The Antibiotic Activity of N-Pentylpantothenamide Results from Its Conversion to Ethyldethia-Coenzyme A, a Coenzyme A Antimetabolite (2002)

Pantothenic acid (vitamin B5) is the natural precursor of coenzyme A (CoA), an essential cofactor in all organisms. The pantothenic acid antimetaboliteN-pentylpantothenamide inhibits the growth ofEscherichia coli with a minimum inhibitory concentration of 2 μm. In this study, we examine the mechanism of this inhibition. Using the last five enzymes of the CoA biosynthetic pathway in E. coli we demonstrate thatN-pentylpantothenamide does not inhibit the CoA biosynthetic enzymes but instead acts as an alternative substrate, forming the CoA analog ethyldethia-CoA. We show thatN-pentylpantothenamide is converted to ethyldethia-CoA 10.5 times faster than CoA is biosynthesized from pantothenic acid, demonstrating that ethyldethia-CoA biosynthesis can effectively compete with CoA biosynthesis in the cell. We conclude that the mechanism of toxicity of N-pentylpantothenamide is most likely due to its biosynthetic conversion to the CoA analog ethyldethia-CoA, which may act as an inhibitor of CoA- and acetyl-CoA-utilizing enzymes. Pantothenic acid (vitamin B5) is the natural precursor of coenzyme A (CoA), an essential cofactor in all organisms. The pantothenic acid antimetaboliteN-pentylpantothenamide inhibits the growth ofEscherichia coli with a minimum inhibitory concentration of 2 μm. In this study, we examine the mechanism of this inhibition. Using the last five enzymes of the CoA biosynthetic pathway in E. coli we demonstrate thatN-pentylpantothenamide does not inhibit the CoA biosynthetic enzymes but instead acts as an alternative substrate, forming the CoA analog ethyldethia-CoA. We show thatN-pentylpantothenamide is converted to ethyldethia-CoA 10.5 times faster than CoA is biosynthesized from pantothenic acid, demonstrating that ethyldethia-CoA biosynthesis can effectively compete with CoA biosynthesis in the cell. We conclude that the mechanism of toxicity of N-pentylpantothenamide is most likely due to its biosynthetic conversion to the CoA analog ethyldethia-CoA, which may act as an inhibitor of CoA- and acetyl-CoA-utilizing enzymes. Coenzyme A (CoA) 1The abbreviations used are: CoA, coenzyme A; MIC, minimum inhibitory concentration; HPLC, high performance liquid chromatography; ESI-MS, electrospray ionization mass spectroscopy; CoaA, pantothenate kinase; CoaBC, phosphopantothenoylcysteine synthetase/decarboxylase; CoaD, phosphopantetheine adenylyltransferase; CoaE, dephospho-CoA kinase; DTT, dithiothreitol. 1a is an essential cofactor in numerous metabolic pathways involving Claisen/aldol condensations and acyl substitutions, while the 4′-phosphopantetheine moiety of CoA is used as a covalently bound cofactor in fatty acid, polyketide, and non-ribosomal peptide biosynthesis. Because of this metabolic centrality, the development of new antibiotics designed as inhibitors of CoA-utilizing enzymes (i.e. as CoA antivitamins or antimetabolites) may be feasible. However, while several CoA analogs have been characterized as inhibitors of CoA-utilizing enzymes in vitro (1Mishra P.K. Drueckhammer D.G. Chem. Rev. 2000; 100: 3283-3309Google Scholar), these analogs are not useful as antibiotics because bacteria are unable to transport CoA across the cell membrane (2Jackowski F.C.S. Neidhardt F.C. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 1. American Society of Microbiology, Washington, D. C.1996: 687-694Google Scholar, 3Jackowski S. Rock C.O. J. Bacteriol. 1984; 158: 115-120Google Scholar). Therefore the design of CoA antimetabolites must be based on pantothenic acid 2a, the most advanced cell-permeable biosynthetic intermediate (4Vallari D.S. Rock C.O. J. Bacteriol. 1985; 164: 136-142Google Scholar, 5Jackowski S. Alix J.H. J. Bacteriol. 1990; 172: 3842-3848Google Scholar), or its precursors (see Fig. 1). Two pantothenic acid analogs have been described previously: pantoyltaurine, in which the carboxylate of pantothenic acid 2a is replaced by a sulfonate, does not inhibit the growth of Escherichia coli because it is not transported by pantothenate permease (6Vallari D.S. Rock C.O. J. Bacteriol. 1985; 162: 1156-1161Google Scholar); andN-pentylpantothenamide 2b, which inhibitsE. coli with a minimum inhibitory concentration (MIC) of ∼2 μm (7Clifton G. Bryant S.R. Skinner C.G. Arch. Biochem. Biophys. 1970; 137: 523-528Google Scholar). In this study we considered two mechanisms for the antibiotic activity of N-pentylpantothenamide 2b. In the first mechanism, inhibition of CoA biosynthesis byN-pentylpantothenamide 2b or one of its metabolites would result in a decreased amount of CoA being formed in the cell and thus would retard bacterial cell growth. In the second mechanism, we considered the possibility that 2b is biosynthetically converted to the CoA analog ethyldethia-CoA, which might function as a CoA antimetabolite and inhibit CoA and acetyl-CoA-utilizing enzymes. Since the five enzymes required for the conversion of pantothenic acid to CoA (Fig. 1) have all recently been overexpressed and characterized (8Geerlof A. Lewendon A. Shaw W.V. J. Biol. Chem. 1999; 274: 27105-27111Google Scholar, 9Kupke T. Uebele M. Schmid D. Jung G. Blaesse M. Steinbacher S. J. Biol. Chem. 2000; 275: 31838-31846Google Scholar, 10Strauss E. Kinsland C. Ge Y. McLafferty F.W. Begley T.P. J. Biol. Chem. 2001; 276: 13513-13516Google Scholar, 11Mishra P.K. Park P.K. Drueckhammer D.G. J. Bacteriol. 2001; 183: 2774-2778Google Scholar), we were able to differentiate between these two mechanisms in this paper. All chemicals and buffer components were purchased from Aldrich, Sigma or Fisher Scientific and used without further purification. 4′-Phosphopantetheine 5 was synthesized as previously described (12Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1961; 83: 663-675Google Scholar). 1H NMR spectra were measured on a Varian INOVA 400 MHz instrument. All HPLC analyses were performed on a HP series 1100 HPLC system with HPLC grade solvents using a Supelcosil LC-18-T 3 μm, 15 cm × 4.6 mm ID column (Supelco). ESI-MS analyses were performed at the Cornell Biotechnology Resource Center on a Bruker Esquire-LC ESI-ion trap mass spectrometer by direct infusion of the analyte mixture into the instrument at a rate of 1 μl/min. Sodium pantothenate (2.0 g, 8.3 mmol) was dissolved in deionized water, and the solution was passed through an Amberlite IR-120 (H+) column. The column was subsequently washed with two column volumes of deionized water, and the combined eluates were lyophilized. The free acid obtained in this manner was dissolved in dry dimethyl formamide (10 ml), and amylamine (1.16 ml, 10 mmol) and diphenylphosphoryl azide (2.24 ml, 10 mmol) were added. The solution was cooled to 0 °C and triethylamine (1.39 ml, 10 mmol) was added. The solution was stirred at 0 °C for 2 h, followed by stirring at room temperature overnight. The volume of solvent was reduced by rotary evaporation with external heating, and the residue applied to a short silica gel column, eluting with 95:5 CH2Cl2/methanol and concentrating the eluatein vacuo to remove final traces of dimethyl formamide. The product was purified by flash column chromatography on silica gel with 95:5 CH2Cl2/methanol, and the fractions containing 2b were collected and were concentrated in vacuo yielding the product as a colorless oil. The oil was dissolved in deionized water and lyophilized to give 2b as a white powder (1.56 g, 65% yield). 1H NMR (400 MHz, CDCl3): δ 7.45 (b, 1H), 6.14 (b, 1H), 3.98 (s, 1H), 3.56 (m, 2H), 3.47 (s, 2H), 3.21 (dt, 2H), 2.45 (t, 2H), 1.48 (m, 2H), 1.28 (m, 4H), 0.99 (s, 3H), 0.91 (s, 3H), 0.87 (t, 3H). Dibenzylchlorophosphate was preparedin situ by reacting dibenzyl phosphite (1.97 g, 6.0 mmol) and N-chlorosuccinimide (0.80 g, 6.0 mmol) in dry benzene (8 ml) for 2 h at room temperature. The reaction mixture was filtered and added drop-wise to a solution of 2b (577 mg, 2.0 mmol) 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 allowed to warm to room temperature and was subsequently 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) and 1 mNaHCO3 (2 × 5 ml), saturated Na2SO4 (1 × 5 ml), and dried (Na2SO4), and then the solvent was removed. The product was purified by flash column chromatography on silica gel with 95:5 CH2Cl2/methanol, and the fractions containing 7 were collected and concentrated in vacuo to give the product as a colorless oil (271 mg, 25% yield).1H NMR (400 MHz, CDCl3): δ7.33 (m, 10H), 7.25 (b, 1H), 6.15 (b, 1H), 5.03 (m, 4H), 3.98 (dd, 1H), 3.86 (s, 1H), 3.53 (m, 3H), 3.19 (dt, 2H), 2.41 (m, 2H), 1.46 (m, 2H), 1.27 (m, 4H), 1.04 (s, 3H), 0.86 (t, 3H), 0.78 (s, 3H). Pd/C (10%, 20 mg) was added to a solution of7 (200 mg, 365 μmol) in 9:1 methanol/water (10 ml), and the suspension was hydrogenated at atmospheric pressure and room temperature for 2 h. The solution was filtered, and the solvent removed in vacuo to give 3b as a clear glass (110 mg, 82% yield). The product was dissolved in H2O, titrated to pH ∼6.0 with 1 m NaOH and stored as frozen aliquots of a stock solution (60 mm) at −20 °C.1H NMR (400 MHz, D2O): δ3.83 (s, 1H), 3.63 (dd, 1H), 3.39 (dd, 1H), 3.30 (m, 2H), 2.95 (t, 2H), 2.76 (t, 2H), 1.28 (m, 2H), 1.08 (m, 4H), 0.77 (s, 3H), 0.69 (s, 3H), 0.67 (t, 3H). Minimal medium (0.8 mm MgSO4, 10.0 mm citric acid, 60 mmK2HPO4, 20 mmNaNH4HPO4, 0.5% glucose) (50 ml) was inoculated with 50 μl of an overnight culture of E. coli B and subsequently grown at 37 °C in the presence of varying concentrations of 2b. The A600was periodically determined until the stationary growth phase was reached in each respective culture. The minimal concentration of2b that retarded the onset of logarithmic growth under these conditions was regarded as the minimal inhibitory concentration (MIC). Pure pantothenate kinase fromE. coli (CoaA) was a gift from Suzanne Jackowski. Phosphopantothenoylcysteine synthetase/decarboxylase from E. coli (CoaBC) was purified as previously described (10Strauss E. Kinsland C. Ge Y. McLafferty F.W. Begley T.P. J. Biol. Chem. 2001; 276: 13513-13516Google Scholar). Phosphopantetheine adenylyltransferase from E. coli (CoaD) has previously been overexpressed from plasmidpUC/coaD (a gift from D. Drueckhammer) and purified (8Geerlof A. Lewendon A. Shaw W.V. J. Biol. Chem. 1999; 274: 27105-27111Google Scholar). However, as this plasmid did not encode a His-tagged protein,pUC/coaD was treated with NdeI andBamHI, and the resulting coaD-containing fragment was cloned into NdeI/BamHI-digested pET28a expression vector (Novagen). The sequence of the resulting plasmid, designated pESC106, was verified by automated DNA sequencing (Cornell Biotechnology Resource Center) and was transformed intoE. coli BL21(DE3) (Novagen). To obtain pure protein E. coli BL21(DE3) pESC106 was grown at 37 °C in 500 ml of LB broth supplemented with 15 μg/ml kanamycin sulfate toA600 ∼0.6 and induced with 1 mmisopropyl-1-thio-β-d-galactopyranoside. After growing overnight at 37 °C, the cells were harvested, suspended in sonication buffer (5 mm imidazole, 0.5 mNaCl, and 20 mm Tris-HCl, pH 7.9, 10 ml/g cell paste), disrupted by sonication, and centrifuged at 35,000 × gfor 30 min to clarify the cell-free extract. This was applied to a 2-ml His-Bind column (Novagen). Weakly bound proteins were removed by washing with sonication buffer, followed by sonication buffer containing 60 mm imidazole. CoaD was eluted by increasing the imidazole concentration to 0.5 m. The chromatography was monitored at A280. Dephospho-CoA kinase from E. coli (CoaE) has previously been overexpressed as a non-fusion protein usingpET/coaE (a gift from D. Drueckhammer) (11Mishra P.K. Park P.K. Drueckhammer D.G. J. Bacteriol. 2001; 183: 2774-2778Google Scholar). To obtain the His-tagged protein, the coaE (previously yacE) gene was amplified by PCR using as the forward primer: 5′-CCGGGAATATAACATATGAGGTATATAGTTGCC-3′, to introduce anNdeI site (underlined) at the start of the gene, and as the reverse primer: 5′-AAAGGACCTGGATCCGCATTACGG-3′ to introduce a BamHI site (underlined) at the end of the gene. The resulting PCR product was cloned intoNdeI/BamHI-digested pET28a expression vector (Novagen). The sequence of the resulting plasmid, designatedpESC124, was verified by automated DNA sequencing (Cornell Biotechnology Resource Center) and was transformed into E. coli BL21(DE3) (Novagen). To obtain pure protein, E. coli BL21(DE3) pESC124 was grown at 37 °C in 500 ml of LB broth supplemented with 15 μg/ml kanamycin sulfate toA600 ∼0.6, and induced with 100 μm isopropyl-1-thio-β-d-galactopyranoside. After growing overnight at 37 °C, the cells were harvested, suspended in sonication buffer (5 mm imidazole, 0.5m NaCl, and 20 mm Tris-HCl, pH 7.9, 10 ml/g cell paste), disrupted by sonication, and centrifuged at 35,000 ×g for 30 min to clarify the cell-free extract. This was applied to a 2.5-ml His-Bind column (Novagen). Weakly bound proteins were removed by washing with sonication buffer, followed by sonication buffer containing 60 mm imidazole. CoaE was eluted by increasing the imidazole concentration to 0.5 m. The chromatography was monitored at A280. HPLC analysis of reaction mixtures containing CoA analogs was performed using 100 mm potassium phosphate buffer, pH 6.6, containing increasing amounts of methanol as follows: 0–3 min, isocratic, 5% MeOH; 3–5 min, 5–10% MeOH; 5–10 min, 10–15% MeOH; 10–13 min, 15–30% MeOH; 13–26 min, isocratic, 30% MeOH. Chromatography was monitored at 254 nm. Retention times: CTP, CDP, CMP, 1.6–1.9 min; ATP, ADP, AMP, 2.3–3.0 min; coenzyme A1a, 12.0 min; 3′-dephospho-coenzyme A 6a, 16.6 min; ethyldethia-coenzyme A 1b, 19.7 min; and 3′-dephospho-ethyldethia-coenzyme A 6b, 26.6 min. Each 150-μl reaction mixture contained ATP (5.0 mm), CTP (1.0 mm), l-cysteine (1.0 mm), DTT (2.0 mm), MgCl2 (5.0 mm), CoaA (15 μg), CoaBC (15 μg), CoaD (15 μg), and CoaE (15 μg) in 50 mm Tris-HCl buffer (pH 7.6). Reactions were initiated by addition of the natural substrate 2a (0.5 mm), the analog 2b (0.5 mm), or by addition of water to the negative control samples. Reactions were incubated for 20 min at 37 °C and stopped by transferring the reaction to °C for 5 min, and the protein was removed by × 5 The (25 was the HPLC column for analysis using the conditions described A reaction mixture mm), ATP mm), MgCl2 (5.0 mm), CoaD μg) and CoaE μg) in 50 mm Tris-HCl buffer (pH 7.6). Reactions were initiated by addition of the biosynthetic incubated for 2 h at 37 °C and stopped by transferring the reaction to °C for 5 min, and the protein was removed by × 5 The was a column (1 × with (50 mm), and the column was eluted with a of The chromatography was monitored The product eluted as the last from the column at The fractions were combined and dissolved in water and lyophilized This was until a of product was 1H NMR (400 MHz, D2O): (s, 1H), (s, 1H), 1H), (s, 1H), (s, 1H), (s, 1H), (m, 3H), (m, 1H), (m, 2H), (m, 1H), (t, 2H), (t, 2H), (m, 2H), 1.04 (m, 4H), 0.67 (s, 3H), (t, 3H), (s, 3H). for A reaction mixture contained 3b mm), ATP mm), MgCl2 (5.0 mm), CoaD μg) and (2 in 50 buffer (pH 7.6). Reactions were initiated by addition of the biosynthetic enzymes. reaction mixtures were treated as for the of 1b, combined and a column (1 × with (50 The column was eluted with a of The chromatography was monitored at The product eluted as the first from the column at mm The fractions were combined and dissolved in water and lyophilized This was until a of product was 1H NMR (400 MHz, D2O): (s, 1H), (s, 1H), 1H), (t, 1H), (t, 1H), (s, 1H), (s, 1H), (s, 1H), (m, 1H), (m, 1H), (t, 2H), (t, 2H), (t, 2H), (m, 2H), (m, 4H), (s, 3H), (t, 3H), (s, 3H). for All were based on the of as monitored by in at nm. of was used for Reactions were performed at °C in a were determined by the obtained to the using 6.0 kinase activity was determined using a that the of to the of Each reaction mixture contained ATP mm), mm), (0.5 mm), MgCl2 (10 mm), mm), kinase (5 (5 and CoaA (5 μg) in 50 buffer (pH 7.6). The reaction was initiated by addition of substrate or 2b, at concentrations between 10 and The phosphopantothenoylcysteine activity of CoaBC was by the of Biochem. using the from Sigma that the of to the of Each reaction mixture contained μl of (50 CTP (1.0 mm), l-cysteine (1.0 mm), DTT mm), and MgCl2 (2.0 mm) in 20 buffer (pH 7.6). The reaction was initiated by addition of of pure To the inhibitory it was in increasing concentrations to mm), and the rate of reaction was with the control Phosphopantetheine adenylyltransferase activity was by the of Biochem. using the from Sigma that the of to the of Each reaction mixture contained μl of ATP mm), DTT (1.0 mm), MgCl2 (10 mm), mm), and CoaD (5 μg) in 50 mm Tris-HCl buffer (pH 7.6). The reaction was initiated by addition of substrate (5 or at concentrations between 10 and μm. Dephospho-CoA kinase activity was determined by a of the (11Mishra P.K. Park P.K. Drueckhammer D.G. J. Bacteriol. 2001; 183: 2774-2778Google Scholar). Each reaction mixture contained ATP (5 mm), (0.5 mm), MgCl2 (10 mm), mm), kinase (5 (5 and CoaE (5 μg) in 50 buffer (pH 7.6). The reaction was initiated by addition of substrate or 6b, at concentrations between and 2.0 The biosynthesis of 1a was monitored as follows: the biosynthetic reaction in the containing 2a (0.5 (0.5 mm), ATP (5.0 mm), CTP (1.0 mm), l-cysteine (1.0 mm), DTT (2.0 mm), MgCl2 mm), CoaA (25 μg), CoaBC (25 μg), CoaD (25 μg), and CoaE (25 μg) in mm Tris-HCl buffer (pH was at 37 °C, and the reaction was initiated by addition of the biosynthetic enzymes. aliquots were and placed in a to After 5 min at °C, the was centrifuged × 5 and 30 μl of the was added to μl of mm in 0.5 m as The solution was and μl was the HPLC column for analysis using the conditions as described The concentrations of 1a and were determined using a obtained using amounts of pure CoA with the The pantothenic acid analog and its phosphate 3b were obtained by the in Fig. We that 2b the growth of E. coli in minimal medium 2 as previously (7Clifton G. Bryant S.R. Skinner C.G. Arch. Biochem. Biophys. 1970; 137: 523-528Google Scholar). of the growth rate of E. coli grown in minimal medium and in the presence of varying amounts of 2b that the inhibitor retarded the onset of logarithmic but did not it (Fig. of the amount of inhibitor all to final cell these show that is than and coli can the inhibitory for E. coli B in minimal medium at 37 °C containing increasing amounts of the pantothenic acid analog N-pentylpantothenamide of 2b 1 2 3 μm, 15 We considered two for the toxicity of it inhibits CoA thus the amount of CoA to or it is biosynthetically converted to ethyldethia-CoA, a CoA antimetabolite that inhibit CoA enzymes. To differentiate between these mechanisms the last five enzymes in CoA biosynthesis were from and incubated with the natural precursor 2a or its and the reaction were by The from these reaction mixtures are in Fig. 2a, the natural biosynthetic was used as the substrate, the new product formed was as CoA it with an of 1a (Fig. the was used as the substrate, a new eluting with a than CoA, was formed (Fig. To the of the new was using 3b as the which its from reaction mixtures in by The of the obtained in this was as being ethyldethia-CoA by 1H NMR and ESI-MS The first by HPLC analysis was thus to be ethyldethia-CoA by with this In the was by the CoaE in the reaction mixtures with and the used to the of a eluting min, by demonstrate that the E. coli CoA biosynthetic enzymes can the conversion of N-pentylpantothenamide 2b to ethyldethia-CoA To the to which the CoA biosynthetic enzymes differentiate between the natural and respective we have for of each This was using kinase for CoaA and CoaE, while an based on was used in the of demonstrate in the of CoaA the is the analog is a substrate than the natural In the of CoaD, 3b is times faster than while CoaE a with the of to give ethyldethia-CoA 4.6 times faster than the of In of a mixture of the natural substrate (50 and the analog 3b with the phosphopantothenoylcysteine synthetase/decarboxylase (10Strauss E. Kinsland C. Ge Y. McLafferty F.W. Begley T.P. J. Biol. Chem. 2001; 276: 13513-13516Google to inhibition of the activity of this with concentrations of the analog as high as 3 as determined using the demonstrate that inhibition of the CoA biosynthetic enzymes be the of antibiotic of of CoA biosynthetic enzymes with natural and substrate analogs based on was from this analysis it does not a reaction with analog from the inhibitor CoaBC was from this analysis it does not a reaction with analog from the inhibitor 2b. in a new To the from the in that the biosynthesis of ethyldethia-CoA is faster than the biosynthesis of CoA, 2a and 2b were incubated with purified and the of 1a and was followed by HPLC (Fig. on the biosynthesis of ethyldethia-CoA 10.5 times faster than the biosynthesis of Since pantothenate kinase does not differentiate between 2a and 2b, this rate is due to the the 6a, and the that the conversion to two (CoaBC) than the conversion of to while the concentrations of the biosynthetic enzymes and the of 2a to 2b in is likely to be from was used this that the biosynthesis of ethyldethia-CoA is likely to compete effectively with the biosynthesis of CoA in the cell. In show that the CoA biosynthetic enzymes are not by N-pentylpantothenamide this as a substrate of these as by its conversion to ethyldethia-CoA in the This that the toxicity from the inhibition of CoA-utilizing enzymes by the CoA analog ethyldethia-CoA The antimetabolite activity of is by the that which from in the of the of the inhibits and P.K. Biochem. Biophys. Scholar). for the of of 2b is to the recently mechanism of of which is converted in to a S. J. J. Begley T.P. Chem. 2001; Scholar). The in conversion to ethyldethia-CoA the of CoA analogs into as a may be a useful for the of cells of CoA and for on enzymes as fatty acid and non-ribosomal all of which the 4′-phosphopantetheine We Drueckhammer for Suzanne of for CoaA, for and (Cornell Biotechnology Resource Center) for with the ESI-MS