Abstract: The biosynthesis of thiamine in Escherichia coli requires the formation of an intermediate thiazole from tyrosine, 1-deoxy-d-xylulose-5-phosphate (Dxp), and cysteine using at least six structural proteins, ThiFSGH, IscS, and ThiI. We describe for the first time the reconstitution of thiazole synthase activity using cell-free extracts and proteins derived from adenosine-treated E. coli 83-1 cells. The addition of adenosine or adenine to growing cultures of Aerobacter aerogenes, Salmonella typhimurium, and E. coli has been shown previously to relieve the repression by thiamine of its own biosynthesis and increase the expression levels of the thiamine biosynthetic enzymes. By exploiting this effect, we show that the in vitro thiazole synthase activity of cleared lysates or desalted proteins from E. coli 83-1 cells is dependent upon the addition of purified ThiGH-His complex, tyrosine (but not cysteine or 1-deoxy-d-xylulose-5-phosphate), and an as yet unidentified intermediate present in the protein fraction from these cells. The activity is strongly stimulated by the addition of S-adenosylmethionine and NADPH. The biosynthesis of thiamine in Escherichia coli requires the formation of an intermediate thiazole from tyrosine, 1-deoxy-d-xylulose-5-phosphate (Dxp), and cysteine using at least six structural proteins, ThiFSGH, IscS, and ThiI. We describe for the first time the reconstitution of thiazole synthase activity using cell-free extracts and proteins derived from adenosine-treated E. coli 83-1 cells. The addition of adenosine or adenine to growing cultures of Aerobacter aerogenes, Salmonella typhimurium, and E. coli has been shown previously to relieve the repression by thiamine of its own biosynthesis and increase the expression levels of the thiamine biosynthetic enzymes. By exploiting this effect, we show that the in vitro thiazole synthase activity of cleared lysates or desalted proteins from E. coli 83-1 cells is dependent upon the addition of purified ThiGH-His complex, tyrosine (but not cysteine or 1-deoxy-d-xylulose-5-phosphate), and an as yet unidentified intermediate present in the protein fraction from these cells. The activity is strongly stimulated by the addition of S-adenosylmethionine and NADPH. Thiamine pyrophosphate (TPP), 1The abbreviations used are: TPP, thiamine pyrophosphate; Dxp, 1-deoxy-d-xylulose-5-phosphate; ThiGH-His, ThiGH in which ThiH has a C-terminal hexahistidine-tag; TP, thiamine monophosphate; Hmp, 4-amino-5-hydroxymethyl-2-methylpyrimidine; Hmp-P and Hmp-PP, 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate and pyrophosphate, respectively; Thz-P, 4-methyl-5-(β-hydroxyethyl) thiazole phosphate; AIR, 5-aminoimidazole ribotide; Dxs-His, 1-deoxy-d-xylulose-5-phosphate synthase containing a hexahistidine tag; HPLC, high performance liquid chromatography; DTT, dithiothreitol; AdoMet, S-adenosylmethionine. 1The abbreviations used are: TPP, thiamine pyrophosphate; Dxp, 1-deoxy-d-xylulose-5-phosphate; ThiGH-His, ThiGH in which ThiH has a C-terminal hexahistidine-tag; TP, thiamine monophosphate; Hmp, 4-amino-5-hydroxymethyl-2-methylpyrimidine; Hmp-P and Hmp-PP, 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate and pyrophosphate, respectively; Thz-P, 4-methyl-5-(β-hydroxyethyl) thiazole phosphate; AIR, 5-aminoimidazole ribotide; Dxs-His, 1-deoxy-d-xylulose-5-phosphate synthase containing a hexahistidine tag; HPLC, high performance liquid chromatography; DTT, dithiothreitol; AdoMet, S-adenosylmethionine. also known as vitamin B1, is an essential cofactor for several enzymes involved in carbohydrate metabolism. All organisms able to produce the vitamin initially assemble the correspondent monophosphate (TP) by coupling 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (Hmp-PP) and 4-methyl-5-(β-hydroxyethyl) thiazole phosphate (Thz-P) (Fig. 1) (1Begley T.P. Downs D.M. Ealick S.E. McLafferty F.W. Van Loon A. Taylor S. Campobasso N. Chiu H.J. Kinsland C. Reddick J.J. Xi J. Arch. Microbiol. 1999; 171: 293-300Crossref PubMed Scopus (234) Google Scholar). TP is then converted to the biologically active form, TPP, either by direct phosphorylation (in enteric bacteria) or by de-phosphorylation to thiamine followed by pyrophosphorylation (in aerobic bacteria and yeast) (2Spenser I.D. White R.L. Angew. Chem. Int. Ed. Engl. 1997; 36: 1033-1046Crossref Scopus (52) Google Scholar). The two heterocyclic precursors of thiamine pyrophosphate, Hmp-PP and Thz-P, are biosynthesized through independent pathways. In Escherichia coli and other enteric bacteria, Hmp-PP derives from 5-aminoimidazole ribotide (AIR), a precursor shared between thiamine and de novo purine biosyntheses (3Estramareix B. Therisod M. J. Am. Chem. Soc. 1984; 106: 3857-3860Crossref Scopus (51) Google Scholar, 4Estramareix B. David S. Biochim. Biophys. Acta. 1990; 1035: 154-160Crossref PubMed Scopus (33) Google Scholar). ThiC is the only enzyme known so far to be required for the rearrangement of AIR to Hmp-P (5Vanderhorn P.B. Backstrom A.D. Stewart V. Begley T.P. J. Bacteriol. 1993; 175: 982-992Crossref PubMed Google Scholar), which is subsequently further phosphorylated to Hmp-PP by ThiD. The Thz-P is assembled from Tyr (6Estramareix B. Therisod M. Biochim. Biophys. Acta. 1972; 192: 375-380Crossref Scopus (23) Google Scholar, 7Bellion E. Kirkley D.H. Faust J.R. Biochim. Biophys. Acta. 1976; 437: 229-237Crossref PubMed Scopus (25) Google Scholar, 8White R.H. Rudolph F.B. Biochim. Biophys. Acta. 1978; 542: 340-347Crossref PubMed Scopus (31) Google Scholar), Cys (9Tazuya K. Yamada K. Nakamura K. Kumaoka H. Biochim. Biophys. Acta. 1987; 924: 210-215Crossref PubMed Scopus (20) Google Scholar), and Dxp (10David S. Estramareix B. Fischer J.-C. Therisod M. J. Chem. Soc. Perkin Trans. 1982; 1: 2131-2137Crossref Google Scholar, 11Himmeldirk K. Kennedy I.A. Hill R.E. Sayer B.G. Spenser I.D. Chem. Commun. 1996; : 1187-1188Crossref Scopus (47) Google Scholar). ThiF-SGH (1Begley T.P. Downs D.M. Ealick S.E. McLafferty F.W. Van Loon A. Taylor S. Campobasso N. Chiu H.J. Kinsland C. Reddick J.J. Xi J. Arch. Microbiol. 1999; 171: 293-300Crossref PubMed Scopus (234) Google Scholar, 5Vanderhorn P.B. Backstrom A.D. Stewart V. Begley T.P. J. Bacteriol. 1993; 175: 982-992Crossref PubMed Google Scholar), ThiI (12Webb E. Claas K. Downs D.M. J. Bacteriol. 1997; 179: 4399-4402Crossref PubMed Google Scholar, 13Palenchar P.M. Buck C.J. Cheng H. Larson T.J. Mueller E.G. J. Biol. Chem. 2000; 275: 8283-8286Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), and IscS (14Lauhon C.T. Kambampati R. J. Biol. Chem. 2000; 275: 20096-20103Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) are all involved in the thiazole formation.Cysteine has been identified as the precursor of the thiazole sulfur atom. The sulfur atom is initially transferred to IscS, a pyridoxal phosphate-dependent cysteine desulfurase (15Zheng L.M. White R.H. Cash V.L. Dean D.R. Biochemistry. 1994; 33: 4714-4720Crossref PubMed Scopus (351) Google Scholar), in the form of a persulfide of an active site Cys residue (16Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). It is likely that in vivo the sulfur atom is then passed to ThiI (12Webb E. Claas K. Downs D.M. J. Bacteriol. 1997; 179: 4399-4402Crossref PubMed Google Scholar) as a persulfide of residue Cys-456 (13Palenchar P.M. Buck C.J. Cheng H. Larson T.J. Mueller E.G. J. Biol. Chem. 2000; 275: 8283-8286Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The sulfur atom is then transferred to the C terminus of ThiS, converting it to a thiocarboxylate (14Lauhon C.T. Kambampati R. J. Biol. Chem. 2000; 275: 20096-20103Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 17Taylor S.V. Kelleher N.L. Kinsland C. Chiu H.J. Costello C.K. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). This final sulfur transfer reaction requires the activation of ThiS as its acyladenylate (17Taylor S.V. Kelleher N.L. Kinsland C. Chiu H.J. Costello C.K. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The adenylation reaction is catalyzed by ThiF, which has been observed to form a complex with ThiS (17Taylor S.V. Kelleher N.L. Kinsland C. Chiu H.J. Costello C.K. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In addition, an unusual acylpersulfide link between ThiS thiocarboxylate and Cys-184 of ThiF has been observed, and mutagenesis studies suggest it may be important for thiazole formation (18Xi J. Ge Y. Kinsland C. McLafferty F.W. Begley T.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8513-8518Crossref PubMed Scopus (102) Google Scholar).Recent studies with purified Bacillus subtilis proteins have resulted in the first demonstration of thiazole biosynthesis in a bacterial cell-free system (19Park J.H. Dorrestein P.C. Zhai H. Kinsland C. McLafferty F.W. Begley T.P. Biochemistry. 2003; 42: 12430-12438Crossref PubMed Scopus (120) Google Scholar). Although B. subtilis shares some metabolic components with E. coli, including some of the biosynthetic proteins (ThiFSG, ThiI, and IscS) and two metabolic precursors (Dxp and Cys), the biosynthetic pathways also differ significantly. Whereas B. subtilis depends upon ThiO, an oxygen-dependent flavoenzyme, to utilize glycine to provide the C-2–N-3 fragment of the thiazole (1Begley T.P. Downs D.M. Ealick S.E. McLafferty F.W. Van Loon A. Taylor S. Campobasso N. Chiu H.J. Kinsland C. Reddick J.J. Xi J. Arch. Microbiol. 1999; 171: 293-300Crossref PubMed Scopus (234) Google Scholar, 20Settembre E.C. Dorrestein P.C. Park J.H. Augustine A.M. Begley T.P. Ealick S.E. Biochemistry. 2003; 42: 2971-2981Crossref PubMed Scopus (97) Google Scholar), E. coli and other enteric organisms substitute ThiO with an oxygen-sensitive protein, ThiH (21Leonardi R. Fairhurst S.A. Kriek M. Lowe D.J. Roach P.L. FEBS Lett. 2003; 539: 95-99Crossref PubMed Scopus (49) Google Scholar, 22Skovran E. Downs D.M. J. Bacteriol. 2000; 182: 3896-3903Crossref PubMed Scopus (67) Google Scholar, 23Gralnick J. Webb E. Beck B. Downs D. J. Bacteriol. 2000; 182: 5180-5187Crossref PubMed Scopus (33) Google Scholar) and use tyrosine to provide the C-2–N-3 unit.Although genetic studies have identified ThiG and ThiH as essential for thiazole biosynthesis in E. coli, incubation of overexpressed ThiFSGH and ThiI with ThiS thiocarboxylate, [U-14C]tyrosine, and Dxp has failed to produce any detectable amount of thiazole product (24Begley T.P. Xi J. Kinsland C. Taylor S. McLafferty F. Curr. Opin. Chem. Biol. 1999; 3: 623-629Crossref PubMed Scopus (84) Google Scholar). Under these conditions, tyrosine was cleaved to p-hydroxybenzyl alcohol, a known by-product of TPP biosynthesis (25White R.H. Biochim. Biophys. Acta. 1979; 583: 55-62Crossref PubMed Scopus (23) Google Scholar), but sulfur transfer from 35S-ThiS thiocarboxylate did not occur (1Begley T.P. Downs D.M. Ealick S.E. McLafferty F.W. Van Loon A. Taylor S. Campobasso N. Chiu H.J. Kinsland C. Reddick J.J. Xi J. Arch. Microbiol. 1999; 171: 293-300Crossref PubMed Scopus (234) Google Scholar).Analysis of the ThiH sequence has identified a conserved cysteine triad motif, thought to be involved in Fe-S cluster binding and characteristic of the "radical AdoMet" family (26Sofia H.J. Chen G. Hetzler B.G. Reyes-Spindola J.F. Miller N.E. Nucleic Acids Res. 2001; 29: 1097-1106Crossref PubMed Scopus (781) Google Scholar). Recently, hexahistidine-tagged ThiH (ThiH-His) has been isolated in a complex with ThiG and shown to contain a highly oxygen-sensitive Fe-S cluster (21Leonardi R. Fairhurst S.A. Kriek M. Lowe D.J. Roach P.L. FEBS Lett. 2003; 539: 95-99Crossref PubMed Scopus (49) Google Scholar), which might explain the difficulty in obtaining in vitro activity under aerobic conditions. The ThiGH complex is likely to be involved in the last steps of the thiazole biosynthesis, a cyclization reaction that could conceivably require sulfur transfer to Dxp from either the ThiS thiocarboxylate or the ThiFS conjugate, condensation with Tyr (or a derivative), and cyclization to the thiazole product.The addition of adenosine or adenine during the growth phase of enteric bacteria, such as Aerobacter aerogenes (27Moyed H.S. J. Bacteriol. 1964; 88: 1024-1029Crossref PubMed Google Scholar), Salmonella typhimurium (28Newell P.C. Tucker R.G. Biochem. J. 1966; 100: 512-516Crossref PubMed Scopus (27) Google Scholar, 29Newell P.C. Tucker R.G. Biochem. J. 1966; 100: 517-524Crossref PubMed Scopus (26) Google Scholar), and E. coli (6Estramareix B. Therisod M. Biochim. Biophys. Acta. 1972; 192: 375-380Crossref Scopus (23) Google Scholar, 30Kawasaki T. Iwashima A. Nose Y. J. Biochem. (Tokyo). 1969; 65: 407-416Crossref PubMed Scopus (20) Google Scholar, 31Kawasaki T. Nose Y. J. Biochem. (Tokyo). 1969; 65: 417-425Crossref PubMed Scopus (20) Google Scholar), is known to release the repression by thiamine pyrophosphate of its own biosynthesis by decreasing the intracellular concentration of the vitamin. 5-AIR is a shared precursor of Hmp-PP and purines (32Newell P.C. Tucker R.G. Biochem. J. 1968; 106: 279-287Crossref PubMed Scopus (79) Google Scholar). Adenosine and adenine are negative regulators of de novo purine biosynthesis (33Zalkin H. Nygaard P. Neidhardt F.C. Curtiss R.R. Ingraham J.L. Lin E.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. Vol. 1. American Society for Microbiology, Washington, D. C.1996: 561-579Google Scholar) and decrease the concentration of AIR, which results in a reduction in Hmp-PP biosynthesis and hence a decrease in intracellular TPP (28Newell P.C. Tucker R.G. Biochem. J. 1966; 100: 512-516Crossref PubMed Scopus (27) Google Scholar). Thiamine has been identified recently (34Winkler W. Nahvi A. Breaker R.R. Nature. 2002; 419: 952-956Crossref PubMed Scopus (948) Google Scholar) as one of the cellular metabolites with the potential to regulate its own production by directly binding to regulatory regions upstream of thiC and thiM within polycistronic mRNAs. A decrease in intracellular thiamine might therefore be expected to result in higher levels of biosynthetic enzymes (30Kawasaki T. Iwashima A. Nose Y. J. Biochem. (Tokyo). 1969; 65: 407-416Crossref PubMed Scopus (20) Google Scholar), thus providing a potential source of enzymes suitable for in vitro studies of thiazole biosynthesis.As a pre-requisite to elucidating the function of the ThiGH complex and the mechanism of the thiazole cyclization in E. coli, we report here the reconstitution of thiazole biosynthetic activity in vitro using a cell-free system containing extracts from adenosine-treated E. coli 83-1 cells (6Estramareix B. Therisod M. Biochim. Biophys. Acta. 1972; 192: 375-380Crossref Scopus (23) Google Scholar) and purified ThiGH-His under anaerobic conditions.EXPERIMENTAL PROCEDURESMaterials—Materials were purchased from the following suppliers: aldolase, triose-phosphate isomerase (TPI) (both from rabbit muscle), and all other reagent grade chemicals from Sigma unless otherwise stated; adenosine, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, and 4-aminobenzoic acid from Across; d-glucose from BDH; HPLC grade solvents, K2HPO4 and KH2PO4, from Fisher; K3[Fe(CN)6] and β-l-arabinose from Avocado; polyacrylamide/bispolyacrylamide solution (30% w/v, 37.5:1) from Amresco. Hmp and Thz-P were prepared as described (35Andersag H. Westphal K. Chem. Ber. 1937; 70: 2035-2044Crossref Google Scholar, 36Leder I.G. Methods Enzymol. 1970; 18: 166-167Crossref Scopus (9) Google Scholar). S-Adenosylmethionine (AdoMet) was a generous gift from Dr. H. Schroeder (BASF, Ludwigshafen, Germany). For TLC analysis, aluminum-backed TLC plates (Silica Gel 60, F-254, 250 μm) were used. A Gilson 321 HPLC system connected to a Shimadzu RF-10Axl fluorimeter and/or to a Gilson UV-visible detector was used for HPLC analysis. 1H NMR spectra were recorded using a Bruker AC300 (300 MHz) spectrometer.Bacterial Strains, Plasmids, and Proteins—E. coli 83-1 cells, which are Tyr/Trp/Phe auxotrophs, were the generous gifts from Prof. R. Azerad and Prof. M. Therisod (6Estramareix B. Therisod M. Biochim. Biophys. Acta. 1972; 192: 375-380Crossref Scopus (23) Google Scholar). BL21(DE3) cells transformed with a pET-23b-derived plasmid encoding for a hexahistidine-tagged 1-deoxy-d-xylulose-5-phosphate synthase (Dxs-His) were kindly donated by Prof. A. Boronat (37Lois L.M. Campos N. Putra S.R. Danielsen K. Rohmer M. Boronat A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2105-2110Crossref PubMed Scopus (345) Google Scholar). Dxs-His was expressed from these cells and purified to ∼80% purity by nickel-affinity and gel filtration chromatographies. ThiG and ThiH-His were purified from E. coli BL21(DE3) pRL1020 as described previously (21Leonardi R. Fairhurst S.A. Kriek M. Lowe D.J. Roach P.L. FEBS Lett. 2003; 539: 95-99Crossref PubMed Scopus (49) Google Scholar). Protein concentration was routinely estimated by the method of Bradford (38Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213510) Google Scholar); ThiG and ThiH-His concentrations in the ThiGH-His complex were determined by SDS-PAGE analysis and gel densitometry (Syngene Gene Genius imaging system) against bovine serum albumin standards.HPLC Purification of Dxp—Dxp was enzymatically synthesized as described by Taylor et al. (39Taylor S.V. Vu L.D. Begley T.P. Schorken U. Grolle S. Sprenger G.A. Bringer-Meyer S. Sahm H. J. Org. Chem. 1998; 63: 2375-2377Crossref Google Scholar), and the progress of the reaction was monitored by TLC analysis (40Hecht S. Kis K. Eisenreich W. Amslinger S. Wungsintaweekul J. Herz S. Rohdich F. Bacher A. J. Org. Chem. 2001; 66: 3948-3952Crossref PubMed Scopus (34) Google Scholar). When the reaction was judged to be complete, the proteins were removed by ultrafiltration (10-kDa molecular weight cut-off, Millipore), and Dxp was purified by semipreparative HPLC (PerkinElmer Life Sciences Prep-10 Octyl column, 250 × 10.0 mm). Dxp was eluted with H2O at 2 ml/min, and the column eluate was fractionated from t = 6.0 to t = 8.0 min. The TPP-containing fractions were identified by TLC analysis and discarded. The TPP-free fractions were pooled and freeze-dried. The Dxp content of the resultant white solid (which also contained residual salts) was determined by 1H NMR, and a stock solution of Dxp (50 mm) was prepared in 50 mm Tris-HCl, pH 8.0, 5 mm MgCl2. The absence of contaminating TPP was confirmed with the thiochrome assay (41Gerrits J. Eidhof H. Brunnekreeft J.W.I. Hessels J. Methods Enzymol. 1997; 279: 74-82Crossref PubMed Scopus (19) Google Scholar).Cell Culture Media—The following media were used to grow or resuspend E. coli 83-1: DM medium, Davis and Mingioli medium (42Davis B.D. Mingioli E.S. J. Bacteriol. 1950; 60: 17-27Crossref PubMed Google Scholar) without glucose; DM1 medium, DM medium supplemented with glucose (0.2%, w/v), Tyr and Phe (0.2 mm each), Trp (0.1 mm), 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, and 4-aminobenzoic acid (10 μm each) (6Estramareix B. Therisod M. Biochim. Biophys. Acta. 1972; 192: 375-380Crossref Scopus (23) Google Scholar). Combinations of Tyr (0.2 mm), Hmp (14 μm), and Thz-P (14 μm) were added to the DM medium where stated. DM and DM1 media were supplemented with ampicillin (100 μg/ml) in all the experiments with E. coli 83-1 pRL1020.Time Course of the Adenosine Derepression—E. coli 83-1 was derepressed in DM1 medium containing adenosine (300 μg/ml), essentially as described previously (6Estramareix B. Therisod M. Biochim. Biophys. Acta. 1972; 192: 375-380Crossref Scopus (23) Google Scholar). The TPP content was determined in duplicate by the thiochrome assay (41Gerrits J. Eidhof H. Brunnekreeft J.W.I. Hessels J. Methods Enzymol. 1997; 279: 74-82Crossref PubMed Scopus (19) Google Scholar) and expressed as nanograms of TPP/mg of wet cells. To be able to use the "wet weight" of the cell pellets in the calculations, the published procedure was slightly modified. Single colonies were used to inoculate DM1 medium (10 ml) and the cells grown for 8–10 h; the resultant culture was used as a 0.3% inoculum into fresh DM1 medium (300 ml) and grown overnight at 37 °C. Cells were harvested by centrifugation (Beckman JA-14, 12,000 rpm, 10 min at 4 °C), resuspended in DM medium (10 ml), transferred to pre-weighed tubes (15 ml), and centrifuged (Beckman JA-14 with adaptors, 7000 rpm, 10 min at 4 °C). After having drained most of the medium, residual liquid was removed by gently wiping the walls of each tube with a clean tissue. Tubes were then re-weighed, and the wet weight was obtained by subtraction. The weighed cell pellets were resuspended in DM medium to yield 24 mg/ml suspensions, pooled, used as inoculum (1.3 ml) in DM1 medium (100 ml/flask, 12 flasks), and aerated in a rotary shaker. β-l-Arabinose (0.2%) was added to the DM1 medium during derepression experiments with E. coli 83-1 pRL1020, where indicated. To provide duplicate data, a pair of flasks were withdrawn at t = 0 and every 30 min for 2.5 h, rapidly cooled in icy water, and the cells harvested by centrifugation. The cell pellets were then resuspended and washed with sterile 0.9% NaCl (two times, 10 ml), and the wet weight determined as described above. Cell pellets (27–180 mg) were stored at –80 °C until analyzed.The thiochrome method (41Gerrits J. Eidhof H. Brunnekreeft J.W.I. Hessels J. Methods Enzymol. 1997; 279: 74-82Crossref PubMed Scopus (19) Google Scholar) was adapted to estimate the TPP content in cell pellets. Briefly, the frozen cell pellets were thawed on ice for 10 min and then resuspended in a 1:1 mixture of H2O/7.2% HClO4 (2 ml for cell pellets of up to 80 mg of wet cells, 4 ml for heavier cell pellets); the white suspensions were sonicated on ice and the precipitated proteins removed by centrifugation. The supernatants (0.5 ml) were oxidized with a K3[Fe(CN)6] (12 mm in 3.35 m NaOH) solution (100 μl) (41Gerrits J. Eidhof H. Brunnekreeft J.W.I. Hessels J. Methods Enzymol. 1997; 279: 74-82Crossref PubMed Scopus (19) Google Scholar) and then adjusted to pH 7.0 with phosphoric acid, and an aliquot (50 μl) was analyzed by reverse phase HPLC using a Phenomenex ODS column (150 × 4.6 mm, 5-μm particle size) and eluted with buffer A (140 mm K2HPO4, 12% MeOH, 1.5% N,N-dimethylformamide, 0.3 mm tetrabutylammonium hydroxide, pH 7.0) and buffer B (70% MeOH, 30% H2O). The compounds were eluted at 0.8 ml/min with linear gradients between the following time points: t = 0 min, 0% buffer B; t = 2 min, 0% buffer B; t = 5 min, 20% buffer B; t = 10 min, 20% buffer B; t = 12 min, 40% buffer B; t = 15 min, 40% buffer B; and detected by fluorescence (excitation at 360 nm and emission at 454 nm). TPP, TP, and unphosphorylated thiamine solutions of known concentration were derivatized and analyzed by HPLC by the same method. The retention times of the thiochrome derivatives of TPP, TP, and unphosphorylated thiamine were determined to be 7.5, 9.0, and 15.0 min, respectively. TP and TPP calibration curves (0–1.3 μm) were constructed and used to quantify the thiamine derivatives present in samples. Unphosphorylated thiamine was not detected in any of the in vivo or in vitro enzyme assays, but the retention time was determined to exclude the possibility of unphosphorylated thiamine contributing to the total amount of thiamine derivatives present.TPP Production by Washed Cell Suspensions—Experiments were carried out in duplicate. Cultures of E. coli 83-1 or 83-1 pRL1020 (100 ml/flask, 14 flasks) were incubated with adenosine as described above, and samples withdrawn at t = 0, 1.0, and 2.5 h to monitor the derepression phase. At the end of this phase, the cultures were quickly cooled in icy water, and the cells were harvested by centrifugation (Beckman JA-14, 12,000 rpm, 10 min at 4 °C). The cell pellets were then washed with sterile 0.9% NaCl (two times, 10 ml) and transferred to pre-weighed tubes to be weighed. The pellets were resuspended in DM medium to yield 30 mg/ml suspensions, pooled, and used as a 10% inoculum into DM medium (10 ml) containing Hmp and either Tyr or Thz-P or no supplement. The 10-ml cultures were shaken at 37 °C, and the cells were harvested every 30 min for 2 h by centrifugation. The cell pellets were washed with NaCl solution, weighed, and stored at –80 °C until analyzed. The TPP content was estimated as described above.Large Scale Growth of E. coli 83-1 pRL1020 in the Presence of Adenosine—A single colony was used to inoculate DM1 medium (10 ml), and cells were grown at 37 °C for 8–10 h. This culture (3 ml) was used to inoculate fresh DM1 medium (800 ml). After overnight incubation at 37 °C, the cells were harvested by centrifugation (Beckman JA-14, 12000 rpm, 10 min at 4 °C). The cell pellets were weighed and resuspended in DM medium to yield 100 mg/ml suspensions, and then the resultant cultures were pooled and used as inocula (5 ml) in DM1 medium (four times, 1.25 liters) supplemented by the addition of adenosine (300 mg/liter). Cells were grown at 37 °C for 2.5 h, then cooled down by swirling in icy water, and harvested. The typical yield of cell paste ranged between 5.5 and 7.5 g from 5 liters. The cell paste was stored at –80 °C until used.Preparation of Cell-free Extracts Under Anaerobic Conditions— Where possible cell samples were manipulated under anaerobic conditions using a Belle glove box (<2 ppm of O2). Cell-free extracts from derepressed E. coli 83-1 pRL1020 or BL21(DE3) pRL1020 were prepared by introducing weighed amounts of cell paste (0.8–1.0 g) into the glove box and resuspending it in anaerobic reaction buffer (1.6–2.0 ml, 50 mm Tris-HCl, pH 8.0, 20 mm MgCl2, 5 mm DTT, 12.5% (w/v) glycerol). The suspensions were then withdrawn from the anaerobic box and rapidly lysed by sonication. The lysates were returned to the box and allowed to degas for 10 min and then cleared by centrifugation (Beckman JA-14 with appropriate adaptors, 7000 rpm, 15 min at 4 °C) in gas-tight tubes. The protein concentration in these lysates was typically 35–45 mg/ml. The cleared lysate prepared from E. coli BL21(DE3) pRL1020 cells (grown in 2YT medium) was routinely gel-filtered through a NAP-10 column (AP Biotech), equilibrated in anaerobic reaction buffer to remove nearly all of the low molecular weight molecules, including TPP, and is referred to as the "ThiGH-His-enriched fraction." Cell-free lysates and desalted protein fractions were prepared by similar methods from derepressed E. coli 83-1 pRL1020 and are termed "derepressed 83-1 lysate" and "derepressed 83-1 protein fraction," respectively.Measurement of the in Vitro Thiazole Synthase Activity by Enzymatic Coupling to Hmp-PP and Thiochrome Assay—Unless otherwise specified, the term "thiamine" is used to indicate any vitamin form, regardless of the extent of phosphorylation. A typical reaction mixture contained the following: 100 μl of derepressed 83-1 lysate or protein fraction, 100 μl of ThiGH-His-enriched protein fraction or purified ThiGH-His (8.0–10.0 mg/ml, total protein concentration) in anaerobic reaction buffer, ATP (20 mm), Tyr (1.5 mm), Cys (1.5 mm), Dxp (1.0 mm), and Hmp (0.6 mm). Depending on the experiment, the total volume varied between 250 and 350 μl, as required. Tyr stock solutions (40 mm) were prepared in 100 mm HCl. Cys stock solutions (80 mm) were freshly prepared by dissolving the solid in anaerobic reaction buffer. Dxp and Hmp stock solutions (50 and 14 mm, respectively) were prepared in 50 mm Tris-HCl, pH 8.0, 5 mm MgCl2. The ATP stock solutions (250 mm) were prepared in 300 mm Tris-HCl, pH 8.8. All solutions were prepared within the glove box using anaerobic buffer or allowed to degas in the glove box (30 min) before being added to the reaction mixtures. To determine the background level of TP or TPP present in the assays, control reactions were prepared and immediately stopped for analysis. The remaining reactions were incubated for 4 h at 37 °C, then stopped by adding HClO4 (7.2%, 500 μl), and the total volume adjusted to 1 ml with H2O. The precipitated proteins were removed by centrifugation and the supernatant (500 μl) assayed for TP and TPP. Due to the presence of DTT, the sensitivity of the thiochrome assay had to be optimized by doubling the final K3[Fe(CN)6] concentration. For each set of conditions, the amounts of TP and TPP were estimated separately, the respective background levels subtracted, and the results expressed as pmol/mg of protein/h.Effect of S-Adenosylmethionine and Reductants—Samples of purified ThiGH-His (100 μl, 113–136 μm in ThiH-His) were incubated for 2 h with a 5-fold molar excess of FeCl3 and Na2S, in the presence of DTT (5 mm), under anaerobic conditions. Thus treated ThiGH-His was added to in vitro reaction mixtures containing S-adenosylmethionine (1 mm) and NADPH (1 mm) in addition to Tyr, Hmp, ATP, and derepressed 83-1 protein fraction and then incubated at 37 °C for 4 h. Alternatively, the reconstituted ThiGH-His samples were anaerobically incubated with sodium dithionite (1 mm) for 30 min at room temperature (43Miller J.R. Busby R.W. Jordan S.W. Cheek J. Henshaw T.F. Ashley G.W. Broderick J.B. Cronan J.E. Marletta M.A. Biochemistry. 2000; 39: 15166-15178Crossref PubMed Scopus (168) Google Scholar) before being transferred to the AdoMet-containing reaction mixtures.RESULTS AND DISCUSSIONEffect of the Adenosine Treatment on Thiamine Biosynthesis in Washed Cell SuspensionsE. coli 83-1 cells are among those enteric bacteria that have been shown to be sensitive to the adenosine dere