Properties and Activity Changes of Chlorogenic Acid:Glucaric Acid

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 caffeoyltransferases. This has been demonstrated by protein preparations catalyzing the formation of isochlorogenic acid, i.e. 3,5-bis-0-caffeoylquinic acid, in sweet potato (10, 37) and of caffeoylglucaric and -galactaric acids in tomato (26). In this paper, we report the purification and characterization of CGT from tomato. We present evidence that this enzyme is strictly donor specific in the acylation of glucaric and galactaric acids. Furthermore, we show that the time course of its activity, measured in cotyledons of the seedling and in the first and the second leaf of the young plant, correlates well with the accumulation patterns of caffeoylglucaric and -galactaric acids in these organs. MATERIALS AND METHODS