Enzymatic Synthesis and Purification of Aromatic Coenzyme a Esters1
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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 Esters1 Till Beuerle and Eran Pichersky2 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 ipate in reductive reactions catalyzed by oxidoreducta- Two recombinant His-tagged proteins, a plant 4-cou- ses (3) and in aldol-type reactions catalyzed by Claisen marate:coenzyme A ligase (EC 6.2.1.12) and a bacterial enzymes (1). benzoate:coenzyme A ligase (EC 6.2.1.25), were ex- The enzyme 4CL catalyzes the formation of CoA pressed in Escherichia coli and purified in a single thioesters of hydroxy-cinnamic acids in a process uti- step using Ni-chelating chromatography. Purified en- lizing ATP (4). These activated hydroxy-cinnamic acids zymes were used to synthesize cinnamoyl-coenzyme A serve as building blocks for numerous secondary com- (CoA), p-coumaroyl-CoA, feruloyl-CoA, caffeoyl-CoA, and benzoyl-CoA. Conversions up to 95% were pounds including flavonoids or anthocyanins (5, 6), achieved. Using a rapid solid-phase extraction proce- lignin (7), and other phenolic products (8–10) which dure, the target CoA esters were isolated with yields of fulfill diverse functions as phytoalexins, cell wall com- up to 80%. Structures were confirmed by analytical ponents, UV protectants, flavor and defense com- comparison with chemically synthesized reference pounds, or pigments. Since 4CL is involved in lignifi- compounds and electrospray ionization–mass spec- cation, a pathway found in practically all plants, this trometry. The recombinant enzymes were stable for enzyme has been extensively characterized from nu- several months at ؊80°C, thus providing a reliable and merous plant species, and many variants of this gene facile method to produce these delicate biological in- are available (11–13). termediates. © 2002 Elsevier Science (USA) BZL catalyzes the analog reaction of benzoic acid Key Words: coenzyme A; electrospray ionization– with CoA. So far this activity has been best described mass spectrometry; affinity chromatography; solid- in the benzoate degradation pathway in microorgan- phase extraction; 4-coumarate:CoA ligase; benzoate: isms (14, 15). In plants, benzoyl-CoA is reported as a CoA ligase; recombinant proteins. substrate in various enzymatic benzoylations in the biosynthesis of natural compounds such as cocaine (16), Taxol (17), dianthramide B (18), benzoylated glu- CoA3 thioesters represent an important class of ac- cosinolate esters in Arabidopsis thaliana (19), or ben- tivated intermediates in various biological pathways. zylbenzoate in Clarkia breweri (20). Although no BZL This type of activation can facilitate the transfer of the has yet been characterized from plants, an enzyme acylated moiety by enzymes known as acyltransferases that catalyzes the formation of 3-hydroxybenzoyl-CoA, (1, 2). Additionally, acylated intermediates can partic- 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 1 This work was supported by a National Science Foundation (21). Grant MCB-9974463 to E.P. and by a DAAD fellowship (Gemein- sames Hochschulprogramm III von Bund und La¨ndern) to T.B. During our investigations of coumaric acid and ben- 2 To whom correspondence and reprint requests should be ad- zoic acid metabolism in plants, we encountered the dressed. Fax: 1-734-647-0884. E-mail: [email protected]. need to generate CoA esters of the above mentioned 3 Abbreviations used: CoA, coenzyme A; BZL, benzoate:CoA ligase; aromatic acids. We initially followed protocols for 4CL, 4-hydroxyxinnamate:CoA ligase; DTE, dithioerythritol; LB me- chemical synthesis of these compounds, but the estab- dium, Luria–Burrous medium; IPTG, isopropyl -D-thiogalactopyr- anoside; SPE, solid-phase extraction; PAL, phenylalanine ammonia- lished protocols have many drawbacks: they involve lyase. multiple steps, they result in low yields with more side 0003-2697/02 $35.00 305 © 2002 Elsevier Science (USA) All rights reserved. 306 BEUERLE AND PICHERSKY products, and they require sophisticated lab equip- structs were transformed into BL21(DE3)pLysS cells ac- ment. An enzymatic approach allows a single-step re- cording to the manufacturer’s instructions. A similar ex- action under mild conditions in an aqueous solvent pression and purification protocol for both proteins was system. established. A published procedure to synthesize some of these A single isolated bacterial colony from freshly chemicals by enzymatic means took advantage of the streaked plates (grown on LB agar medium containing high levels of 4CL in wheat seedlings, but because the 50 gmlϪ1 ampicillin and 34 gmlϪ1 chloramphenicol) investigators used crude seedling extract, the product was used to inoculate 10-ml liquid cultures in LB me- yield was relatively low, due mostly to thioesterase dium containing 50 gmlϪ1 ampicillin and 34 gmlϪ1 activity (22). Here we present a facile and reliable chloramphenicol and grown overnight at 37°C. One method to enzymatically synthesize (hydroxy)cinna- aliquot of 1 ml of each culture was used to inoculate moyl-CoA and benzoyl-CoA esters that results in high 50-ml liquid cultures containing 50 gmlϪ1 ampicillin. yields (up to 80%) and high specific activities, taking Once the cultures reached a cell density of 0.4–0.5 advantage of cloned 4CL and BZL and an Escherichia OD600, recombinant protein expression was induced by coli expression system. the addition of 0.8 mM isopropyl -D-thiogalactopyr- anoside (IPTG), and the culture was grown for 20–24 h MATERIALS AND METHODS at room temperature. Cells were harvested by centrif- ugation at 3000g for 10 min at 4°C. Pellets were resus- Chemicals, Solvents, and Reagents pended in 10 times the volume with buffer of 50 mM Chemicals, solvents, and reagents were purchased Bis–Tris, pH 7.0, containing 10% glycerol, 2 mM DTE, from Sigma, Fluka, and Aldrich (St. Louis, MO) unless 1 mM EDTA, 10 mM NaCl. After three cycles of freeze/ otherwise stated. Benzoyl-CoA, coenzyme A sodium thaw at Ϫ80°C/37°C, cells were disrupted by three 20-s 14 salt, and L-[U- C]phenylalanine with a specific activity intervals of sonication on ice. The resulting homoge- of 460 mCi/mmol and [7-14C]benzoic acid with a specific nate was centrifuged at 20,000g for 10 min to pellet the activity of 16.6 mCi/mmol were purchased from Sigma. debris. The supernatant was assayed for activity and Acetonitrile UV-grade and liquid scintillation cock- stored at Ϫ80°C prior to protein purification. tail Econo-Safe were purchased from Burdick & Jack- Since Ni2ϩ chelating chromatography is incompatible son (Muskegon, MI) and Research Products Interna- with sulfur-containing reducing agents and EDTA, the tional (Mount Prospect, Il), respectively. buffer was exchanged to binding buffer conditions (5 mM imidazole, 500 mM NaCl, 20 mM Tris–HCl, pH Source of 4CL and BZL Genes 7.9) using PD-10 columns (Amersham Pharmacia, Pis- cataway, NJ). Plasmids pQE-19(11) and pPE204(14), containing Hi-Trap chelating columns of 1 ml bed vol (Amer- the coding region of tobacco 4CL and BZL, were the sham Pharmacia) were conditioned with 10 ml of wa- kind gifts of Drs. C. J. Douglas and C. S. Harwood, ter, 5 ml of charging buffer (50 mM NiSO ),and5mlof respectively. 4 binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris–HCl, pH 7.9). After loading the protein solution Cloning, Expression, and Purification of 4CL and BZL (3.5 ml in binding buffer) the column was rinsed with Primers were designed to remove the native stop 10 ml of binding buffer and 8 ml of washing buffer (80 codon and place the gene of interest in frame with the mM imidazole, 500 mM NaCl, 20 mM Tris–HCl, pH DNA encoding a C-terminal peptide containing a poly- 7.9). His-tagged 4CL was eluted with elution buffer histidine region. The gene for 4CL was amplified from (400 mM imidazole, 500 mM NaCl, 20 mM Tris–HCl pQE-19 using the primer pair 4CL-CT-His 5Ј-ATG- pH 7.9). His-tagged BZL was eluted with elution buffer GAGAAAGATACAAAACAGG and 4CL-CT-His 3Ј-AT- containing1Mimidazole. Fractions of 2.5 ml were TTGGAAGCCCAGCAGCC and the following PCR pro- collected throughout the procedure, but only the first gram: 94°C 1 min, 30 cycles 94°C 0.5 min, 54°C 0.5 400mMand1Mimidazole fractions contained 4CL min, 72°C 2 min, and then 72°C for 7 min. The gene for and BZL, respectively. For long-time storage, the BZL was amplified from pPE204 in a reaction mix buffer was changed to 50 mM Bis–Tris pH 7.0, 10% containing 10% dimethyl sulfoxide using the primer glycerol, 2 mM DTE using PD-10 columns, and the pair BZL-CT-His 5Ј-ATGAATGCAGCCGCGGTCAC and sample was stored at Ϫ80°C. BZL-CT-His 3Ј-GCCCAACACACCCTCGCG and follow- ing the PCR program: 96°C 1 min, 30 cycles 96°C 0.5 min, Enzyme Activity Assays 54°C 0.5 min, 72°C 2 min, and then 72°C for 7 min. The amplified genes were transferred into the pCRT7/ Cinnamic and (hydroxy)cinnamic acid:CoA ligase ac- CT-TOPO expression vector (Invitrogen Inc., Carlsbad, tivity assay. Enzyme activity was measured spectro- CA) according to the manufacturer’s instructions. Con- photometrically at room temperature with a 1-ml mix- SYNTHESIS OF COENZYME A ESTERS 307 ture containing 100 mM Tris–HCl, pH 7.5, 2.5 mM terminated by extraction (3ϫ) with ethylacetate (phase MgCl2, 2.5 mM ATP, 0.2 mM (hydroxy)cinnamic acids separation achieved by centrifugation). CoA esters and 0.2 mM CoA.