Enzymatic Synthesis and Purification of Aromatic Coenzyme A Esters

Beuerle, Till; Pichersky, Eran

doi:10.1006/abio.2001.5574

Cited by 134 publications

(128 citation statements)

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“…FeruloylCoA was generated in situ by adding a recombinant tobacco 4-coumarate:coenzyme A ligase (Beuerle and Pichersky, 2002) plus ferulate, ATP, and CoASH to the reaction mixture. Primary alcohols of various chain lengths (C12-C18) and 15-hydroxypentadecanoic acid methyl ester were tested as acyl acceptors.…”

Section: Recombinant Asftp Functions As a Feruloyl Transferasementioning

confidence: 99%

“…A single bacterial colony was used to inoculate a liquid culture, which was in turn used to inoculate a subculture of Luria-Bertani medium containing 100 mg mL 21 carbenicillin, which was grown at 20°C with shaking. When OD = 0.6, isopropyl b-D-thiogalactopyranoside (0.8 mM) was added for induction of protein expression, and culture was grown for 24 h. Procedures for protein extraction and subsequent purification are described elsewhere (Beuerle and Pichersky, 2002). The soluble recombinant protein was purified by Ni +2 chelating chromatography, and the presence of the fusion protein was confirmed by SDS-PAGE and western blot.…”

Section: Recombinant Protein Expressionmentioning

confidence: 99%

“…Feruloyl-CoA substrate was generated by incubating a reaction mixture (1 mL) containing 50 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 , 2.5 mM ATP, 0.2 mM ferulic acid, 0.2 mM CoA, and 10 mL of purified recombinant 4-coumarate ligase (Beuerle and Pichersky, 2002) for 1 h at 30°C. To initiate ester synthesis, 10 mL of recombinant ASFT-tagged protein, 1 mM dithiothreitol, and 1 mM fatty alcohol or methyl 15-hydroxypentadecanoate were added.…”

Section: Feruloyl Transferase Assaymentioning

confidence: 99%

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Identification of an Arabidopsis Feruloyl-Coenzyme A Transferase Required for Suberin Synthesis

et al. 2009

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All plants produce suberin, a lipophilic barrier of the cell wall that controls water and solute fluxes and restricts pathogen infection. It is often described as a heteropolymer comprised of polyaliphatic and polyaromatic domains. Major monomers include v-hydroxy and a,v-dicarboxylic fatty acids, glycerol, and ferulate. No genes have yet been identified for the aromatic suberin pathway. Here we demonstrate that Arabidopsis (Arabidopsis thaliana) gene AT5G41040, a member of the BAHD family of acyltransferases, is essential for incorporation of ferulate into suberin. In Arabidopsis plants transformed with the AT5G41040 promoter:YFP fusion, reporter expression is localized to cell layers undergoing suberization. Knockout mutants of AT5G41040 show almost complete elimination of suberin-associated ester-linked ferulate. However, the classic lamellar structure of suberin in root periderm of at5g41040 is not disrupted. The reduction in ferulate in at5g41040-knockout seeds is associated with an approximate stoichiometric decrease in aliphatic monomers containing v-hydroxyl groups. Recombinant AT5G41040p catalyzed acyl transfer from feruloyl-coenzyme A to v-hydroxyfatty acids and fatty alcohols, demonstrating that the gene encodes a feruloyl transferase. CYP86B1, a cytochrome P450 monooxygenase gene whose transcript levels correlate with AT5G41040 expression, was also investigated. Knockouts and overexpression confirmed CYP86B1 as an oxidase required for the biosynthesis of very-long-chain saturated a,v-bifunctional aliphatic monomers in suberin. The seed suberin composition of cyp86b1 knockout was surprisingly dominated by unsubstituted fatty acids that are incapable of polymeric linkages. Together, these results challenge our current view of suberin structure by questioning both the function of ester-linked ferulate as an essential component and the existence of an extended aliphatic polyester.

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Section: Recombinant Asftp Functions As a Feruloyl Transferasementioning

confidence: 99%

Section: Recombinant Protein Expressionmentioning

confidence: 99%

Section: Feruloyl Transferase Assaymentioning

confidence: 99%

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Identification of an Arabidopsis Feruloyl-Coenzyme A Transferase Required for Suberin Synthesis

et al. 2009

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“…[U-13 C]benzoyl-CoA was prepared as described (Beuerle and Pichersky, 2002) with the exception that benzoic acid was isotopically labeled. Another 8 mg of purified EcCS was added and left to react at room temperature for 12 h. The solution was basified using 20 mL of 1 N NaOH and extracted three times with 500 mL of chloroform.…”

Section: Synthesis Of [ 13 C 7 ]Cocainementioning

confidence: 99%

The Last Step in Cocaine Biosynthesis Is Catalyzed by a BAHD Acyltransferase

et al. 2014

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ORCID IDs: 0000-0003-1007-4700 (J.J.); 0000-0002-4865-3938 (J.C.D.).The esterification of methylecgonine (2-carbomethoxy-3b-tropine) with benzoic acid is the final step in the biosynthetic pathway leading to the production of cocaine in Erythoxylum coca. Here we report the identification of a member of the BAHD family of plant acyltransferases as cocaine synthase. The enzyme is capable of producing both cocaine and cinnamoylcocaine via the activated benzoyl-or cinnamoyl-Coenzyme A thioesters, respectively. Cocaine synthase activity is highest in young developing leaves, especially in the palisade parenchyma and spongy mesophyll. These data correlate well with the tissue distribution pattern of cocaine as visualized with antibodies. Matrix-assisted laser-desorption ionization mass spectral imaging revealed that cocaine and cinnamoylcocaine are differently distributed on the upper versus lower leaf surfaces. Our findings provide further evidence that tropane alkaloid biosynthesis in the Erythroxylaceae occurs in the above-ground portions of the plant in contrast with the Solanaceae, in which tropane alkaloid biosynthesis occurs in the roots.

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“…To monitor protein elution from columns during purification, we developed a radiochemical assay for CST and CPLT 14 C]-labeled hydroxycinnamic acid using recombinant tobacco (Nicotiana tabacum) 4-coumarate:CoA ligase (4CL) as described by Beuerle and Pichersky (2002). The enzymatic hydroxycinnamoyl-CoA ester synthesis proceeded at room temperature in Tris-HCl buffer (50 mm, pH 7.5) or in potassium phosphate buffer (50 mm, pH 7) with near equal efficiency.…”

Section: Radiolabeled Acyltransferase Assaymentioning

confidence: 99%

Differential Production of meta Hydroxylated Phenylpropanoids in Sweet Basil Peltate Glandular Trichomes and Leaves Is Controlled by the Activities of Specific Acyltransferases and Hydroxylases

et al. 2002

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Sweet basil (Ocimum basilicum) peltate glandular trichomes produce a variety of small molecular weight phenylpropanoids, such as eugenol, caffeic acid, and rosmarinic acid, that result from meta hydroxylation reactions. Some basil lines do not synthesize eugenol but instead synthesize chavicol, a phenylpropanoid that does not contain a meta hydroxyl group. Two distinct acyltransferases, p-coumaroyl-coenzyme A:shikimic acid p-coumaroyl transferase and p-coumaroyl-coenzyme A:4-hydroxyphenyllactic acid p-coumaroyl transferase, responsible for the production of p-coumaroyl shikimate and of p-coumaroyl 4-hydroxyphenyllactate, respectively, were partially purified and shown to be specific for their substrates. p-Coumaroyl-coenzyme A:shikimic acid p-coumaroyl transferase is expressed in basil peltate glands that are actively producing eugenol and is not active in glands of noneugenol-producing basil plants, suggesting that the levels of this activity determine the levels of synthesis of some meta-hydroxylated phenylpropanoids in these glands such as eugenol. Two basil cDNAs encoding isozymes of cytochrome P450 CYP98A13, which meta hydroxylates p-coumaroyl shikimate, were isolated and found to be highly similar (90% identity) to the Arabidopsis homolog, CYP98A3. Like the Arabidopsis enzyme, the basil enzymes were found to be very specific for p-coumaroyl shikimate. Finally, additional hydroxylase activities were identified in basil peltate glands that convert p-coumaroyl 4-hydroxyphenyllactic acid to its caffeoyl derivative and p-coumaric acid to caffeic acid.Basil (Ocimum basilicum) plants synthesize significant quantities of phenylpropanoid derivatives that contain an hydroxyl group at the meta position on the aromatic ring. Several of these compounds, such as eugenol, rosmarinic acid, and caffeic acid (Fig. 1), are found in high concentrations in specialized structures that are located on the surface of the aerial parts of the plant and are known as peltate glandular trichomes (glands; Gang et al., 2001). These specialized metabolites have been found in other plant species as well, but the nature of the enzymes that catalyze the meta hydroxylation has so far remained poorly understood.One of the difficulties in identifying the enzymes catalyzing the 3-hydroxylations in the phenylpropanoid pathways is that they appear to be found at very low abundance in the tissues examined (Petersen, 1997). We have recently shown (Gang et al., 2001) that, as in peppermint (Mentha ϫ piperita; Gershenzon et al., 1992;McCaskill and Croteau, 1995), the basil gland cells can be removed from the plant and studied in isolation from the rest of the plant, greatly facilitating biochemical and molecular investigations of a single, fully differentiated cell type (Gershenzon et al., 1992;McCaskill et al., 1992;McCaskill and Croteau, 1995;Lange et al., 2000;Gang et al., 2001). Furthermore, because these gland cells are so highly specialized for production of plantspecialized metabolites, the enzymes in these metabolic pathways are highly express...

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Enzymatic Synthesis and Purification of Aromatic Coenzyme A Esters

Cited by 134 publications

References 28 publications

Identification of an Arabidopsis Feruloyl-Coenzyme A Transferase Required for Suberin Synthesis

Identification of an Arabidopsis Feruloyl-Coenzyme A Transferase Required for Suberin Synthesis

The Last Step in Cocaine Biosynthesis Is Catalyzed by a BAHD Acyltransferase

Differential Production of meta Hydroxylated Phenylpropanoids in Sweet Basil Peltate Glandular Trichomes and Leaves Is Controlled by the Activities of Specific Acyltransferases and Hydroxylases

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