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J Biol Chem, Vol. 273, Issue 26, 16555-16560, June 26, 1998
From the Department of Chemistry, Cornell University, Baker
Laboratory, Ithaca, New York 14853
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. The
thiI gene plays an essential role in the formation of the thiocarboxylate because ThiS isolated from a
thiI The thiamin biosynthetic pathway in Escherichia coli is
outlined in Fig. 1 (1-3). This pathway
involves the separate synthesis of the thiazole
(5-methyl-4-(
Thiamin Biosynthesis in Escherichia coli
IDENTIFICATION OF ThiS THIOCARBOXYLATE AS THE IMMEDIATE SULFUR
DONOR IN THE THIAZOLE FORMATION*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-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 (4-7). The thiazole is
derived from tyrosine (8-10), cysteine (11, 12), and
1-deoxy-D-xylulose-5-phosphate1
(13, 14). 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 (GenBankTM accession number U00006;
Ref. 15).
View larger version (27K):
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Fig. 1.
The generally accepted pathway for thiamin
biosynthesis in E. coli. Salvage pathways are not
shown.
The thiC gene complements pyrimidine (4-amino-5-hydroxymethylpyrimidine) requiring mutants, the thiFGH genes complement thiazole-requiring mutants, and thiE codes for thiamin phosphate synthase (16). An additional gene (thiI) required for thiazole biosynthesis in Salmonella typhimurium has recently been identified (17). This gene maps at 9.5 min on the E. coli chromosome and complements a nuvC mutation.2,3 NuvC is required for the biosynthesis of thiouridine in tRNA (18) as well as the thiazole moiety of thiamin (19) (Fig. 2). This suggests that ThiI may play a role in the sulfur transfer chemistry involved in the thiazole biosynthesis.
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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.4 Although the anomalous mass of ThiG was previously noted (15), ThiS was missed in the original reading frame assignment because of errors in both published sequences (15, 21) and also because of the difficulty of detecting ThiS, which stains poorly with Coomassie Blue and migrates at the dye front during normal SDS-PAGE5 analysis. The carboxyl terminus of ThiS has the -Gly-Gly sequence found at the carboxyl terminus of human erythrocyte ubiquitin (GenBankTM accession number 1070588). This sequence similarity was striking because ThiF shows significant sequence similarity to the ubiquitin-activating enzyme (GenBankTM 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.
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EXPERIMENTAL PROCEDURES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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LB broth was purchased in dehydrated form from Life
Technologies, Inc. Tryptose blood agar base was purchased from Difco
(Detroit, MI). Ampicillin and
isopropyl--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. WizardTM PCR preps
were purchased from Promega (Madison, WI). BL21(DE3) and the 
DE3
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 (22). 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).
Construction of thiI Strain, VJS2890--
A
nuvC 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 Ryals
et al. (19). Plasmid pVJS728, which was able to complement this nuvC mutant, was isolated from an E. coli
K-12 HindIII library in pHG165 (23). The
thiI456::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. coli
chromosome by recombination in the sbcBC strain VJS2889 (24,
25). Genetic crosses were performed via bacteriophage P1
kc-mediated transduction (26).
Phenotype Analysis of VJS2890--
The VJS2890 strain was tested
for growth with 5-methyl-4-(-hydroxyethyl)thiazole and screened for
UV sensitivity (nuvC+ phenotype). VJS2890 was
grown in thiamin-limiting defined medium supplemented with 0.2%
glucose (26) and then plated as a lawn on defined medium. 1 µl of 100 µM 5-methyl-4-(-hydroxyethyl)thiazole was spotted on
the plate, and growth around or between the
5-methyl-4-(-hydroxyethyl)thiazole spots was determined after
overnight incubation at 37 °C. UV sensitivity was determined as
described previously by Ryals et al. (19).
Construction of thiI Overexpression Strain,
VJS2890(DE3)--
Site-specific integration of the DE3 prophage
into the chromosome of the ThiI mutant, VJS2890, was accomplished using
the DE3 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--D-thiogalactopyranoside-induced expression of
T7 RNA polymerase was chosen for use as an overexpression strain.
Construction of the ThiFS Overexpression Vector-- The 3 base pairs immediately upstream of the ThiF start codon on plasmid pVJS716 (15) 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 WizardTM PCR preps (Promega) and the NdeI/SalI fragment cloned into the corresponding sites on pET-22b (Novagen) to yield plasmid pG201. The MscI/SalI fragment (549 base pairs) was exchanged with the corresponding DNA from pVJS720 (15). A representative plasmid was named pCAC111. The remaining PCR-derived DNA (520 base pairs) was sequenced, and no mutations were observed.
Overexpression and Purification of ThiFS from E. coli BL21(DE3)
(thiI+)--
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 A595 of
approximately 0.6. Expression was induced by adding isopropyl--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)2SO4
with 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 dialyzed (12,000-14,000 molecular weight cut-off) for 12 h at 4 °C against 4 liters of loading buffer to exchange out
(NH4)2SO4 from the solution. This was then passed through a 0.45-µm filter and loaded onto a
Protein-Pak DEAE HR 8-µm 1000 Å column (20 × 100 mm) (Waters).
The ThiF/ThiS protein mixture was purified by gradient elution (flow
rate was 4 ml/min). The column with bound protein was washed with
loading buffer for 10 min and then taken from 0% elution buffer (50 mM Tris, 2 mM DTT, 2 mM EDTA, 1 M NaCl, pH 7.5) to 30% elution buffer over 70 min. Under
these conditions, ThiF and ThiS eluted together at 15% elution buffer,
suggesting that ThiS is bound to ThiF. The ThiFS-containing fractions
were identified by SDS-PAGE analysis, pooled, concentrated, exchanged
into gel filtration buffer (50 mM Tris, 2 mM
DTT, 2 mM EDTA, 20 mM NaCl) using a YM10 Amicon ultrafiltration membrane, and loaded onto a Superdex 75 gel filtration column (Amersham Pharmacia Biotech). At a flow rate of 1.2 ml/min, ThiF
and ThiS again eluted together after 1.5 h. The ThiFS-containing fractions were identified by SDS-PAGE analysis and pooled,
concentrated, and exchanged into 50 mM Tris, 2 mM DTT, pH 7.5, using a YM10 Amicon ultrafiltration
membrane (Millipore). The protein solution was diluted by adding
glycerol to 10% and frozen at a final concentration of 16 mg/ml until
use. SDS-PAGE analysis of the purification is shown in Fig.
4A.
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Overexpression and Purification of ThiFS from E. coli
VJS2890(DE3) (thiI)--
The purification of ThiFS from
E. coli VJS2890(DE3) (thiI) was
identical to the purification of ThiFS from E. coli
BL21(DE3) (thiI+). SDS-PAGE analysis of the
purification is shown in Fig. 4B.
Pyrophosphate Assay-- Pyrophosphate was assayed using the Enz-Chek pyrophosphate assay kit (27). The assay volume was 1 ml. The reaction buffer was 20 mM Tris-HCl, 1 mM MgCl2, pH 7.5. 75 µl of ThiFS isolated from BL21(DE3) (thiI+) (approximate concentration 16 mg/ml; the molar ratio of ThiF to ThiS was not determined) was added to initiate the reaction. ATP was added to a concentration of 2.5 mM. Assays were run for 1 min.
Mass Spectrometric Detection of a Covalent ThiS-AMP
Adduct--
Reaction buffer (50 µl, 10 mM ATP, 10 mM MgCl2, 50 mM Tris, 2 mM DTT, pH 7.5) was added to ThiFS (25 µl of a 16 mg/ml
solution) isolated from E. coli VJS2890(DE3)
(thiI) incubated at 25 °C for 10 min and
then acidified to pH 3 by addition of glacial acetic acid (2 µl). The
assay mixture was diluted with water (100 µl) and loaded onto a
reverse-phase peptide trap (Michrom BioResources, Inc.). The trap was
washed with 1 ml of 98% H2O/1% MeOH/1% acetic acid, and
the protein was eluted with 150 µl of 80% methanol/20% acetic acid
(the first 50 µl was discarded). 2 µl of this eluent was loaded
into a borosilicate glass capillary (1.5 mm inner diameter) pulled to a
2-µm tip at one end (28). A platinum wire inserted into the distal
end of the glass capillary made contact with the solution and a voltage of 0.7-1.4 kV on the wire determined the flow rate (
25-75 nl/min). Droplets formed by ESI were sampled by the heated metal capillary (110 °C) of a 6T Fourier transform mass spectrometer described previously (29, 30). Briefly, ions are guided through five stages of
differential pumping by a skimmer and three quadrupole ion guides into
the magnet bore held at 109 torr. Ions of interest were
isolated in the trapped ion cell by the stored wave form inverse
Fourier transform (SWIFT) technique (31) and collisionally activated by
sustained off-resonance irradiation (SORI) (32) in the presence of
106 torr N2.
Mass Spectrometric Detection of ThiS Thiocarboxylate--
ThiFS
(25 µl, 16 mg/ml), isolated from E. coli BL21(DE3)
(thiI+) was thawed and acidified to pH 3 by the
addition of glacial acetic acid (2 µl). The mixture was diluted with
water (100 µl) and loaded onto a reverse-phase peptide trap (Michrom
BioResources, Inc.). The trap was washed with 1 ml of 98%
H2O/1% MeOH/1% acetic acid, and the protein was eluted
with 150 µl of 80% methanol/20% acetic acid (the first 50 µl was
discarded). 2 µl of this eluent was loaded into a borosilicate glass
capillary and electrosprayed as described above. ThiFS isolated from
E. coli VJS2890(DE3) (thiI) was
analyzed in an identical manner.
Iodoacetic Acid Derivatization of ThiS Thiocarboxylate-- ThiFS (25 µl, 16 mg/ml) isolated from E. coli BL21(DE3) (thiI+) was thawed, and iodoacetic acid was added (25 µl of a 10 mM solution in 25 mM Tris, pH 7.5). This mixture was incubated for 5 min at 25 °C and then acidified to pH 3 by addition of glacial acetic acid (2 µl). The mixture was diluted with water (100 µl) and loaded onto a reverse-phase peptide trap (Michrom BioResources, Inc.). The trap was washed with 1 ml of 98% H2O/1% MeOH/1% acetic acid, and the protein was eluted with 150 µl of 80% methanol/20% acetic acid (the first 50 µl was discarded). 2 µl of this eluent was loaded into a borosilicate glass capillary and electrosprayed as described above.
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RESULTS |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Purification of ThiFS-- The purification of ThiFS is summarized in Fig. 4. Both of the ThiFS overexpression strains provided high levels of soluble protein. The average yield of purified (>90%) ThiFS was 5 mg/liter of cell culture. ThiF and ThiS copurify through several steps, demonstrating that ThiF and ThiS form a stable complex. ThiS stains poorly with Coomassie Blue and is not visible on the gel. The mass spectra discussed below clearly demonstrate its presence in all ThiFS samples examined.
Pyrophosphate Production by ThiFS Isolated from E. coli BL21(DE3) (thiI+)-- ThiFS catalyzed the formation of pyrophosphate from ATP (Fig. 5). This reaction required Mg2+ as a cofactor. Pyrophosphate production reached a plateau, presumably because of the complete consumption of ThiS, after the formation of approximately 23 nmol pyrophosphate. If the ratio of ThiF:ThiS was 1:1, 35 nmol of pyrophosphate would be expected. This suggests that the ThiF:ThiS stoichiometry is 1:0.66, and that during purification 34% of ThiS dissociates from the ThiF. It was not possible to determine whether ThiF on its own would catalyze the hydrolysis of ATP because overexpression of ThiF in the absence of ThiS gave only inclusion bodies. The overexpression of ThiS using the pET-16B vector was also unsuccessful.
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Mass Spectrometric Detection of a Covalent ThiS-AMP
Adduct--
The ESI/FTMS of ATP-treated ThiFS isolated from E. coli VJS2890(DE3) (thiI) is shown in Fig.
6. In addition to showing ions that can
be assigned to [ThiS]5+ (m/z = 1463.15), the spectrum shows ions at m/z = 1528.97 that can be assigned to [ThiS-COAMP]5+.
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Mass Spectrometric Characterization of ThiS Isolated from E. coli
BL21(DE3) (thiI+) and from E. coli VJS2890(DE3)
(thiI)--
ESI/FTMS on ThiFS isolated from E. coli BL21(DE3) (thiI+) demonstrated that
the predominant ThiS ion had an additional mass of 16 Da compared with
ThiS isolated from E. coli VJS2890(DE3) (thiI) (Fig. 7,
A and B, respectively). This is consistent with
the replacement of one oxygen with sulfur on the protein. The ratio of
ThiS to ThiS+16 in the (thiI+) sample was
approximately 1:9. SWIFT isolation and SORI activation of the
[ThiS+16]6+ ions gave 10 fragment ions; two of these had
masses of 7235.64 and 7178.69 and were assigned as b65 and
b64 ions (fragments that contain the NH2
terminus of the protein) (33). These represent the loss of 91 Da
(COOH-terminal glycine + 16 Da) and 148 Da (COOH-terminal glycine + 16 Da + internal glycine), respectively, from the COOH terminus of the
ThiS+16 adduct and strongly suggested that the COOH-terminal glycine
carboxylate of ThiS has been converted to a thiocarboxylate in the
(thiI+) strain (Fig.
8).
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4, corresponding to
[ThiS+16-H+CH2COOH] (Fig.
9A). SWIFT isolation of the 6+
charge state of the sodium adduct at m = 7406.7 (formed
in a separate sample ionization), followed by SORI activation gave a
series of 5+ fragments (Fig. 9B). All of these fragments
were assigned either as b ions or y ions (fragments that contain the
COOH terminus of the protein) (33). All of the observed b ions through
b63 had a mass identical to that predicted from the
unmodified ThiS sequence, and the y62, y63, and
y64 ions (sodium adducts) all contained the modified amino
acid. This localized the site of modification to the carboxyl-terminal
Ala64, Gly65, and Gly66 residues
(Fig. 9C). Because the only functional group in these three
residues is the Gly66 carboxylate, ThiS isolated from E. coli BL21(DE3) (thiI+) must contain a
thiocarboxylate at the COOH terminus.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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The sequence similarity between ThiF/ThiS and the
ubiquitin-activating enzyme E1/ubiquitin suggested that ThiF might
catalyze the adenylation of ThiS and that ThiS might be the sulfur
carrier 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 soluble ThiFS from
E. coli BL21(DE3) (thiI+) and from
E. coli VJS2890(DE3) (thiI) and the
partial purification of these proteins (>90%) has made it possible to
test these hypotheses (Fig. 3).
When ThiFS isolated from E. coli BL21(DE3) (thiI+) was incubated with ATP, a rapid initial burst of pyrophosphate formation was observed (Fig. 5). The reaction then stops presumably because of the complete conversion of ThiS to ThiS-COAMP. It was possible to directly detect this species using ESI/FTMS (Fig. 6). This demonstrates that ThiF catalyzes the adenylation of ThiS on an oxygen nucleophile.
Analysis of ThiFS isolated from E. coli BL21(DE3) (thiI+) by ESI/FTMS demonstrated the presence of a ThiS+16 adduct as the major form of isolated ThiS (Fig. 7A). This could be because of methionine oxidation during isolation or to the replacement of an oxygen by sulfur. Partial sequencing of ThiS+16 (Fig. 8) and the product resulting from alkylation of ThiS+16 by iodoacetate (Fig. 9A) unambiguously identified the +16 adduct as an oxygen to sulfur replacement and uniquely localized the site of modification to the carboxylic acid group of the carboxyl-terminal glycine residue.
MS analysis of ThiS isolated from E. coli VJS2890(DE3)
(thiI) demonstrated that the ThiS-COSH was not
formed in this strain (Fig. 7B). This suggests that ThiI
plays an essential role in the conversion of ThiS-COAMP to
ThiS-COSH.
The detection of ThiS-COSH was surprising. None of the previous mechanistic proposals for the formation of the thiazole moiety of thiamin predicted the existence of such an intermediate (1).
The mechanistic enzymology of the sulfur transfer chemistry involved in the biosynthesis of thiamin, molybdopterin, biotin, and lipoic acid is still poorly understood. The immediate sulfur source for the formation of biotin and lipoic acid is unknown, and the mechanism of the sulfur insertion chemistry is not yet clear (34-38). The sulfur transfer chemistry involved in thiamin and molybdopterin biosynthesis is likely to be similar. A three-enzyme system (MoeB, MoaD, and MoaE) for the sulfur transfer to precursor Z in molybdopterin biosynthesis has been reconstituted (39). ThiF and MoeB (GenBankTM accession number 127233) show high sequence similarity, and both MoaD (GenBankTM accession number 2507065) and ThiS contain the carboxyl-terminal GG residues and bind sulfur as a thiocarboxylate (20, 40). With the identification of the immediate sulfur donor in thiamin biosynthesis and the development of an overexpression strain yielding milligram quantities of ThiS-COSH,6 it should now be possible to reconstitute the biosynthesis of the thiazole moiety. Efforts along these lines are in progress.
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FOOTNOTES |
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* This work was supported by Grants DK44083 (to T. P. B.) and GM16609 (to F. W. M.) from the National Institutes of Health, a fellowship from the ACS Analytical Chemistry Division sponsored by the Perkin-Elmer Corp. (to N. L. K.), a National Institutes of Health Training Grant (to S. V. T. and N. L. K.), and a National Science Foundation Fellowship (to C. L. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed.
1 This compound has also been referred to as 1-deoxy-D-threo-2-pentulose-5-phosphate.
2 C. Kinsland and T. P. Begley, unpublished results.
3 Mueller, E. G., Buck, C. J., Palenchar, P. M., Barnhart, L. E., and Paulson, J. L. (1998) Nucleic Acids Res., in press.
4 Kelleher, N. L., Taylor, S. V., Grannis, D., Kinsland, C., Chiu, H.-J., Begley, T. P., and McLafferty, F. W. (1998) Protein Sci., in press.
5 The 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.
6 Kinsland, C., Taylor, S. V., Kelleher, N. L., McLafferty, F. W., and Begley, T. P. (1998) Protein Sci., in press.
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REFERENCES |
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Abstract
Introduction
Procedures
Results
Discussion
References
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M. M. Wuebbens and K. V. Rajagopalan Mechanistic and Mutational Studies of Escherichia coli Molybdopterin Synthase Clarify the Final Step of Molybdopterin Biosynthesis J. Biol. Chem., April 11, 2003; 278(16): 14523 - 14532. [Abstract] [Full Text] [PDF]
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D. A. Rodionov, A. G. Vitreschak, A. A. Mironov, and M. S. Gelfand Comparative Genomics of Thiamin Biosynthesis in Procaryotes. NEW GENES AND REGULATORY MECHANISMS J. Biol. Chem., December 6, 2002; 277(50): 48949 - 48959. [Abstract] [Full Text] [PDF]
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J. Miranda-Rios, M. Navarro, and M. Soberon A conserved RNA structure (thi box) is involved in regulation of thiamin biosynthetic gene expression in bacteria PNAS, July 19, 2001; (2001) 161168098. [Abstract] [Full Text] [PDF]
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J. Xi, Y. Ge, C. Kinsland, F. W. McLafferty, and T. P. Begley Biosynthesis of the thiazole moiety of thiamin in Escherichia coli: Identification of an acyldisulfide-linked protein-protein conjugate that is functionally analogous to the ubiquitin/E1 complex PNAS, June 28, 2001; (2001) 141226698. [Abstract] [Full Text] [PDF]
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G. C. McGhee and A. L. Jones Complete Nucleotide Sequence of Ubiquitous Plasmid pEA29 from Erwinia amylovora Strain Ea88: Gene Organization and Intraspecies Variation Appl. Envir. Microbiol., November 1, 2000; 66(11): 4897 - 4907. [Abstract] [Full Text]
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J. Gralnick, E. Webb, B. Beck, and D. Downs Lesions in gshA (Encoding gamma -L-Glutamyl-L-Cysteine Synthetase) Prevent Aerobic Synthesis of Thiamine in Salmonella enterica Serovar Typhimurium LT2 J. Bacteriol., September 15, 2000; 182(18): 5180 - 5187. [Abstract] [Full Text]
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,
~35 µM ThiFS + 2.5 mM ATP; 
, 2.5 mM ATP.
