Phosphopantothenoylcysteine Synthetase from Escherichia coli

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 thedfp 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. 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 thedfp 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)1fulfils 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 (1Lee C.H. Chen A.F. Everse J. Anderson B. You K. The Pyridine Nucleotide Coenzymes. Academic Press, Inc., New York1982: 189Crossref Google Scholar). 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 (2Begley T.P. Kinsland C. Strauss E. Litwack G. Begley T.P. Vitamins and Hormones. Academic Press, Inc., New York2001: 157-171Google Scholar, 3Geerlof A. Lewendon A. Shaw W.V. J. Biol. Chem. 1999; 274: 27105-27111Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). 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(4Yang H. Abeles R.H. Biochemistry. 1987; 26: 4076-4081Crossref PubMed Scopus (22) Google Scholar). 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 (5Spitzer E.D. Weiss B. J. Bacteriol. 1985; 164: 994-1003Crossref PubMed Google Scholar, 6Spitzer E.D. Jimenez-Billini H.E. Weiss B. J. Bacteriol. 1988; 170: 872-876Crossref PubMed Google Scholar). 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. (7Kupke T. Uebele M. Schmid D. Jung G. Blaesse M. Steinbacher S. J. Biol. Chem. 2000; 275: 31838-31846Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) also identified Dfp as havingPPC-DC activity by sequence comparison with EpiD, a lantibiotic-synthesizing protein catalyzing the decarboxylation of peptidylcysteine intermediates. We now report that the protein encoded by dfp also catalyzes the formation of PPanCys from 4′-phosphopantothenate andl-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 (4Yang H. Abeles R.H. Biochemistry. 1987; 26: 4076-4081Crossref PubMed Scopus (22) Google Scholar). 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. 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′-14Ccystine (100 mCi/mmol, 1 mCi/ml) and 14CNaHCO3 (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. 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 (TableI). 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.Table IPurification of PPC-DC activity from E. coli B cellsPurification stepProteinActivitySpecific activityYieldPurificationmgnmol/minnmol/min/mg%-foldCrude extract1-aThe data were obtained from 38 g of frozen cell paste. Special precautions had to be followed during the purification, see “Experimental Procedures” for details.29278450.289100130–50% (NH4)2SO4fraction11527670.665912.3HiPrep 16/60 Sephacryl S-2004294801.12573.9DEAE-Sepharose Fast Flow15.724015.32853Hydroxyapatite (CHT Type I)3.3210331.012107HiTrap Blue0.4536.780.94.3280Superdex 200 prep grade0.0269.53711.112801-a The data were obtained from 38 g of frozen cell paste. Special precautions had to be followed during the purification, see “Experimental Procedures” for details. Open table in a new tab ThecoaBC gene was amplified by PCR using as the forward primer: 5′-CCTACAGGAAAAATCCATATGAGCCTGGCCGG-3′, introducing anNdeI 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). E. coliTuner(DE3) pCLK1210 was grown in LB broth supplemented with 15 μg/ml kanamycin sulfate at 37 °C toA595 ∼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, 0.5m 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 atA280 and A450. 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 mH2SO4 (2 × 5 ml), 1 mNaHCO3 (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): δ 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 addingS-propyl thiopantothenate 4′-(dibenzyl phosphate) (50 mg, 93 μmol) (Ref. 8Martin D.P. Bibart R.T. Drueckhammer D.G. J. Am. Chem. Soc. 1994; 116: 4660-4668Crossref Scopus (42) Google Scholar, with phosphorylation step modified as above) tol-cysteine (12.4 mg, 102 μmol) dissolved in 102 μl of 1m NaOH and 900 μl of methanol. After stirring for 4 h, the solvent was removed under a stream of argon, 500 μl of 1m 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): δ 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-1-14CCys, was similarly synthesized on the same scale as above usingl-1-14Ccysteine (∼0.4 mCi/mmol), prepared from l-1,1′-14Ccystine andl-cysteine. The CO2 release assay was performed as described previously (4Yang H. Abeles R.H. Biochemistry. 1987; 26: 4076-4081Crossref PubMed Scopus (22) Google Scholar), 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 mmMgCl2, 50 mm Tris, pH 7.6, enzyme (∼2 μg) and either 3.0 mmPPan-1-14CCys or 3.5 mml-1-14Ccysteine 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 the14C-labeled substrate (10 μl of stock solution,i.e. 21 mm cysteine or 18 mmPPanCys) to the rest of the assay components in 50 μl and stopped after 10 min at room temperature by addition of 100 μl of 5m H2SO4. The amount of CO2 released was determined as reported previously (4Yang H. Abeles R.H. Biochemistry. 1987; 26: 4076-4081Crossref PubMed Scopus (22) Google Scholar). Efficiency of 14CO2 trapping was determined over the same concentration range as for the assay, using 14CNaHCO3 as standard. Assay results were adjusted using the resulting standard curve (y = 0.70x −0.08, R2 = 0.99). 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 (9Beu S.C. Senko M.W. Quinn J.P. Wampler F.M. McLafferty F.W. J. Am. Soc. Mass Spectrom. 1993; 4: 557-565Crossref PubMed Scopus (182) Google Scholar), with nanospray sample injection (10Wilm M. Mann M. Anal. Chem. 1996; 68: 1-8Crossref PubMed Scopus (1719) Google Scholar). Specific ions were isolated using stored waveform inverse Fourier transform (SWIFT) (11Loo J.A. Smith R.D. Rapid Commun. Mass Spectrom. 1988; 2: 207-221Crossref Scopus (253) Google Scholar), and assignment of the fragment masses and compositions was made using the computer program THRASH (12Little D.P. Speir J.P. Senko M.W. O'Connor P.B. McLafferty F.W. Anal. Chem. 1994; 66: 2809-2815Crossref PubMed Scopus (673) Google Scholar). Spectra were calibrated externally using bovine ubiquitin (Mr = 8,564.64). HPLC analysis was performed on reaction mixtures containing 1.5 mm4′-phosphopantothenate, 3.5 mm CTP, 3.5 mml-cysteine, 25 mm Tris, 2.5 mmMgCl2, 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. Our procedure for the purification ofPPC-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 of a of proteins the major being gene as determined by NH2-terminal the final having a activity of the purification we that substrate and not PPC-DC activity in partially purified the high of PPanCys to the enzyme, combined with the reported to in cell extracts J. Biol. Chem. Full Text PDF PubMed Google Scholar), was not the substrate for the protein catalyzing its The was to the and we CTP, as We that 14CO2 was released from the reaction cysteine was not in the of the Our of CTP as activating nucleoside rather than the ATP was on J. Biol. Chem. Full Text PDF PubMed Google Scholar) that had demonstrated that crude extracts from E. coli used CTP for 4′-phosphopantothenoylcysteine synthetase activity. However, it was not possible to these PPC-DC and in our partially purified were on the same or on proteins were until the enzyme was purified to The proteins from the purified with the highest activity were by and to a of the NH2-terminal sequence of the band at by a J. Biol. PubMed Scopus Google Scholar) to to of the protein for by thedfp gene, as a DNA and pantothenate metabolism in an of the dfp gene the from the E. coli by PCR and of the product into pET28a as the resulting of this plasmid into the Tuner(DE3) cell a mutant of into cells and of allowed the of Dfp at 37 °C with 200 μm in the strain to protein under a of The presence of an NH2-terminal × made the purification of the protein possible by imidazole from a resin the purification procedure and had to be performed at an temperature as the protein at 4 °C. of the protein could be concentrated by centrifugation through a membrane if the procedure was at 15 °C. The report of the first purification of PPC-DC from E. coli (4Yang H. Abeles R.H. Biochemistry. 1987; 26: 4076-4081Crossref PubMed Scopus (22) Google Scholar) that the enzyme contained a pyruvoyl We to we could a by analysis of the The analysis of Dfp by high that the protein had a molecular of This to the of the protein with the NH2-terminal removed and the that PPC-DC a pyruvoyl group or The purified protein exhibited a of with a at and The of the was previously to be by analysis of the protein and comparison to (5Spitzer E.D. Weiss B. J. Bacteriol. 1985; 164: 994-1003Crossref PubMed Google Scholar). We this by the cofactor from enzyme and it to The isolated product had an of corresponding to (Mr for the of the molecular this corresponding to molecular ions could be our on partially purified mixtures of PPC-DC that it was activity, we our with the purified Dfp This enzyme catalyzes the release in the presence of CTP, and not when of these was of the reaction mixture by demonstrated the formation of of the nucleoside and could for CTP in the coupling reaction at as high as 10 The of in the formation of intermediates in the biosynthesis of and has been these enzymes CTP to form activated or (Ref. C.H. S. C. 1999; Full Text Full Text PDF PubMed Scopus Google and However, the of CTP by enzymes to acyl in a to our not been it has been reported that of the of be identified by the presence of the and being the C.H. S. C. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). However, Dfp from E. coli does not contain a or that has been with HPLC analysis of the of the coupling reaction that CTP was to This the formation of an activated is by cysteine This of with that of the partially purified Scopus Google Scholar), was shown to and phosphate as of the coupling reaction, the formation of an intermediate for that on the release of 14CO2 by the Dfp protein demonstrated a of CO2 for the first 10 min of We have therefore that the of a reaction quenched this of is a of the of The of the was hampered by a number of It was that increasing the enzyme concentration above μg/ml led to a in activity. Protein from the had been removed by exhibited not leading to the of this is the in activity is only at of enzyme concentration above to under normal However, it does the of the enzyme to a at The enzyme also activity over this be by the presence of high of NaCl in the enzyme its presence the of the these for were determined at μg/ml enzyme, at of at 5 × substrate at high The of with that this intermediate is not the and of the and PPC-DC of are for the and and decarboxylation are for the and and decarboxylation Open table in a new tab The Dfp protein from E. to and PPC-DC from PPC-DC has been partially purified from E. B. M. J. PubMed Scopus (22) Google Scholar) and Scopus Google Scholar), and in the of the it was shown that the enzyme is 1987; PubMed Scopus Google from ATP as the activating of CTP, and an intermediate as by the formation of and phosphate Scopus Google Scholar). These the key CoA biosynthetic enzymes in bacteria and the of the of the enzyme in the of new Our identification of the Dfp protein from E. coli as having and PPC-DC to the of the dfp gene as coaBC, in with the for CoA biosynthetic enzymes and to the of in and for and This completes the identification of all the enzymes involved in the biosynthesis of coenzyme A in bacteria.