Enzymatic Synthesis and Purification of Aromatic Coenzyme A Esters

Analytical Biochemistry 302, 305–312 (2002) doi:10.1006/abio.2001.5574, available online at http://www.idealibrary.com on

Enzymatic Synthesis and Purification of Aromatic Coenzyme A Esters 1 Till Beuerle and Eran Pichersky 2 Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 North University Street, Ann Arbor, Michigan 48109-1048

Received November 14, 2001; published online February 13, 2002

Two recombinant His-tagged proteins, a plant 4-coumarate:coenzyme A ligase (EC 6.2.1.12) and a bacterial benzoate:coenzyme A ligase (EC 6.2.1.25), were expressed in Escherichia coli and purified in a single step using Ni-chelating chromatography. Purified enzymes were used to synthesize cinnamoyl-coenzyme A (CoA), p-coumaroyl-CoA, feruloyl-CoA, caffeoyl-CoA, and benzoyl-CoA. Conversions up to 95% were achieved. Using a rapid solid-phase extraction procedure, the target CoA esters were isolated with yields of up to 80%. Structures were confirmed by analytical comparison with chemically synthesized reference compounds and electrospray ionization–mass spectrometry. The recombinant enzymes were stable for several months at ⴚ80°C, thus providing a reliable and facile method to produce these delicate biological intermediates. © 2002 Elsevier Science (USA) Key Words: coenzyme A; electrospray ionization– mass spectrometry; affinity chromatography; solidphase extraction; 4-coumarate:CoA ligase; benzoate: CoA ligase; recombinant proteins.

CoA 3 thioesters represent an important class of activated intermediates in various biological pathways. This type of activation can facilitate the transfer of the acylated moiety by enzymes known as acyltransferases (1, 2). Additionally, acylated intermediates can partic1 This work was supported by a National Science Foundation Grant MCB-9974463 to E.P. and by a DAAD fellowship (Gemeinsames Hochschulprogramm III von Bund und La¨ndern) to T.B. 2 To whom correspondence and reprint requests should be addressed. Fax: 1-734-647-0884. E-mail: [email protected]. 3 Abbreviations used: CoA, coenzyme A; BZL, benzoate:CoA ligase; 4CL, 4-hydroxyxinnamate:CoA ligase; DTE, dithioerythritol; LB medium, Luria–Burrous medium; IPTG, isopropyl ␤-D-thiogalactopyranoside; SPE, solid-phase extraction; PAL, phenylalanine ammonialyase.

0003-2697/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

ipate in reductive reactions catalyzed by oxidoreductases (3) and in aldol-type reactions catalyzed by Claisen enzymes (1). The enzyme 4CL catalyzes the formation of CoA thioesters of hydroxy-cinnamic acids in a process utilizing ATP (4). These activated hydroxy-cinnamic acids serve as building blocks for numerous secondary compounds including flavonoids or anthocyanins (5, 6), lignin (7), and other phenolic products (8 –10) which fulfill diverse functions as phytoalexins, cell wall components, UV protectants, flavor and defense compounds, or pigments. Since 4CL is involved in lignification, a pathway found in practically all plants, this enzyme has been extensively characterized from numerous plant species, and many variants of this gene are available (11–13). BZL catalyzes the analog reaction of benzoic acid with CoA. So far this activity has been best described in the benzoate degradation pathway in microorganisms (14, 15). In plants, benzoyl-CoA is reported as a substrate in various enzymatic benzoylations in the biosynthesis of natural compounds such as cocaine (16), Taxol (17), dianthramide B (18), benzoylated glucosinolate esters in Arabidopsis thaliana (19), or benzylbenzoate in Clarkia breweri (20). Although no BZL has yet been characterized from plants, an enzyme that catalyzes the formation of 3-hydroxybenzoyl-CoA, an intermediate in the biosynthesis of xanthone, in cell cultures of Centaurium erythraea has been reported, but the gene encoding this enzyme was not isolated (21). During our investigations of coumaric acid and benzoic acid metabolism in plants, we encountered the need to generate CoA esters of the above mentioned aromatic acids. We initially followed protocols for chemical synthesis of these compounds, but the established protocols have many drawbacks: they involve multiple steps, they result in low yields with more side 305

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products, and they require sophisticated lab equipment. An enzymatic approach allows a single-step reaction under mild conditions in an aqueous solvent system. A published procedure to synthesize some of these chemicals by enzymatic means took advantage of the high levels of 4CL in wheat seedlings, but because the investigators used crude seedling extract, the product yield was relatively low, due mostly to thioesterase activity (22). Here we present a facile and reliable method to enzymatically synthesize (hydroxy)cinnamoyl-CoA and benzoyl-CoA esters that results in high yields (up to 80%) and high specific activities, taking advantage of cloned 4CL and BZL and an Escherichia coli expression system. MATERIALS AND METHODS

Chemicals, Solvents, and Reagents Chemicals, solvents, and reagents were purchased from Sigma, Fluka, and Aldrich (St. Louis, MO) unless otherwise stated. Benzoyl-CoA, coenzyme A sodium salt, and L-[U- 14C]phenylalanine with a specific activity of 460 mCi/mmol and [7- 14C]benzoic acid with a specific activity of 16.6 mCi/mmol were purchased from Sigma. Acetonitrile UV-grade and liquid scintillation cocktail Econo-Safe were purchased from Burdick & Jackson (Muskegon, MI) and Research Products International (Mount Prospect, Il), respectively. Source of 4CL and BZL Genes Plasmids pQE-19(11) and pPE204(14), containing the coding region of tobacco 4CL and BZL, were the kind gifts of Drs. C. J. Douglas and C. S. Harwood, respectively. Cloning, Expression, and Purification of 4CL and BZL Primers were designed to remove the native stop codon and place the gene of interest in frame with the DNA encoding a C-terminal peptide containing a polyhistidine region. The gene for 4CL was amplified from pQE-19 using the primer pair 4CL-CT-His 5⬘-ATGGAGAAAGATACAAAACAGG and 4CL-CT-His 3⬘-ATTTGGAAGCCCAGCAGCC and the following PCR program: 94°C 1 min, 30 cycles 94°C 0.5 min, 54°C 0.5 min, 72°C 2 min, and then 72°C for 7 min. The gene for BZL was amplified from pPE204 in a reaction mix containing 10% dimethyl sulfoxide using the primer pair BZL-CT-His 5⬘-ATGAATGCAGCCGCGGTCAC and BZL-CT-His 3⬘-GCCCAACACACCCTCGCG and following the PCR program: 96°C 1 min, 30 cycles 96°C 0.5 min, 54°C 0.5 min, 72°C 2 min, and then 72°C for 7 min. The amplified genes were transferred into the pCRT7/ CT-TOPO expression vector (Invitrogen Inc., Carlsbad, CA) according to the manufacturer’s instructions. Con-

structs were transformed into BL21(DE3)pLysS cells according to the manufacturer’s instructions. A similar expression and purification protocol for both proteins was established. A single isolated bacterial colony from freshly streaked plates (grown on LB agar medium containing 50 ␮g ml ⫺1 ampicillin and 34 ␮g ml ⫺1 chloramphenicol) was used to inoculate 10-ml liquid cultures in LB medium containing 50 ␮g ml ⫺1 ampicillin and 34 ␮g ml ⫺1 chloramphenicol and grown overnight at 37°C. One aliquot of 1 ml of each culture was used to inoculate 50-ml liquid cultures containing 50 ␮g ml ⫺1 ampicillin. Once the cultures reached a cell density of 0.4 – 0.5 OD 600, recombinant protein expression was induced by the addition of 0.8 mM isopropyl ␤-D-thiogalactopyranoside (IPTG), and the culture was grown for 20 –24 h at room temperature. Cells were harvested by centrifugation at 3000g for 10 min at 4°C. Pellets were resuspended in 10 times the volume with buffer of 50 mM Bis–Tris, pH 7.0, containing 10% glycerol, 2 mM DTE, 1 mM EDTA, 10 mM NaCl. After three cycles of freeze/ thaw at ⫺80°C/37°C, cells were disrupted by three 20-s intervals of sonication on ice. The resulting homogenate was centrifuged at 20,000g for 10 min to pellet the debris. The supernatant was assayed for activity and stored at ⫺80°C prior to protein purification. Since Ni 2⫹ chelating chromatography is incompatible with sulfur-containing reducing agents and EDTA, the buffer was exchanged to binding buffer conditions (5 mM imidazole, 500 mM NaCl, 20 mM Tris–HCl, pH 7.9) using PD-10 columns (Amersham Pharmacia, Piscataway, NJ). Hi-Trap chelating columns of 1 ml bed vol (Amersham Pharmacia) were conditioned with 10 ml of water, 5 ml of charging buffer (50 mM NiSO 4), and 5 ml of binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris–HCl, pH 7.9). After loading the protein solution (3.5 ml in binding buffer) the column was rinsed with 10 ml of binding buffer and 8 ml of washing buffer (80 mM imidazole, 500 mM NaCl, 20 mM Tris–HCl, pH 7.9). His-tagged 4CL was eluted with elution buffer (400 mM imidazole, 500 mM NaCl, 20 mM Tris–HCl pH 7.9). His-tagged BZL was eluted with elution buffer containing 1 M imidazole. Fractions of 2.5 ml were collected throughout the procedure, but only the first 400 mM and 1 M imidazole fractions contained 4CL and BZL, respectively. For long-time storage, the buffer was changed to 50 mM Bis–Tris pH 7.0, 10% glycerol, 2 mM DTE using PD-10 columns, and the sample was stored at ⫺80°C. Enzyme Activity Assays Cinnamic and (hydroxy)cinnamic acid:CoA ligase activity assay. Enzyme activity was measured spectrophotometrically at room temperature with a 1-ml mix-

SYNTHESIS OF COENZYME A ESTERS

ture containing 100 mM Tris–HCl, pH 7.5, 2.5 mM MgCl 2, 2.5 mM ATP, 0.2 mM (hydroxy)cinnamic acids and 0.2 mM CoA. The assay was started by the addition of CoA. The change in absorbance of the reaction mixture was monitored at the wavelengths of 311, 333, 345, 346, and 352 nm according to the reported absorption maxima for cinnamoyl-CoA, p-coumaroyl-CoA, feruloyl-CoA, caffeoyl-CoA, and sinapoyl-CoA, respectively (23, 24). Benzoyl:CoA ligase activity assay. Radioisotopic assays were performed with 100 ␮l buffer containing 100 mM Tris–HCl, pH 7.5, 2.5 mM MgCl 2, 2.5 mM ATP, 0.2 mM CoA, and 10,000 to 50,000 dpm [7- 14C]benzoic acid. The assay was started by the addition of CoA and kept at room temperature for 15 to 120 min. The reaction was stopped by the addition of 5 ␮l 50% trichloracetic acid and 180 ␮l ethyl acetate, vortexed, and phase separated by a 1-min centrifugation at 14,000g. The upper organic phase was removed and the ethyl acetate extraction was repeated. The remaining aqueous phase was counted in a liquid scintillation counter. The amount of radioactivity in the aqueous phase indicated the amount of synthesized benzoyl-CoA. Thioesterase activity assay. Reactions (total sample volume 1 ml) containing 100 mM Tris–HCl, 2.5 mM MgCl 2, 0.05 mM cinnamoyl-CoA, and either 50 ␮g crude supernatant of BL21(DE)LysS cells containing an empty vector or 50 ␮g purified 4CL were incubated at room temperature. The reaction was started by addition of cinnamoyl-CoA. The decrease in absorbance at 311 nm over time was recorded. Thioesterase activity for benzoyl-CoA was measured in a similar assay to that described above. Released radiolabeled benzoic acid was extracted with ethyl acetate and counted in a liquid scintillation counter. Chemical synthesis and purification of cinnamoylCoA, p-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA, and benzoyl-CoA. Coenzyme A esters of cinnamic and several (hydroxy)cinnamic acids were synthesized by a modified imidazolide method. Imidazolides of the corresponding acids were prepared as previously described (25). The reaction was monitored for purity and completion by TLC (Polygram Sil G/UV, MachereyNagel, Easton, PA), developed in diethylether acidified with 1% acetic acid, and visualized under UV light. Reactions were allowed to proceed until the disappearance of the acid on the TLC. The acid imidazolides were used without further purification for the next reaction step. Corresponding CoA esters were obtained by a modification of the method of Pabsch et al. (25). Acid imidazolides were used in twofold excess compared to the CoA-sodium salt. The reaction was monitored with TLC (Silicagel) and a solvent system of n-butanol:acetic acid:water 63:10:27. CoA esters were identified with delayed nitroprusside reaction (26). The reaction was

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terminated by extraction (3⫻) with ethylacetate (phase separation achieved by centrifugation). CoA esters were purified using solid-phase extraction cartridges (1000 mg Chromabond C 18 ec, Macherey-Nagel) preconditioned with consecutive washes of methanol, dH 2O, and 4% ammonium acetate solution (5 column vol each). After evaporation of the organic solvent, ammonium acetate was added to the water phase to a final concentration of 4%, and the mixture was loaded onto the SPE cartridge. The column was rinsed with 4% ammonium acetate solution until the flowthrough showed the absence of free CoA (determined by spectrophotometry). The CoA esters were recovered by elution with distilled water. Fractions containing the CoA esters were identified by their UV spectrum and lyophilized overnight. Yields varied between 15 and 45%. The purity was checked by TLC and reverse-phase HPLC analysis with UV detection at 216 nm on a Nova-Pak column (Nova-Pak C 18 60A 4 ␮m, 3.9 ⫻ 300 mm, Waters, Milford, MA) and was higher than 90%. TLC was performed on Silicagel plates using the solvent system 1-butanol:water:acetic acid (60:35:25), R f’s of 0.37, 0.4, 0.45, 0.54, and 0.56 were observed for caffeoyl-CoA, p-coumaroyl-CoA, feruloyl-CoA, cinnamoyl-CoA, and benzoyl-CoA, respectively. For HPLC, a flow of 1 ml/min and a gradient of solvent A (acetonitrile) and solvent B (20 mM KH 2PO 4, pH 2.9) were applied: 0 –5 min, 5% A; 5–32 min, 5–38% A linear; 32–35 min, 38 –75% A linear; 35– 40 min, 75–5% A linear; 40 – 45 min 5% A. R t’s of 16.1, 18.4, 19.2, 19.8, and 21.9 min were recorded for caffeoyl-CoA, benzoylCoA, p-coumaroyl-CoA, feruloyl-CoA, and cinnamoylCoA, respectively. CoA esters were stored in solution at pH 6 or lyophilized and stored over Drierite for several months at 80°C without noticeable degradation. Electrospray ionization–mass spectrometry (ESI-MS) analysis of hydroxy-cinnamoyl and benzoyl-CoA esters. For ESI-MS analysis, purified CoA esters were diluted in distilled water at a concentration of 0.1 mM. Samples were analyzed on a Micromass Quattro LC (Micromass, Beverly, MA) by direct infusion with a flow rate of 8 ␮l min ⫺1. Samples were ionized in positive and negative mode with a capillary voltage of 2.9 and 3.5 V, respectively. Nitrogen was used for nebulization (65 liters h ⫺1) and desolvation gas (420 liters h ⫺1, 250°C). The source temperature was set at 120°C and cone voltages between 20 to 30 V were applied. Standard mass spectra were recorded for a total of 30 s with a total scan time of 2 s over the mass range from 200 to 1000 m/z. Data acquisition and evaluation were conducted with MassLynx version 3.5 software (Micromass, Manchester, UK) on a personal computer. Preparative enzymatic synthesis of p-coumaroyl-CoA. To synthesize coumaroyl-CoA, 3.3 mg coumaric acid, 2 mg CoA, and 6.9 mg ATP were dissolved in a total

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volume of 10 ml of 50 mM Tris–HCl buffer containing 2.5 mM MgCl 2. The reaction was started by the addition of 0.25 mg purified 4CL. After 5 h at room temperature, 6.9 mg ATP, 2 mg CoA, and 0.25 mg enzyme were added and the reaction continued. After an additional 12 h, 0.4 g of ammonium acetate was added and the crude enzyme reaction was loaded on preconditioned 1000 mg C 18 SPE cartridge and purified as described above. Four 5-ml fractions containing p-coumaroyl-CoA were lyophilized and 3.4 mg p-coumaroylCoA were obtained, which represented 80% yield based on CoA used. p-Coumaroyl-CoA was characterized and stored as described above. Enzymatic synthesis of [U- 14C]cinnamic acid. L-[U14 C]Phenylalanine was dissolved in up to 600 ␮l of 30 mM Tris–HCl buffer, pH 8.5, containing 0.05 Units of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5, Sigma) at 30°C. Small aliquots of the reaction mixture were acidified and extracted with ethyl acetate to check for reaction completion. The ratio of counts in the organic phase (cinnamic acid) vs the aqueous phase (phenylalanine) indicated the conversion of L-phenylalanine to cinnamic acid. Reaction times varied from 18 to 72 h. The reaction was performed on different scales of 10, 25, and 250 ␮Ci, with yields between 85 and 93%. The mixture was acidified with 20 ␮l of 6 N HCl and extracted (3⫻) with 300 ␮l each to isolate [U- 14C]cinnamic acid. The organic phases were combined and concentrated to dryness and then redissolved in a small volume of 25% ethanol and stored at 4°C. Chemical and radiochemical purity were checked with TLC (Silicagel, R f 0.73) using the solvent system pentane: diethylether:acetic acid (100:100:1) and by reversephase HPLC analysis with UV detection at 216 and 270 nm. The HPLC was operated at a flow rate of 1 ml/min and a gradient of solvent A (acetonitrile) and solvent B (water, pH 2.9, adjusted with H 2SO 4) was applied: 0 –5 min, 5% A; 5– 45 min, 5–75% A linear; 45–50 min, 75–100% A linear; 50 –53 min, 100% A linear; 53–55 min, 100 –5% A linear; 55– 60 min, 5% A. Fractions were collected every minute and counted in a liquid scintillation counter. [U- 14C]cinnamic acid coeluted with an authentic reference compound at R t 25.7 min. Enzymatic synthesis of [U- 14C]cinnamoyl-CoA. An almost complete enzymatic conversion of cinnamic acid to cinnamoyl-CoA was achieved after 90 min at room temperature. The reaction mixture contained 50 mM Tris–HCl, pH 7.5, 2.5 mM MgCl 2, 2.5 mM ATP, 0.2 mM CoA, 25 ␮Ci [ 14C-U]cinnamic acid, and 50 ␮g of purified 4CL in a total volume of 500 ␮l. The conversion rate was checked as described above. The ratio of counts in the aqueous phase vs the organic phase was calculated as conversion into cinnamoyl-CoA. The reaction mixture was acidified with 50 ␮l 6 N HCl and extracted

three times with 250 ␮l diethylether to remove residual cinnamic acid. After evaporating the remaining organic solvent, ammonium acetate was added to a final concentration of 4% (w/v). The aqueous solution was loaded on a preconditioned 100-mg SPE cartridge (Chromabond C 18 ec, Macherey-Nagel) as described above. The column was successively rinsed with 5 ml of 4% ammonium acetate solution to remove unreacted CoA and protein and with 5 ml of distilled water to elute the CoA ester. Fractions of 1 ml were collected. Most of the radioactivity was recovered in one fraction. The overall yield in this procedure was ⬎80% based on radioactivity. Chemical and radiochemical purity of more 95% was verified with HPLC and TLC (Silicagel, R f 0.4) and authentic nonlabeled standards, as described above. In addition, purity was also checked by hydrolysis of the reaction product at pH 12 (NaOH) for 30 min at 60°C and detection of liberated cinnamic acid with HPLC and TLC as descried above. Enzymatic synthesis of [7- 14C]benzoyl-CoA. A total volume of 500 ␮l of 100 mM Tris–HCl, pH 8.5, containing 2.5 mg CoA, 1.7 mg ATP, 25 ␮Ci [7- 14C]benzoic acid (16.6 mCi/mmol, Sigma), and 50 ␮g purified BZL was incubated at room temperature. The conversion was checked every hour by extracting a small portion as described above. After 4 h, 95% was converted to benzoyl-CoA and the reaction was stopped by the addition of 75 ␮l 6 N HCl, followed by 3⫻ extraction with 350-␮l portions of diethylether. Benzoyl-CoA was purified from the aqueous phase with SPE as described for [U- 14C]cinnamoyl-CoA. Chemical and radiochemical purity of more 95% was verified with HPLC and TLC (Silicagel, R f 0.35) as described for [U- 14C]cinnamoylCoA. The overall yield was 75%, based on recovered radioactivity. RESULTS

Thioesterase Activity in Crude Extracts of E. coli The soluble protein fraction of cell-free extracts of E. coli BL-21 cells expressing 4CL or BZL showed high activity of the corresponding CoA ligase activity. However, attempts to use the crude extracts for the synthesis of CoA esters resulted in unsatisfactory yields of ⬍40%, and the yield could not be increased by varying the reaction parameters. Incubation of cinnamoyl-CoA with crude extracts of BL-21 cells containing an empty expression vector indicated that soluble thioesterase activity (0.26 nkatal/mg crude protein extract) was present in these cells. It appeared that poor yields using crude enzyme preparations might therefore reflect a balance between CoA ligase and thioesterase activity. Crude enzyme extracts might also contain other ATP-utilizing enzymes which compete for the limited amount of ATP. Once the supply of ATP is used up, no new ligation

309

SYNTHESIS OF COENZYME A ESTERS

FIG. 1. SDS–PAGE analysis of purified recombinant BZL and 4CL. (Lane A) Molecular mass markers; (lane B) soluble extract from uninduced BL21(DE3)pLysS cells harboring the His-tagged BZL gene construct (10 ␮g protein); (lane C) soluble extract from BL21(DE3)pLysS cells harboring the His-tagged BZL gene construct 20 h after induction with IPTG (10 ␮g protein); (lane D) recombinant BZL after Ni 2⫹-chelating chromatography (2 ␮g protein); (lane E) soluble extract from uninduced BL21(DE3)pLysS cells harboring the His-tagged 4CL gene construct (10 ␮g protein); (lane F) soluble extract from BL21(DE3)pLysS cells harboring the His-tagged 4CL gene construct 20 h after induction with IPTG (20 ␮g protein); (lane G) recombinant 4CL after Ni 2⫹-chelating chromatography (2 ␮g protein).

reaction occurs, while the already formed CoA esters continue to be cleaved by the thioesterase. Since 4CL is inhibited at ATP concentrations higher than 5 mM, this problem cannot be simply solved by adding large amounts of ATP. Purification of 4CL and BZL by Affinity Chromatography To circumvent these difficulties, we used His-tagging and affinity chromatography to purify the expressed heterologous proteins. Most importantly, no thioesterase activity was detectable after the affinity chromatography step. When 2 ␮g of the purified protein fractions was analyzed by SDS–PAGE and Coomassie blue staining of the gel, the purified BZL and 4CL samples both were shown to contain only one major band of the expected size for these ligases (Fig. 1). The molecular weight of the purified His-tagged BZL protein was estimated from the SDS–PAGE analysis to be 61.5 kDa, and that of the His-tagged 4CL to be 63 kDa (Fig. 1), corresponding to the calculated molecular weights of 60,115 and 62,900 Da, respectively. In both samples, one additional, unidentified protein band of approximately 75 kDa was barely detected (in this system, the minimum detection level is 50 ng protein per band). In our purification procedure, 50-ml cultures typically yielded 3.4 mg of recombinant 4CL and 3.5 mg recombinant BZL. Expression in E. coli and enzyme purification were repeated several times with similar results. The enzymes were stored at ⫺80°C in a buffer containing 10% glycerol and were stable for several month and multiple freeze/thaw cycles. Recombinant 4CL was also tested with cinnamic, ferulic, caffeic, and sinapic acid as substrates. While

the preferred substrate of 4CL has been reported to be ferulic acid, followed by p-coumaric acid, cinnamic acid, and caffeic acid (11), the His-tagged protein had a slightly different specificity, being more active with ferulic acid than with p-coumaric acid (Table 1). This observed difference in relative activity might be due of the introduction of the C-terminal His-tag, or the original report may be in error as a result of the presence of bacterial thioesterase activity in crude extracts that may have degraded the hydroxy-cinnamoyl-CoA esters at different rates. At any rate, the difference in activity of 4CL for the substrates can be neglected in practice, because it can be easily overcome by adding more enzyme to the reaction. Preparative Enzymatic Synthesis of p-Coumaroyl-CoA We chose the synthesis of p-coumaroyl-CoA to demonstrate the use of an enzymatic approach to generate CoA esters. Our procedure, detailed under Materials and Methods, gave a 80% yield after purification, based on the amount CoA utilized (Table 2A). Enzymatic Synthesis of Labeled Cinnamoyl-CoA Since radiolabeled cinnamic acid was not commercially available, we combined two enzymatic steps to synthesize U- 14C-labeled cinnamoyl-CoA. Starting from L-[U- 14C]phenylalanine, [U- 14C]cinnamic acid was obtained utilizing the enzyme PAL (EC 4.3.1.5) from Rhodotorula glutinis. Subsequent conversion to cinnamoyl-CoA was achieved using purified recombinant 4CL. The product obtained showed high chemical and radiochemical purity (Table 2A). Enzymatic Synthesis of [7- 14C]Benzoyl-CoA The radiolabeled benzoic acid that we used had a relative low specific activity. Since we limited ourselves for practical reasons to a relatively small, easy to han-

TABLE 1

Substrate Specificity of Recombinant Purified His-Tagged Tobacco 4CL Compared to Crude Extracts of E. coli Expressing Recombinant Tobacco 4CL (11)

Substrate p-Coumaric acid Ferulic acid Cinnamic acid Caffeic acid Sinapic acid

Relative activity (%) of Relative activity (%) of recombinant His-tagged recombinant tobacco 4CL tobacco 4CL as reported in (11) 100 109 74 64 0

100 60 25 20 0

Note. The specific enzyme activity of purified 4CL with coumaric acid as substrate was 31.3 nkatal/mg protein.

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BEUERLE AND PICHERSKY TABLE 2 Acid moiety

Yield (%)

Amount (mg)

Purity (%)

A. Yields, amount, and purity of enzymatically synthesized CoA esters [U- 14C]Cinnamic acid p-Coumaric acid [7- 14C]Benzoic acid

80 a 80 c 75 a

0.032 b 3.5 0.275 b

⬎95 ⬎95 ⬎95

B. Yields, amount, and purity of CoA esters chemically synthesized via imidazolide activation Cinnamic acid p-Coumaric acid Benzoic acid Caffeic acid Ferulic acid a b c

45 15 43 43 42

16 5 15 16 16

⬎95 ⬎90 ⬎95 90 ⬎90

Yield based radioactivity. Calculated, based on radioactivity. Yield based on CoA.

dle volume, we had to use higher concentrations of benzoic acid in our reaction (3 mM). Since BZL shows product inhibition (80% at 0.1 mM benzoyl-CoA) (14), we tested several conditions to optimize the conversion. Even though CoA esters are more likely to be cleaved in basic solutions, it proved to be necessary to raise the pH to 8.5 to increase the conversion rate. Despite product inhibition, using excess of protein forced the reaction toward high yields (Table 2A). Thus, using larger volumes should provide a facile route for a preparative synthesis of benzoyl-CoA. Chemical Synthesis of CoA Esters To verify the authenticity of products from the enzymatic reactions, reference compounds were synthesized using a method of imidazolide activation for the acids, recently published for caffeoyl-CoA (25). Yields and purity of all chemically synthesized CoA esters are summarized in Table 2B. Solid-Phase Extraction Procedure C 18 SPE using cartridges was found to be most efficient and convenient method to purify the products. Aromatic CoA esters bind to the column in the presence of ammonium acetate, and rinsing the columns with 4% ammonium acetate solutions washes off the free and oxidized CoA and proteins. The CoA esters were recovered by rinsing the column with distilled water. The progress of each rinsing step can be easily followed using a spectrophotometer. Under the above described conditions, the excess hydroxy-cinnamic acids and benzoic acid were retained by the C 18 material, thus separate from the target compounds. An additional advantage of this method is that CoA esters were

obtained in aqueous solution ready to use in enzymatic assays or in in vivo labeling experiments. The products of the large-scale syntheses of unlabeled CoA esters were lyophilized overnight and the compounds were obtained as fluffy powders. Aqueous solutions and dry powders of CoA esters were stored at ⫺80°C over Drierite without noticeable degradation for several months. ESI-MS of Hydroxy-cinnamoyl-CoA’s and Benzoyl-CoA At cone voltages between 20 and 30 V in both ionization modes (ESI positive and ESI negative), mass spectra of hydroxy-cinnamoyl-CoA and benzoyl-CoA esters were dominated by ions that stand in good agreement with calculated singly and doubly charged pseudo-molecular ions of the corresponding CoA ester (Fig. 2 and Table 3). At ESI negative mode (cone 30 V), in-source fragmentation occurred and an ion with a mass of 80 m/z lower than [M ⫺ H] ⫺ was observed (Fig. 2B, ion m/z 790.2), most likely representing loss of HPO 3 from the phosphoadenosine moiety of the CoA ester. The results of ESI-MS analysis of all hydroxycinnamoyl and benzoyl-CoA esters described here are summarized in Table 3. ESI-MS spectra of enzymatically synthesized CoA esters were identical to those CoA esters obtained via the chemical method. DISCUSSION

Despite some reports over failed synthesis of ␣,␤unsaturated CoA esters using 1-acyl imidazolide activation (29), the chemical method used to synthesize caffeoyl-CoA via the imidazolide (25) can be applied generally to synthesize other hydroxy-cinnamoyl-CoA and benzoyl-CoA esters. Nevertheless, multiple steps are necessary and our yields did not exceed 45%. Several reports have recently appeared describing the use of crude enzyme extracts of various source to generate hydroxy-cinnamoyl-CoA esters (22, 27). As previously noted, and as we learned from our own experiments (unpublished data), crude plant extracts contain thioesterase activities that result in the undesired cleavage of hydroxy-cinnamoyl and benzoyl-CoA esters. This makes it difficult to predict reaction conditions or maximize conversion rates, and therefore yields are low and do not exceed 40% (22, 27). The use of purified enzymes expressed in E. coli is a way to circumvent this problem. The tobacco 4CL protein we employed in our syntheses allowed us to generate preparative amounts of cinammoyl-CoA and coumaroyl-CoA and, based on our preliminary experiments (Table 1), should also be useful in the generation of preparative amounts of the caffeic and ferulic acid CoA esters. The Rhodopseudo-

311

SYNTHESIS OF COENZYME A ESTERS

monas palustris BZL gene was reported to use benzoic acid, halogenated benzoic acid derivatives, cyclohexencarboxylic acids, and pyridine carboxylic acids as substrates (14), and therefore it is likely that our method should be capable of generating CoA esters of these acids as well. Together, the one-step enzymatic synthesis and the one-step SPE purification described here are simple and efficient, providing an excellent method especially

TABLE 3

Dominant Ions in ESI Mass Spectra of Hydroxy-cinnamoyl and Benzoyl-CoA Ester in Positive and Negative Mode

Compound

Mr

ESI negative a m/z of [M ⫺ H] ⫺/ [M ⫺ 2H] 2⫺/ [M ⫺ H ⫺ HPO 3] ⫺

Benzoyl-CoA Cinnamoyl-CoA p-Coumaroyl-CoA Caffeoyl-CoA Feruloyl-CoA

871.6 897.7 913.7 929.7 943.7

870.2/434.7/790.2 896.2/447.7/816.2 912.3/455.8/832.3 928.2/463.7/848.2 942.2/470.7/862.2

a b

ESI positive b m/z of [M ⫹ H] ⫹/ [M ⫹ 2H] 2⫹ 872.2/436.8 898.2/449.7 914.2/457.8 930.3/465.8 944.3/472.8

Cone voltage 30 V. Cone voltage 20 V.

for the generation of radiolabeled CoA esters, where a minimum of handling is desirable. Furthermore, we were able to isolate these CoA esters using SPE to high purity in aqueous solvents, making them immediately usable in enzyme assays and in in vitro feeding experiments. We also used a two-step enzymatic method, starting with L-[U- 14C]phenylalanine, to make radiolabeled cinnamoyl-CoA. Since PAL is also active with tyrosine (28), an analogous approach starting with radiolabeled L-tyrosine would lead to p-coumaroyl-CoA. The enzymatic syntheses as well as the isolation procedure can be easily scaled up to generate milligram amounts of CoA esters. Considering the fact that CoA is the most costly component and that the enzymatic method described here uses much less CoA per product formed, this method is superior to the chemical method. ACKNOWLEDGMENTS We thank Drs. C. J. Douglas and C. S. Harwood for their kind gifts of 4CL and BZL, respectively.

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FIG. 2. Formular and ESI-MS spectra of benzoyl CoA. (A) Formula and calculated molecular weight of benzoyl-CoA; (B) ESI-MS of benzoyl-CoA in negative mode (cone 30 V) with singly and doubly charged pseudo molecular ion [M ⫺ H] ⫺ at m/z 870.2 and [M ⫺ 2H] 2⫺ 434.7, respectively, as well as the fragment ion [M ⫺ H ⫺ HPO 3] ⫺; (C) ESI-MS of benzoyl-CoA in positive mode (cone 20 V) with singly and doubly charged pseudo molecular ion [M ⫹ H] ⫹ at m/z 872.2 and [M ⫹ 2H] 2⫹ at m/z 436.8.

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