Thiamin Biosynthesis in Escherichia coli
ThiFSGH and ThiI are required for the biosynthesis of the thiazole moiety of thiamin in Escherichia coli. The overproduction, purification, and characterization of ThiFS and the identification of two of the early steps in the biosynthesis of the thiazole moiety of thiamin are described here. ThiS isolated from E. coli thiI+ is post-translationally modified by converting the carboxylic acid group of the carboxyl-terminal glycine into a thiocarboxylate. ThethiI gene plays an essential role in the formation of the thiocarboxylate because ThiS isolated from athiI− strain does not contain this modification. ThiF catalyzes the adenylation by ATP of the carboxyl-terminal glycine of ThiS. This reaction is likely to be involved in the activation of ThiS for sulfur transfer from cysteine or from a cysteine-derived sulfur donor. ThiFSGH and ThiI are required for the biosynthesis of the thiazole moiety of thiamin in Escherichia coli. The overproduction, purification, and characterization of ThiFS and the identification of two of the early steps in the biosynthesis of the thiazole moiety of thiamin are described here. ThiS isolated from E. coli thiI+ is post-translationally modified by converting the carboxylic acid group of the carboxyl-terminal glycine into a thiocarboxylate. ThethiI gene plays an essential role in the formation of the thiocarboxylate because ThiS isolated from athiI− strain does not contain this modification. ThiF catalyzes the adenylation by ATP of the carboxyl-terminal glycine of ThiS. This reaction is likely to be involved in the activation of ThiS for sulfur transfer from cysteine or from a cysteine-derived sulfur donor. The thiamin biosynthetic pathway in Escherichia coli is outlined in Fig. 1 (1Begley T.P. Nat. Prod. Rep. 1996; 13: 177-186Crossref PubMed Scopus (50) Google Scholar, 2Estramareix B. David S. New J. Chem. 1996; 20: 607-629Google Scholar, 3Spenser I.D. White R.L. Angew. Chem. Int. Ed. Engl. 1997; 36: 1032-1046Crossref Scopus (52) Google Scholar). This pathway involves the separate synthesis of the thiazole (5-methyl-4-(β-hydroxyethyl)thiazole phosphate) and the pyrimidine (4-amino-5-hydroxymethylpyrimidine pyrophosphate) moieties, which are then coupled to give thiamin phosphate. The pyrimidine is derived from 5aminoimidazole ribotide (4Himmeldirk K. Sayer B.G. Spenser I.D. J. Am. Chem. Soc. 1998; 120: 3581-3589Crossref Scopus (24) Google Scholar, 5Estramareix B. David S. Biochim. Biophys. Acta. 1990; 1035: 154-160Crossref PubMed Scopus (33) Google Scholar, 6Estramareix B. David S. Biochem. Biophys. Res. Commun. 1986; 134: 1136-1141Crossref PubMed Scopus (26) Google Scholar, 7Tazuya K. Tanaka M. Morisaki M. Yamada K. Kumaoka H. Biochem. Int. 1987; 14: 769-777Google Scholar). The thiazole is derived from tyrosine (8Estramareix B. Therisod M. Biochim. Biophys. Acta. 1972; 273: 275-282Crossref PubMed Scopus (39) Google Scholar, 9Bellion E. Kirkley D.H. Faust J.R. Biochim. Biophys. Acta. 1976; 437: 229-237Crossref PubMed Scopus (25) Google Scholar, 10White R.H. Rudolph F.B. Biochim. Biophys. Acta. 1978; 542: 340-347Crossref PubMed Scopus (31) Google Scholar), cysteine (11Tazuya K. Yamada K. Nakamura K. Kumaoka H. Biochim. Biophys. Acta. 1987; 924: 210-215Crossref PubMed Scopus (20) Google Scholar, 12DeMoll E. Shive W. Biochem. Biophys. Res. Commun. 1985; 132: 217-222Crossref PubMed Scopus (25) Google Scholar), and 1-deoxy-d-xylulose-5-phosphate 1This compound has also been referred to as 1-deoxy-d-threo-2-pentulose-5-phosphate. 1This compound has also been referred to as 1-deoxy-d-threo-2-pentulose-5-phosphate.(13David S. Estramareix B. Fischer J.C. Therisod M. J. Chem. Soc. Perkin Trans. I. 1982; : 2131-2137Crossref Scopus (69) Google Scholar, 14Sprenger G.A. Schörken U. Wiegert T. Grolle S. de Graaf A.A. Taylor S.V. Begley T.P. Bringer-Meyer S. Sahm H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12857-12862Crossref PubMed Scopus (411) Google Scholar). The mechanistic enzymology of the thiazole and the pyrimidine formation is still poorly understood. A five gene operon (thiCEFGH) involved in thiamin biosynthesis has been cloned and characterized (GenBank™ accession number U00006; Ref. 15Vander Horn P.B. Backstrom A.D. Stewart V. Begley T.P. J. Bacteriol. 1993; 175: 982-992Crossref PubMed Google Scholar). The thiC gene complements pyrimidine (4-amino-5-hydroxymethylpyrimidine) requiring mutants, thethiFGH genes complement thiazole-requiring mutants, andthiE codes for thiamin phosphate synthase (16Backstrom A.D. McMordie R.A.S. Begley T.P. J. Am. Chem. Soc. 1995; 117: 2351-2352Crossref Scopus (42) Google Scholar). An additional gene (thiI) required for thiazole biosynthesis inSalmonella typhimurium has recently been identified (17Webb E. Claas K. Downs D.M. J. Bacteriol. 1997; 179: 4399-4402Crossref PubMed Google Scholar). This gene maps at 9.5 min on the E. coli chromosome and complements a nuvCmutation. 2C. Kinsland and T. P. Begley, unpublished results., 3Mueller, E. G., Buck, C. J., Palenchar, P. M., Barnhart, L. E., and Paulson, J. L. (1998) Nucleic Acids Res., in press.NuvC is required for the biosynthesis of thiouridine in tRNA (18Lipsett M. J. Biol. Chem. 1972; 247: 1458-1461Abstract Full Text PDF PubMed Google Scholar) as well as the thiazole moiety of thiamin (19Ryals J. Hsu R.Y. Lipsett M.N. Bremer H. J. Bacteriol. 1982; 151: 899-904Crossref PubMed Google Scholar) (Fig. 2). This suggests that ThiI may play a role in the sulfur transfer chemistry involved in the thiazole biosynthesis. When the gene product, originally assigned as ThiG, was purified from an overexpression strain, mass spectrometry and Edman sequencing both demonstrated that the protein consisted of two subunits. One of the subunits, which we will call ThiS, has a mass of 7310.74 Da. The second subunit, which will retain the ThiG name, has a mass of 26896.5 Da. 4Kelleher, N. L., Taylor, S. V., Grannis, D., Kinsland, C., Chiu, H.-J., Begley, T. P., and McLafferty, F. W. (1998)Protein Sci., in press. 4Kelleher, N. L., Taylor, S. V., Grannis, D., Kinsland, C., Chiu, H.-J., Begley, T. P., and McLafferty, F. W. (1998)Protein Sci., in press. Although the anomalous mass of ThiG was previously noted (15Vander Horn P.B. Backstrom A.D. Stewart V. Begley T.P. J. Bacteriol. 1993; 175: 982-992Crossref PubMed Google Scholar), ThiS was missed in the original reading frame assignment because of errors in both published sequences (15Vander Horn P.B. Backstrom A.D. Stewart V. Begley T.P. J. Bacteriol. 1993; 175: 982-992Crossref PubMed Google Scholar, 21Blattner F.R. Burland V. Plunkett III, G. Sofia H.J. Daniels D.L. Nucleic Acids Res. 1993; 21: 5408-5417Crossref PubMed Scopus (141) Google Scholar) and also because of the difficulty of detecting ThiS, which stains poorly with Coomassie Blue and migrates at the dye front during normal SDS-PAGE 5The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; ESI/FTMS, electrospray ionization Fourier transform mass spectrometry; DTT, dithiothreitol; SWIFT, stored wave form inverse Fourier transform; SORI, sustained off-resonance irradiation; PCR, polymerase chain reaction; ThiS-COAMP, C-terminal carboxy adenylated ThiS. 5The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; ESI/FTMS, electrospray ionization Fourier transform mass spectrometry; DTT, dithiothreitol; SWIFT, stored wave form inverse Fourier transform; SORI, sustained off-resonance irradiation; PCR, polymerase chain reaction; ThiS-COAMP, C-terminal carboxy adenylated ThiS. analysis. The carboxyl terminus of ThiS has the -Gly-Gly sequence found at the carboxyl terminus of human erythrocyte ubiquitin (GenBank™ accession number 1070588). This sequence similarity was striking because ThiF shows significant sequence similarity to the ubiquitin-activating enzyme (GenBank™ accession number 731039) including the ATP-binding site. This suggested that ThiF might catalyze the adenylation of ThiS and that ThiS-COAMP might react with cysteine (or a cysteine-derived sulfur donor) to give ThiS-COSH (Fig. 3). In addition, the dual role of ThiI in thiazole and in 4-thiouridine biosynthesis suggested that ThiI might play a role in this sulfur transfer reaction. In this paper, we describe experiments to test these hypotheses. LB broth was purchased in dehydrated form from Life Technologies, Inc. Tryptose blood agar base was purchased from Difco (Detroit, MI). Ampicillin and isopropyl-β-d-thiogalactopyranoside were from Jersey Lab and Glove Supply (Livingston, NJ). Tris, DTT, EDTA, (NH4)2SO4, and ATP were from Sigma. Sodium chloride was from Fisher (Pittsburgh, PA). Acetic acid and MeOH were from Aldrich. Acrylamide/Bis (37.5:1) was purchased from Bio-Rad. Dialysis membrane was from Spectrum (Houston, TX). All buffers were prepared from distilled, deionized water and were filtered through Millipore type HA 0.45-μm filters before use. Wizard™ PCR preps were purchased from Promega (Madison, WI). BL21(DE3) and the λDE3 Lysogenization kit were purchased from Novagen (Madison, WI). Protein purifications were performed on a Waters 650 chromatography instrument (Milford, MA). Gel filtration column (Superdex 75) was from Amersham Pharmacia Biotech. Reverse-phase mass spectrometric preparations were on Michrom BioResources (Auburn, CA) reverse-phase peptide traps. Protein concentration was assayed using Coomassie Plus protein assay reagent from Pierce. Concentrations were calibrated against a standard curve generated using bovine serum albumin. SDS-PAGE gels were prepared and run using standard methods (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206620) Google Scholar). Pyrophosphate assays were conducted using the Enz-Chek pyrophosphate assay kit from Molecular Probes Inc. (Eugene, OR). Mass values reported correspond to the most abundant isotopic peak (the difference in 1.0034 Da units from the monoisotopic peak is given in italics following the value). All spectra were calibrated externally using bovine ubiquitin as standard (8564.64 −5). AnuvC E. coli B strain was supplied by Hans Bremer (Dept. of Molecular Cell Biology, University of Texas, Dallas, TX). This strain is equivalent to the original nuvC strain, RYH101, described by Ryalset al. (19Ryals J. Hsu R.Y. Lipsett M.N. Bremer H. J. Bacteriol. 1982; 151: 899-904Crossref PubMed Google Scholar). Plasmid pVJS728, which was able to complement this nuvC mutant, was isolated from an E. coliK-12 HindIII library in pHG165 (23Stewart G.S.A.B. Lubinsky-Mink S. Jackson C.G. Cassel A. Kuhn J. Plasmid. 1986; 15: 172-181Crossref PubMed Scopus (142) Google Scholar). ThethiI456::Km (E. coli K-12) mutation was constructed by inserting a kanamycin cassette into the PstI site of pVJS728. This was back crossed to the E. colichromosome by recombination in the sbcBC strain VJS2889 (24Stewart V. Parales Jr., J. J. Bacteriol. 1988; 170: 1589-1597Crossref PubMed Google Scholar,25Winans S.C. Elledge S.J. Krueger J.H. Walker G.C. J. Bacteriol. 1985; 161: 1219-1221Crossref PubMed Google Scholar). Genetic crosses were performed via bacteriophage P1kc-mediated transduction (26Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 433Google Scholar). The VJS2890 strain was tested for growth with 5-methyl-4-(β-hydroxyethyl)thiazole and screened for UV sensitivity (nuvC+ phenotype). VJS2890 was grown in thiamin-limiting defined medium supplemented with 0.2% glucose (26Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 433Google Scholar) and then plated as a lawn on defined medium. 1 μl of 100 μm 5-methyl-4-(β-hydroxyethyl)thiazole was spotted on the plate, and growth around or between the 5-methyl-4-(β-hydroxyethyl)thiazole spots was determined after overnight incubation at 37 °C. UV sensitivity was determined as described previously by Ryals et al. (19Ryals J. Hsu R.Y. Lipsett M.N. Bremer H. J. Bacteriol. 1982; 151: 899-904Crossref PubMed Google Scholar). Site-specific integration of the λDE3 prophage into the chromosome of the ThiI mutant, VJS2890, was accomplished using the λDE3 lysogenization kit (Novagen). The basal and induced expression levels of T7 RNA polymerase in a representative set of 12 lysogens were tested following the protocol provided with the kit. The lysogen (VJS2890(DE3)) that showed the lowest level of basal expression and a high level of isopropyl-β-d-thiogalactopyranoside-induced expression of T7 RNA polymerase was chosen for use as an overexpression strain. The 3 base pairs immediately upstream of the ThiF start codon on plasmid pVJS716 (15Vander Horn P.B. Backstrom A.D. Stewart V. Begley T.P. J. Bacteriol. 1993; 175: 982-992Crossref PubMed Google Scholar) were mutated to insert an NdeI site (CATATG) using PCR. The 5′ primer was 5′-CT GGA AAT TGC AGG AGT TGC ATA TG ATG ACC GTG ACT TTA-3′ and the 3′ primer was 5′-CCA GAT AGC CAC TGG CGG-3′. The desired 1.8-kilobase pair fragment was purified using Wizard™ PCR preps (Promega) and the NdeI/SalI fragment cloned into the corresponding sites on pET-22b (Novagen) to yield plasmid pG201. TheMscI/SalI fragment (549 base pairs) was exchanged with the corresponding DNA from pVJS720 (15Vander Horn P.B. Backstrom A.D. Stewart V. Begley T.P. J. Bacteriol. 1993; 175: 982-992Crossref PubMed Google Scholar). A representative plasmid was named pCAC111. The remaining PCR-derived DNA (520 base pairs) was sequenced, and no mutations were observed. Plasmid pCAC111 was transformed into E. coli strain BL21(DE3), and transformants were selected on tryptose blood agar plates containing ampicillin (200 μg/ml). A single colony of E. coli containing pCAC111 was grown at 37 °C for 12 h in 3 ml of LB broth supplemented with ampicillin (200 μg/ml); 1 ml of this starter culture was diluted into 1 liter of the same medium and grown to an A 595 of approximately 0.6. Expression was induced by adding isopropyl-β-d-thiogalactopyranoside to a final concentration of 0.3 mm, and growth was continued at 37 °C for 12 h. Cells were harvested by centrifugation (8000 × g, 30 min) and stored at −70 °C. A frozen cell pellet from 1 liter of cell culture was thawed and resuspended in 25 ml of loading buffer (50 mm Tris, 2 mm DTT, 2 mm EDTA, pH 7.5), treated with lysozyme (5 mg, 40 min, 4 °C), sonicated at 4 °C for 4 min, and centrifuged (27,000 × g, 15 min). The resulting cell free lysate was taken to 20% (NH4)2SO4 saturation at 4 °C by slow addition of solid (NH4)2SO4 and gentle stirring over 45 min. This mixture was centrifuged (27,000 × g, 15 min), and the supernatant was taken to 50% (NH4)2SO4 saturation at 4 °C by slow addition of solid (NH4)2SO4with gentle stirring over 45 min. This mixture was centrifuged as before, and the resulting pellet was stored at −70 °C. The 20–50% (NH4)2SO4 pellet was thawed, resuspended in 10 ml of loading buffer, and for 12 h at 4 °C against 4 of loading buffer to (NH4)2SO4 from the This was then through a 0.45-μm and a column × 100 The protein mixture was purified by was 4 The column with protein was with loading buffer for 10 min and then taken from buffer (50 mm Tris, 2 mm DTT, 2 mm EDTA, pH to buffer over min. these ThiF and ThiS at buffer, that ThiS is to The were identified by SDS-PAGE exchanged into gel filtration buffer (50 mm Tris, 2 2 mm EDTA, mm using a and a gel filtration column Pharmacia a of ThiF and ThiS after h. The were identified by SDS-PAGE and and exchanged into mm Tris, 2 mm DTT, pH using a membrane The protein was diluted by adding to and frozen at a final concentration of use. SDS-PAGE of the is in Fig. 4 A. The of ThiFS coli was to the of ThiFS from E. SDS-PAGE of the is in Fig. 4 B. Pyrophosphate was assayed using the Enz-Chek pyrophosphate assay kit Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The assay was 1 The reaction buffer was mm 1 pH μl of ThiFS isolated from BL21(DE3) concentration the of ThiF to ThiS was not was to the reaction. ATP was to a concentration of were run for 1 min. buffer (50 10 mm 10 mm mm Tris, 2 mm DTT, pH was to ThiFS μl of a isolated from E. coli at 25 °C for 10 min and then to pH 3 by addition of acid The assay mixture was diluted with water and a reverse-phase peptide The was with 1 ml of and the protein was with μl of acid (the μl was 2 μl of this was into a mm to a at M. M. Int. J. Mass Proc. Scopus Google Scholar). A into the of the with the and a of on the determined the by were by the of a Fourier transform mass described previously S.C. J. Am. Soc. Mass 1993; PubMed Scopus Google Scholar, Chem. Res. Scopus Google Scholar). are through five of by a and into the at of were isolated in the cell by the stored wave form inverse Fourier transform J. Am. Chem. Soc. 1985; Scopus Google Scholar) and by sustained off-resonance Acta. Scopus Google Scholar) in the of ThiFS isolated from E. coli BL21(DE3) was thawed and to pH 3 by the addition of acid The mixture was diluted with water and a reverse-phase peptide The was with 1 ml of and the protein was with μl of acid (the μl was 2 μl of this was into a and as described ThiFS isolated coli was in an ThiFS isolated from E. coli BL21(DE3) was thawed, and acid was μl of a 10 mm in 25 mm Tris, pH This mixture was for min at 25 °C and then to pH 3 by addition of acid The mixture was diluted with water and a reverse-phase peptide The was with 1 ml of and the protein was with μl of acid (the μl was 2 μl of this was into a and as described The of ThiFS is in Fig. of the ThiFS overexpression provided high levels of The yield of purified ThiFS was of cell ThiF and ThiS through that ThiF and ThiS form a ThiS stains poorly with Coomassie Blue and is not on the The mass spectra in ThiFS ThiFS the formation of pyrophosphate from ATP (Fig. This reaction required as a Pyrophosphate a because of the of ThiS, after the formation of approximately the of was of pyrophosphate be This suggests that the is and that during of ThiS from the was not to ThiF on catalyze the of ATP because overexpression of ThiF in the of ThiS The overexpression of ThiS using the was also The of ThiFS isolated from E. coli is in Fig. In addition to that be assigned to the shows at that be assigned to on ThiFS isolated from E. coli BL21(DE3) demonstrated that the ThiS an additional mass of Da with ThiS isolated from E. coli (Fig. and This is with the of with sulfur on the The of ThiS to in the was approximately and activation of the 10 fragment two of these of and and were assigned as and that contain the of the P. J. Mass PubMed Scopus Google Scholar). the of Da glycine and Da glycine Da from the terminus of the and suggested that the glycine of ThiS has been to a thiocarboxylate in the strain (Fig. sequencing of the fragment resulting from and activation of from of protein sites of and The acid as ThiFS from E. coli BL21(DE3) was with of the reaction mixture by demonstrated the formation of a with mass of corresponding to (Fig. of the of the at in a separate by activation a of (Fig. All of these were assigned as or that contain the terminus of the P. J. Mass PubMed Scopus Google Scholar). All of the through a mass to that from the ThiS and the and the modified This the site of to the carboxyl-terminal and (Fig. the group in these is the ThiS isolated from E. coli BL21(DE3) contain a thiocarboxylate at the The sequence similarity between and the ubiquitin-activating enzyme suggested that ThiF might catalyze the adenylation of ThiS and that ThiS might be the sulfur in thiamin biosynthesis. In addition, the dual role of ThiI in both thiazole biosynthesis and in sulfur transfer in 4-thiouridine biosynthesis in tRNA suggested that ThiI may play a role in the sulfur transfer to ThiS. The high level overexpression of ThiFS coli BL21(DE3) and coli and the of these has to test these (Fig. 3). When ThiFS isolated from E. coli BL21(DE3) was with a of pyrophosphate formation was (Fig. The reaction then because of the of ThiS to was to this using (Fig. This that ThiF catalyzes the adenylation of ThiS on an of ThiFS isolated from E. coli BL21(DE3) by demonstrated the of a as the form of isolated ThiS (Fig. This be because of during or to the of an by sequencing of (Fig. and the resulting from of by (Fig. identified the as an to sulfur and the site of to the carboxylic acid group of the carboxyl-terminal glycine of ThiS isolated from E. coli demonstrated that the ThiS-COSH was not in this strain (Fig. This suggests that ThiI plays an essential role in the of ThiS-COAMP to The of ThiS-COSH was of the mechanistic for the formation of the thiazole moiety of thiamin the of an (1Begley T.P. Nat. Prod. Rep. 1996; 13: 177-186Crossref PubMed Scopus (50) Google Scholar). 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Chem. 1993; Full Text PDF PubMed Google Scholar). the identification of the sulfur in thiamin biosynthesis and the of an overexpression strain of C., Taylor, S. V., N. L., McLafferty, F. and Begley, T. P. (1998) Protein Sci., in press. be to the biosynthesis of the thiazole these are in