% trichloroacetic acid followed by 3 washes with 0.5 ml of cold acetone. After the final acetone wash, the pellet was allowed to dry. The precipitated protein was resuspended in 100 μl of 50 mm sodium carbonate, pH 10.5, 1% SDS. Sodium periodate (NaIO4) was added to a final concentration of 10 mm and incubated at room temperature in the dark for 30 min. Ethylene glycol was added to a final concentration of 50 mm to quench periodate. pH was adjusted to 11 with 1m NaOH and the sample was incubated at 50 °C for 1 h. NaOH was then added to a final concentration of 3 m and the sample was incubated at 110 °C for 3 h to hydrolyze the protein. The hydrolysate was neutralized with 10% (v/v) perchloric acid (HClO4) and the precipitate was removed by centrifugation at 13,000 × g (Marathon 13 K/M microcentrifuge, Fisher Scientific) for 5 min. A 5-μl sample of the supernatant was mixed with carrier standard amino acids (phospholysine, phosphohistidine, and phosphoarginine) and analyzed by silica gel TLC (20Cartwright J.L. McLennan A.G. Arch. Biochem. Biophys. 1999; 361: 101-105Crossref PubMed Scopus (12) Google Scholar, 21Tuhackova Z. Krivanek J. Biochem. Biophys. Res. Commun. 1996; 218: 61-66Crossref PubMed Scopus (12) Google Scholar) along with samples of the individual phosphorylated amino acids. Phosphohistidine and phospholysine standards were synthesized as described previously (22Wei Y.F. Matthews H.R. Methods Enzymol. 1991; 200: 388-414Crossref PubMed Scopus (92) Google Scholar), while phosphoarginine was commercially available (Sigma). To generate the cobUH46A mutation a polymerase chain reaction (PCR) that incorporated the phosphorylated mutagenic oligonucleotide (5′-GAGAATTCAGCATGCTAAAGATGGCA-3′) was used (23Michael S.F. BioTechniques. 1994; 16: 410-414PubMed Google Scholar). The bases in bold-face type identify the altered codon. The external primers used in the mutagenesis were the −40 5′-GTTTTCCCAGTCACGAC-3′) and reverse (5′-AGCGGATAACAATTTCACACAGGA-3′) universal primers and the DNA template used was plasmid pJO51 (5O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1995; 270: 23560-23569Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The PCR-amplified product was cloned into pUC119 usingBamHI/HindIII sites and then subcloned into the overexpression vector pT7-7 using NdeI/HindIII restriction sites. The mutated, overexpression plasmid is pCOBU9 (cobUH46A). To generate the cobUH46N, cobUH45A/H46A, andcobUH45A mutations the EcoRI site immediately upstream of the histidine codons was used as a convenient restriction site in each mutagenic primer. Each mutagenic primer was used in a PCR reaction with the −40 primer shown above. The DNA template was pJO51 (5O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1995; 270: 23560-23569Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), which allowed amplification from the internal EcoRI site of cobU to the external −40 primer site. The PCR product was then digested with EcoRI/HindIII and the digested fragment was cloned intoEcoRI/HindIII-digested pJO52. This resulted in the exchange of the wild-type 3′-half of cobU with the mutagenized PCR product. The mutagenic primers used were as follows: H46N (5′-GCGAGAATTCAGCATAATAAAGATGGCAGG-3′), H45A/H46A (5′-GCGAGAATTCAGGCTGCTAAAGATGGCAGG-3′), and H45A (5′-GCGAGAATTCAGGCGCATAAAGATGGCAGG-3′). The bases in bold-face type indicate the altered codon(s). The resulting plasmids carrying the cobUH46N, cobUH45A/H46A,and cobUH45A mutations were pCOBU10, pCOBU11, and pCOBU12, respectively. All mutant clones were sequenced to ensure correct incorporation of the mutated codon, and that no additional PCR-induced mutations were present. Nonradioactive sequencing was performed at the University of Wisconsin, Madison Biotechnology Center. Plasmids pCOBU9-12 were individually transformed into JE2587 ((hisG-cob)Δ299/pGP1-2rpo + T7 RNA polymerasekan +) and used for overexpression and purification of the respective proteins as described above for wild-type CobU. Fig. 2shows a sample of purified CobU following the protocol outlined under “Experimental Procedures.” This protocol resulted in a 10-fold purification of CobU to >95% homogeneity based on densitometry (data not shown). This protocol allowed for a more ra" @default.
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