From the Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York, 14853
Received for publication, January 23, 2001, and in revised form, March 7, 2001
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ABSTRACT |
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Phosphopantothenoylcysteine synthase
catalyzes the formation of
(R)-4'-phospho-N-pantothenoylcysteine from
4'-phosphopantothenate and L-cysteine: this enzyme,
involved in the biosynthesis of coenzyme A (CoA), has not previously
been identified. Recently it was shown that the
NH2-terminal domain of the Dfp protein from bacteria catalyzes the next step in CoA biosynthesis, the decarboxylation of
(R)-4'-phospho-N-pantothenoylcysteine to form
4'-phosphopantetheine (Kupke, T., Uebele, M., Schmid, D., Jung, G.,
Blaesse, M., and Steinbacher, S. (2000) J. Biol.
Chem. 275, 31838-31846). We have partially purified
phosphopantothenoylcysteine decarboxylase from Escherichia
coli and demonstrated that the protein encoded by the
dfp gene, here renamed coaBC, also has
phosphopantothenoylcysteine synthetase activity, using CTP rather than
ATP as the activating nucleoside 5'-triphosphate. This discovery
completes the identification of all the enzymes involved in the
biosynthesis of coenzyme A in bacteria.
Coenzyme A (CoA)1
fulfils a vital role in the normal metabolism of living organisms: it
has been reported that ~4% of all enzymes utilize either CoA, its
thioesters or 4'-phosphopantetheine (1). As an essential cofactor of
fatty acid synthases, polyketide synthases, and nonribosomal peptide
synthases, 4'-phosphopantetheine functions as an acyl group carrier,
activating carboxylic acids for biological Claisen reactions and the
formation of peptides and esters. Acetyl-CoA and succinyl-CoA are key
intermediates in energy metabolism, taking part in the tricarboxylic
acid cycle. It is therefore surprising that three of the CoA
biosynthetic enzymes have only recently been identified in E. coli (2, 3).
Phosphopantothenoylcysteine decarboxylase (PPC-DC) catalyzes
the decarboxylation of 4'-phosphopantothenoylcysteine
(PPanCys) to form 4'-phosphopantetheine (Fig.
1). From a mechanistic perspective this
reaction proves to be particularly interesting as it is unclear how the
stabilization of the carbanion intermediate is achieved. To study this
enzyme we have repeated, with major modifications, the published
purification of PPC-DC from wild-type E. coli
(4). Amino-terminal sequencing identified the dfp gene as
coding for the protein having this activity. The flavin-containing Dfp
protein was first identified from a temperature-sensitive mutant
(dfp-707), believed to be involved in DNA and pantothenate
metabolism (5, 6). However, the conditional lethality of the mutant was
reported not to be a direct result of pantothenate auxotrophy, and its complete characterization was not achieved. While our work was in
progress, Kupke et al. (7) also identified Dfp as having PPC-DC activity by sequence comparison with EpiD, a
lantibiotic-synthesizing protein catalyzing the decarboxylation of
peptidylcysteine intermediates.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Reactions catalyzed by the
PPC-S and PPC-DC activities of
Dfp.
We now report that the protein encoded by dfp also catalyzes
the formation of PPanCys from 4'-phosphopantothenate and
L-cysteine using cytidine 5'-triphosphate (CTP) as the
activating nucleoside 5'-triphosphate. The protein releases CMP as the
product of the coupling reaction, indicating the formation of an
activated acyl-cytidylate intermediate. In addition, we show that the
protein does not contain a covalently linked pyruvoyl group as cofactor
as was reported previously (4). The identification of
phosphopantothenoylcysteine synthase (PPC-S) completes the
identification of all the enzymes involved in the biosynthesis of CoA
in bacteria and prepares the way for its careful study and possible
exploitation in the design of antimicrobial agents.
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EXPERIMENTAL PROCEDURES |
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Materials and Methods-- All chemicals were purchased from Aldrich or Sigma and were of the highest purity. Resins and columns were purchased from Amersham Pharmacia Biotech, except hydroxyapatite (Bio-Rad). Radiolabeled chemicals (L-1,1'-[14C]cystine (100 mCi/mmol, 1 mCi/ml) and [14C]NaHCO3 (50 mCi/mmol, 1 mCi/ml)) were purchased from American Radiolabeled Chemicals (St. Louis, MO). Ecolume (ICN) was used for liquid scintillation counting, performed on a Beckman LS 1801 instrument. Oligonucleotide synthesis and automated DNA sequencing were performed at the Cornell Biotechnology Resource Center. 1H NMR was performed on a Bruker INNOVA 400 MHz instrument.
Purification of the Native Protein-- PPC-DC activity was partially purified from 38 g of frozen E. coli B cells (Fermentation Facility, University of Alabama, Birmingham, AL) using normal protein purification methodology (Table I). However, protein concentration was not accomplished by buffer removal through membranes as this led to large losses of enzymatic activity; instead, solutions were concentrated on short DEAE-Sepharose columns and the concentrated protein eluted in buffer containing 400 mM NaCl when necessary. Buffer exchanges and salt removal were performed by dialysis.
Cloning and Overexpression of the coaBC Gene-- The coaBC gene was amplified by PCR using as the forward primer: 5'-CCTACAGGAAAAATCCATATGAGCCTGGCCGG-3', introducing an NdeI restriction site (underlined), and as the reverse primer: 5'-GCCAGTTCTACGGCGTCCTCGAGACAGGCACGCAGG-3', introducing an XhoI restrictrion site (underlined). The resulting PCR product coding for Dfp from Met25 to the end of the protein was cloned into NdeI/XhoI-digested pET28a expression vector (Novagen). The sequence of the resulting plasmid, designated pCLK1210, was verified by automated DNA sequencing. The expression plasmid was transformed into the E. coli Tuner(DE3) strain (Novagen).
Purification of Recombinant Protein-- E. coli Tuner(DE3) pCLK1210 was grown in LB broth supplemented with 15 µg/ml kanamycin sulfate at 37 °C to A595 ~0.6, and induced with 200 µm IPTG. After growing overnight at 37 °C, the cells were harvested, suspended in sonication buffer (5 mM imidazole, 0.5 M NaCl, and 20 mM Tris, pH 7.9, 10 ml/1 g of cell paste), disrupted by sonication, and centrifuged at 35,000 × g for 30 min to collect the crude cell extract. This was applied to a 5-ml His-Bind column (Novagen). Weakly bound proteins were removed by washing with sonication buffer, followed by sonication buffer containing 100 mM imidazole. The protein of interest was eluted as a yellow band by increasing the imidazole concentration to 0.5 M. Elution was monitored at A280 and A450.
Synthesis of 4'-Phosphopantothenate and
4'-Phosphopantothenoylcysteine--
Benzyl pantothenate was
synthesized by alkylation of sodium pantothenate (1.00 g, 4.15 mmol
dissolved in 15 ml of dry DMF) with benzyl bromide (494 µl, 4.15 mmol) by heating at 70 °C overnight. DMF was removed in
vacuo, ethyl acetate (100 ml) was added, the organic layer was
washed with saturated aqueous NaCl (3 × 20 ml), dried
(Na2SO4), and the solvent removed. The product
was purified by flash column chromatography (silica gel; ethyl
acetate/hexane 2:1 to 4:1; 1.13 g, yield 88%).
Dibenzylchlorophosphate was prepared in situ by reacting
dibenzylphosphite (1.57 g, 6.0 mmol) and N-chlorosuccinimide in dry benzene (8 ml) for 2 h at 25 °C. The reaction mixture
was filtered and added dropwise to benzyl pantothenate (615 mg, 2.0 mmol) dissolved in dry pyridine (9 ml) at 40 °C (dry
ice/acetonitrile). After stirring at
40 °C for 2 h, the
mixture was placed in a
20 °C freezer overnight. The reaction was
warmed to 25 °C and then quenched with water (3 ml). The solvent was
removed in vacuo, and ethyl acetate (25 ml) was added. The
resulting suspension was washed with 1 M
H2SO4 (2 × 5 ml), 1 M
NaHCO3 (2 × 5 ml), and saturated
Na2SO4 (1 × 5 ml), dried
(Na2SO4), and the solvent removed. The
resulting benzyl pantothenate 4'-(dibenzyl phosphate) was purified by
flash column chromatography (silica gel; ethyl acetate/hexane 2:1), to
give a clear oil (576 mg, yield 50%). Benzyl pantothenate 4'-(dibenzyl
phosphate) (250 mg, 439 µmol) was dissolved in 90% aqueous methanol
and hydrogenated at atmospheric pressure for 2 h in the presence
of 10% palladium/carbon. After filtering and solvent
evaporation, the final product (131 mg, quantitative) was dissolved in
H2O, titrated to pH ~6.5 with 1 M NaOH,
and stored as frozen aliquots of a stock solution (50 mM)
at
20 °C. 1H NMR (400 MHz, D2O):
0.70 (s, 3H), 0.77 (s, 3H), 2.43 (t, 2H), 3.31 (t,
2H), 3.43-3.45 (m, 1H), 3.64 (d of d, 1H), 3.82 (s, 1H).
4'-Phosphopantothenoylcysteine was synthesized by adding
S-propyl thiopantothenate 4'-(dibenzyl phosphate) (50 mg, 93 µmol) (Ref. 8, with phosphorylation step modified as above) to
L-cysteine (12.4 mg, 102 µmol) dissolved in 102 µl of 1 M NaOH and 900 µl of methanol. After stirring for 4 h, the solvent was removed under a stream of argon, 500 µl of 1 M HCl was added, and the product was extracted with ethyl
acetate (3 × 500 µl). The combined organic extracts were dried
(Na2SO4) and the solvent evaporated under a
stream of argon. The resulting residue was dissolved in ~2.5 ml of
liquid NH3 and sodium pieces added until the blue color persisted. Methanol (two drops) was then added, and the ammonia was
allowed to evaporate. Ice water (1 ml) was added, followed by a
suspension of Dowex 50WX8-100 resin in deionized water, until the pH
of the solution was acidic. The suspension was loaded on a prepared
Dowex 50WX8-100 column (500 µl) in a plastic pipette, the product
was eluted with 2 ml of water, and the solvent was removed by
lyophilization. The final product (30 mg, 80%) was dissolved in
H2O, titrated to pH ~6.5 with 1 m NaOH, and stored as frozen aliquots of a stock solution (18 mM) at
20 °C. 1H NMR (400 MHz, D2O):
0.64 (s,
3H), 0.79 (s, 3H), 2.40 (d of t, 2H), 2.70 (d,
2H), 3.22 (d of d, 1H), 3.34 (t, 2H), 3.55 (d of d, 1H), 3.91 (s, 1H),
4.19 (t, 1H).
The corresponding 14C-labeled substrate, PPan-1-[14C]Cys, was similarly synthesized on the same scale as above using L-1-[14C]cysteine (~0.4 mCi/mmol), prepared from L-1,1'-[14C]cystine and L-cysteine.
Assay Procedure--
The CO2 release assay was
performed as described previously (4), with modifications. Assays were
performed in unmodified microcentrifuge tubes, and all additions
were made directly to the sample followed by immediate closure of the
tube. Assay mixtures contained 10 mM DTT, 2 mM
MgCl2, 50 mM Tris, pH 7.6, enzyme (~2 µg)
and either 3.0 mM PPan-1-[14C]Cys
or 3.5 mM L-1-[14C]cysteine in a
total volume of 60 µl; if the synthetase activity was being assayed,
mixtures also contained 2.5 mM 4'-phosphopantothenate and
3.5 mM CTP. The reaction was initiated by addition of the 14C-labeled substrate (10 µl of stock solution,
i.e. 21 mM cysteine or 18 mM
PPanCys) to the rest of the assay components in 50 µl and
stopped after 10 min at room temperature by addition of 100 µl of 5 M H2SO4. The amount of
CO2 released was determined as reported previously
(4). Efficiency of 14CO2 trapping was
determined over the same concentration range as for the assay, using
[14C]NaHCO3 as standard. Assay results were
adjusted using the resulting standard curve (y = 0.70x 0.08, R2 = 0.99).
High Resolution Mass Spectrometry-- Protein samples were desalted by loading onto reverse-phase protein traps (Michrom Bioresources, Auburn, CA), washed with 2 ml of 2:96:2 methanol/H2O/acetic acid, and step eluted with 70:26:4 methanol/H2O/acetic acid. Mass spectra were acquired on a modified 6-T Finnigan FTMS with the Odyssey data system described previously (9), with nanospray sample injection (10). Specific ions were isolated using stored waveform inverse Fourier transform (SWIFT) (11), and assignment of the fragment masses and compositions was made using the computer program THRASH (12). Spectra were calibrated externally using bovine ubiquitin (Mr = 8,564.64).
Identification of the CTP Product--
HPLC analysis was
performed on reaction mixtures containing 1.5 mM
4'-phosphopantothenate, 3.5 mM CTP, 3.5 mM
L-cysteine, 25 mM Tris, 2.5 mM
MgCl2, pH 7.6, and 20 µl of purified enzyme (0.95 mg/ml
in 25 mM Tris, 1 mM EDTA, 2 mM DTT,
pH 7.6) in a total volume of 500 µl. After standing at 25 °C for
4 h, protein was removed by centrifugation through a membrane
(Vivaspin 500, Vivascience), and the reaction mixture was analyzed with
a Supelcosil LC-18-T 3 µm, 15 cm × 4.6 mm inner diameter
column (Supelco) on an HP series 1100 HPLC system eluting with 100 mM potassium phosphate buffer, pH 6.8, containing 4 mM tetrabutylammonium hydrogensulfate. Elution was
monitored at 254 nm, and identification was performed by comparison
to CMP, CDP, and CTP as standards.
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RESULTS AND DISCUSSION |
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Purification of the Native Protein and Identification of the Synthetase Activity-- Our procedure for the purification of PPC-DC differed significantly from that previously published; it is summarized in Table I. Throughout the purification the protein exhibited a number of characteristics that hampered its isolation. These included copurification with proteins of high molecular weight and coagulation on concentrating membranes leading to large losses of activity. After solving these problems PPC-DC could be partially purified 1,280-fold as one of a small set of proteins (~15% purity, the major contaminant (~80%) being alcohol dehydrogenase (adhE gene product) as determined by NH2-terminal sequencing), the final fraction having a specific activity of 370 nmol CO2/min/mg.
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During the purification trials we observed that substrate analogs like 4'-phosphopantothenoylalanine and 4'-phosphopantothenoylserine did not inhibit PPC-DC activity in partially purified fractions. Furthermore, the high concentrations of PPanCys needed to saturate the enzyme, combined with the reported failure to detect PPanCys in cell extracts (13), suggested that PPanCys was not the physiological substrate for the protein catalyzing its decarboxylation. The most likely alternative was to consider close interaction between the PPC-S and PPC-DC activities, and subsequently we tested 4'-phosphopantothenate, CTP, and L-1-[14C]cysteine as substrates. We found that 14CO2 was released from the reaction mixture, while cysteine was not decarboxylated in the absence of the other substrates. Our choice of CTP as activating nucleoside triphosphate rather than the more usual ATP was based on previous studies (13) that had demonstrated that crude extracts from E. coli preferentially used CTP for 4'-phosphopantothenoylcysteine synthetase activity. However, it was not possible to determine whether these PPC-DC and PPC-S activities found in our partially purified fractions were located on the same protein, or on two distinct proteins which were copurifying, until the enzyme was purified to homogeneity.
Identification, Cloning, Overexpression, and Purification of Recombinant PPC-DC-- The proteins from the purified fraction with the highest specific activity were separated by SDS-PAGE and subsequently electroblotted to a polyvinylidene difluoride membrane. Determination of the NH2-terminal sequence of the band at 45.0 kDa gave SLAGKKILVLGV, revealed by a BLAST (14) search to correspond to residues 26-36 of the protein coded for by the dfp gene, annotated as a flavoprotein affecting DNA and pantothenate metabolism in an unspecified way.
Amplification of the dfp gene (using ATG25
as the start codon) from the E. coli genome by PCR and
subsequent insertion of the product into pET28a gave
pCLK1210 as the resulting plasmid. Transformation of this
plasmid into the Tuner(DE3) cell strain, a lac permease deletion mutant allowing uniform entry of IPTG into cells and providing
strict concentration-dependent control of induction, allowed the
production of soluble overexpressed Dfp following induction at 37 °C
with 200 µM IPTG (Fig. 2,
lane 1). Expression in the BL21(DE3) host strain failed to
produce soluble protein under a variety of conditions. The presence of
an NH2-terminal 6 × His-tag made the purification of
the recombinant protein possible by imidazole elution from a
Ni2+-chelating affinity resin (Fig. 2, lane 2).
Both the purification procedure and subsequent dialysis had to be
performed at an ambient temperature as the protein precipitated at
4 °C. Solutions of the protein could be concentrated by
centrifugation through a membrane if the procedure was carried out at
15 °C.
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PPC-DC Is Not a Pyruvoyl-dependent Enzyme-- The report of the first purification of PPC-DC from E. coli (4) claimed that the enzyme contained a base labile pyruvoyl group. We set out to determine whether we could identify such a post-translational modification by mass spectrometric analysis of the pure recombinant protein.
The analysis of Dfp by high resolution ESI/FTMS showed that the protein
had a molecular mass of 45,470.6 Da (Fig.
3). This value corresponds exactly to the
calculated mass of the recombinant protein with the
NH2-terminal methionine removed (45,470.4 Da) and excludes
the possibility that PPC-DC contains a pyruvoyl group or any
other covalent post-translational modification.
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Identification of the Flavin Cofactor--
The purified
protein exhibited a UV-visible spectrum characteristic of
flavoenzymes, with a max at 455 and 378 nm. The identity of the flavin was previously found to be flavin
mononucleotide (FMN) by TLC analysis of the denatured protein and
comparison to standards (5). We confirmed this finding by isolating the flavin cofactor from heat-denatured enzyme and subjecting it to ESI/MS
analysis. The isolated product had an m/z value of 457.2 (M + H)+ corresponding to FMN
(Mr 456.3 for the free acid). Further
MS/MS analyses of the molecular ion confirmed this identification. No peaks corresponding to FAD molecular ions could be observed.
4'-Phosphopantothenoylcysteine Synthetase Activity of the Dfp Protein-- Since our initial studies on partially purified mixtures of PPC-DC suggested that it was copurifying with PPC-S activity, we repeated our experiments with the purified Dfp protein. This enzyme catalyzes the release of 14CO2 from L-1-[14C]cysteine in the presence of 4'-phosphopantothenate, CTP, and Mg2+, but not when any one of these cosubstrates was absent. Analysis of the reaction mixture by ESI/MS demonstrated the formation of 4'-phosphopantetheine.
None of the other nucleoside 5'-triphosphates, i.e. ATP, GTP, and UTP, could substitute for CTP in the coupling reaction even at concentrations as high as 10 mM. The function of cytidylyltransferases in the formation of critical intermediates in the biosynthesis of lipids and complex carbohydrates has been studied extensively; these enzymes use CTP to form activated CDP-alcohols or CMP-acid sugars (Ref. 15 and references cited therein). However, the utilization of CTP by enzymes to activate acyl groups in a transient fashion has, to our knowledge, not been reported. Furthermore, it has been reported that members of the family of cytidylyltransferases can be identified by the presence of certain conserved binding motifs, the HXGH and RTXGISTT motifs being the best examples (15). However, Dfp from E. coli does not contain such a motif, or any other motif that has been associated with NTP binding.
HPLC analysis of the products of the coupling reaction showed that CTP was converted exclusively to CMP. This indicates the formation of an activated acyl-cytidylate, which is subsequently attacked by cysteine (Fig. 1). This mode of action contrasts with that of the partially purified rat liver PPC-S (16), which was shown to produce ADP and phosphate as products of the coupling reaction, suggesting the formation of an acyl-phosphate intermediate for that system.
Steady State Kinetic Analysis-- Time course assays based on the release of 14CO2 by the Dfp protein demonstrated a linear increase of CO2 production for the first 10 min of reaction. We have therefore assumed that the average rate of a reaction quenched within this period of time is a close approximation of the initial rates of reaction.
The accurate determination of the kinetic parameters was hampered by a number of factors. It was found that increasing the enzyme concentration above ~30 µg/ml led to a rapid decrease in activity. Protein from which the His-tag had been removed by thrombin cleavage exhibited similar behavior (data not shown). Although leading to practical problems, the physiological significance of this observation is unclear, because the decrease in activity is only observed at levels of enzyme concentration above those expected to occur under normal cellular conditions. However, it does confirm the tendency of the enzyme to aggregate, a process hastened at lower temperatures. The enzyme also looses activity over time; this deterioration may be slowed by the presence of high (~200 µM) concentrations of NaCl in the enzyme sample, but its presence negatively influences the determination of the kinetic parameters.
Despite these problems, preliminary values for Km and kcat were determined at ~30 µg/ml enzyme, while maintaining nonvariable substrates at concentrations of at least 5 × Km (Table II). 4'-Phosphopantothenate shows substrate inhibition at high concentrations. The 2.7-fold higher value of Km(pPanCys), compared with Km(4'-phosphopantothenate), suggests that this intermediate is not the physiological substrate.
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Conclusion--
The Dfp protein from E. coli
bears little resemblance to PPC-S and PPC-DC from
mammalian sources: PPC-DC has been partially purified from
both horse (17) and rat liver (18), and in the case of the former, it
was shown that the enzyme is pyruvoyl-dependent (19).
PPC-S from rat liver uses ATP as the activating nucleotide, instead of CTP, and forms an acyl-phosphate intermediate as suggested by the formation of ADP and phosphate (16). These differences between
the key CoA biosynthetic enzymes in bacteria and higher organisms
raise the possibility of exploiting the selective inhibition of the
bacterial enzyme in the development of new antibiotics. Our
identification of the Dfp protein from E. coli as having
both PPC-S and PPC-DC activities leads us to
suggest the renaming of the dfp gene as coaBC, in
accordance with the nomenclature established for CoA biosynthetic
enzymes and to accommodate the existence of separate orthologs, notably
in Klebsiella pneumoniae and Streptococci, encoding for CoaB and CoaC. This finding completes the identification of all the enzymes involved in the biosynthesis of coenzyme A in bacteria.
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ACKNOWLEDGEMENT |
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We thank Ryan Mehl for assistance with HPLC analysis and for helpful discussions.
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FOOTNOTES |
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* This work was supported by grants from the Petroleum Research Foundation and from SmithKline Beecham.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: Dept. of Chemistry and
Chemical Biology, 120 Baker Laboratory, Cornell University, Ithaca, NY
14853-1401. Tel.: 607-255-7133; Fax: 607-255-4137; E-mail:
tpb2@cornell.edu.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.C100033200
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ABBREVIATIONS |
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The abbreviations used are:
CoA, coenzyme
A;
PPC-DC, phosphopantothenoylcysteine decarboxylase;
PPC-S, phosphopantothenoylcysteine synthetase,
PPanCys,
(R)-4'-phospho-N-pantothenoylcysteine;
PCR, polymerase chain reaction;
IPTG, isopropyl-1-thio--D-galactopyranoside;
DTT, dithiothreitol;
DMF, dimethyl formamide;
ESI/FTMS, electrospray
ionization Fourier transform mass spectrometry;
SWIFT, stored waveform
inverse Fourier transform;
HPLC, high performance liquid
chromatography;
FMN, flavin mononucleotide;
PAGE, polyacrylamide
gel electrophoresis.
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REFERENCES |
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