A novel acyltransferase from cotyledons of tomato (Lycopersicon esculentum Mill.), which catalyzes the transfer of caffeic acid from chlorogenic acid (5-O-caffeoylquinic acid) to glucanc and galactaric acids, was purified with a 2400-fold enrichment and a 4% recovery. The enzyme showed specific activities (theoretical Vi., per milligram of protein) of 625 nanokatals (caffeoylglucaric acid formation) and 310 nanokatals (caffeoylgalactaric acid formation). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis it gave an apparent Mr of 40,000, identical to the value obtained by gel filtration column chromatography. Highest activity was found at pH 5.7, which was constant over a range of 20 to 120 millimolar K-phosphate. The isoelectric point of the enzyme was at pH 5.75. The reaction temperature optimum was at 380C and the apparent energy of activation was calculated to be 57 kilojoules per mole. The apparent K, values were 0.4 millimolar for glucaric acid, 1.7 millimolar for galactaric acid, and with both acceptors as second substrates 20 millimolar for chlorogenic acid. The relative ratio of the V1,,IKm values for glucaric acid and galactaric acid was found to be 100:12. Substratecompetition experiments support the conclusion that one single enzyme is responsible for both the glucaric and galactaric acid ester formation with marked preference for glucaric acid. It is proposed that the enzyme be called chlorogenic acid:glucaric acid O-caffeoyltransferase (EC 2.3.1.-). The three caffeic aciddependent enzyme activities involved in the formation of the glucaric and galactaric acid esters, the chlorogenic acid:glucaric acid caffeoyltransferase as the key activity as well as the caffeic acid:CoA ligase and the caffeoyl-CoA:quinic acid caffeoyltransferase as the preceding activities, were determined. The time course of changes in these activities were followed during development of the seedling in the cotyledons and growth of the young plant in the first and second leaf. The results from tomato seedlings suggest a sequential appearance of these enzymes.have been investigated with regard to their enzymic synthesis. One reason this biochemical area has been neglected to date might be that since the discovery of the mechanism of the biosynthesis of a caffeic acid (3,4-bishydroxycinnamic acid) ester ofquinic acid, chlorogenic acid (5-0-caffeoylquinic acid; 8), this has been widely accepted as the mechanism for all other known HCA esters. For Stockigt and Zenk showed in 1974 (22) that in tobacco cell cultures the formation of chlorogenic acid proceeds via the caffeoyl-CoA thioester as acyl donor. However, it has been shown that beside this commonly found mechanism, l-O-acyl glucosides, whose formation is catalyzed by UDP-glucose-dependent glucosyltransferases, may also act as HCA donors (1). Moreover, alternative esterifications (transacylations) may also be possible. For example, chlorogenic acid, which is common in Asteraceae, Solanaceae, and Rubiaceae (13) may act as an acyl donor molecule for caffeoyltran...

We have isolated an enzyme classified as chlorogenate: glucarate caffeoyltransferase (CGT) from seedlings of tomato (Solanum lycopersicum) that catalyzes the formation of caffeoylglucarate and caffeoylgalactarate using chlorogenate (5-O-caffeoylquinate) as acyl donor. Peptide sequences obtained by trypsin digestion and spectrometric sequencing were used to isolate the SlCGT cDNA encoding a protein of 380 amino acids with a putative targeting signal of 24 amino acids indicating an entry of the SlCGT into the secretory pathway. Immunogold electron microscopy revealed the localization of the enzyme in the apoplastic space of tomato leaves. Southern blot analysis of genomic cDNA suggests that SlCGT is encoded by a single-copy gene. The SlCGT cDNA was functionally expressed in Nicotiana benthamiana leaves and proved to confer chlorogenate-dependent caffeoyltransferase activity in the presence of glucarate. Sequence comparison of the deduced amino acid sequence identified the protein unexpectedly as a GDSL lipase-like protein, representing a new member of the SGNH protein superfamily. Lipases of this family employ a catalytic triad of Ser-Asp-His with Ser as nucleophile of the GDSL motif. Site-directed mutagenesis of each residue of the assumed respective SlCGT catalytic triad, however, indicated that the catalytic triad of the GDSL lipase is not essential for SlCGT enzymatic activity. SlCGT is therefore the first example of a GDSL lipase-like protein that lost hydrolytic activity and has acquired a completely new function in plant metabolism, functioning in secondary metabolism as acyltransferase in synthesis of hydroxycinnamate esters by employing amino acid residues different from the lipase catalytic triad.Plant metabolism is characterized by the formation of a vast number of secondary compounds, brought about by gene families coding for enzymes that modify various phenolic, terpenoid, alkaloid, or polyketide skeletons by oxidation and reduction as well as by methylation, glycosylation, prenylation, and acylation. Most of the phenolic structures in plants are synthesized via the shikimate/hydroxycinnamate pathway, which feeds into different types of hydroxycinnamate (HCA) 4 sidechain reactions (1). Among them are extensions with formation of additional ring systems (e.g. flavonoids or stilbenes), degradation (e.g. hydroxybenzoates), reduction (e.g. hydroxycinnamyl alcohols feeding into lignin biosynthesis), oxidation and lactonization (e.g. coumarins), and conjugation with a wide range of different primary and secondary compounds to form esters or amides. Syntheses of HCA conjugates are catalyzed by hydroxycinnamoyltransferases that play a decisive role in catalyzing the formation of complex patterns of HCA esters (2). Such a pattern, for example, was recently identified from Brassica napus seeds and exhibited a mixture of sinapate esters containing choline, malate, mono-and disaccharides, as well as flavonoid glycosides and an unusual cyclic spermidine amide (3).Tomato (Solanum lycopersicum) leaves accumulate caf...

The phenylpropane metabolism of tomato (Lycopersicon escukntm Mill) cotyledons was investigated. The (10,18) and glucaric acid (3). To date two mechanisms for the formation of such esters have been described (1); one involving the hydroxycinnamoyl-CoA thioester and the other one the l-O-(hydroxycinnamic acid)-acyl glucoside. The former mechanism was first shown to operate in the formation of chlorogenic acid (12), the 5-0-caffeoylquinic acid (6), in Nicotiana cell-suspension cultures and the latter one in the formation ofthe sinapic acid (4-hydroxy-3,5-dimethoxycinnamic acid) ester of malate (17) in Raphanus cotyledons. Both pathways can even lead to the same product, depending on the source of enzyme used, since recently it was shown that Ipomoea root tissue catalyzes the formation of chlorogenic acid from 1-0-caffeoylglucose ( 19,20).We studied the metabolism of hydroxycinnamic acid conjugates in tomato expecting one of the two described biosynthetic mechanisms to be operative in the formation of caffeoylglucaric acid. Earlier work ( 11) had shown that chlorogenic acid in tomato was formed via the caffeoyl-CoA thioester. However, all attempts to find an analogous reaction for the formation of caffeoylglucaric-acid, as well as experiments using 1 -O-caffeoylglucose as possible acyl donor, failed. The latter mechanism was considered possible for the formation of caffeoylglucaric acid in Cestrum 'Supported by the Deutsche Forschungsgemeinschaft. leaves which contain metabolically active l-O-caffeoylglucose (9).Since the accumulation patterns of chlorogenic and caffeoylglucaric acids indicated a precursor-product relationship, we considered chlorogenic acid as the possible acyl donor as was found in the biosynthesis of isochlorogenic acid (3,5-di-O-caffeoylquinic acid) in Ipomoea root tissue (8). Our results show that the enzymic formation of caffeoylglucaric acid in tomato cotyledons proceeds exclusively with chlorogenic acid as the acyl donor.MATERIALS AND METHODS Plant Material. Tomato seeds (Lycopersicon esculentum Mill cv Moneymaker Spezialzucht) were obtained from Waltz, Stuttgart, FRG; seedlings were grown for 3 weeks in a growth chamber with a 14-h day at 24°C and 70% RH in a defined soil as described (15). Adult plants were grown in a greenhouse.Substrates. CoA (free acid), caffeic and chlorogenic acids (5-O-caffeoylquinic acid; IUPAC nomenclature [6]) were purchased from Fluka (Neu-Ulm, FRG), and quinic, glucaric, galactaric and gluconic acids from Merck (Darmstadt, FRG). CaffeoylCoA was synthesized chemically and identified according to Zenk and coworkers (4, 13) and purified by a modification (D Strack, H Keller, G Weissenbock, unpublished data) of a described method (7) involving the hydroxycinnamoyl-N-hydroxysuccinimide ester and subsequent transesterification of the hydroxycinnamoyl moiety onto CoA. Purification was achieved on a polyamide column (details not documented) by stepwise elution with water, methanol and increasing concentrations of aqueous NH40H in methanol (0.01%, 0.05%, 0.1%...

No abstract

Hydroxycinnamoyltransferase activities which catalyze the formation of O-hydroxycinnamoyl- (p-coumaroyl-, feruloyl-, and sinapoyl-)-glucaric acids via the corresponding 1-O-hydroxycinnamoyl- β-glucoses, and O-(p-coumaroyl)-quinic acid and O-(p-coumaroyl)-shikimic acid both via p-coumaroyl-CoA thioester have been isolated from leaves of Cestrum elegáns D. F. L. v. Schlechtendal. The enzymic activities involved could be classified as 1-O-hydroxycinnamoyl-βglucose : glucaric acid hydroxycinnamoyltransferase (EC 2.3.1.- ) and p-coumaroyl-CoA : quinic acid/shikimic acid hydroxycinnamoyltransferase (EC 2.3.1. - ). This is the first time that both the O-glucoside- and the S-CoA-dependent activities in phenolic acid-ester formation were found to be present in the same plant