Chlorogenic Acid Biosynthesis Research Articles

Cloning and Characterization of a cDNA Coding a Hydroxycinnamoyl-CoA Quinate Hydroxycinnamoyl Transferase Involved in Chlorogenic Acid Biosynthesis inLonicera japonica

The flower buds of Lonicera japonica are widely used in Chinese medicine for their anti-inflammatory properties. The reason why L. japonica has potent and significant effects is that it contains various active components, especially chlorogenic acid (CGA). In the present study, we isolated a hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) gene encoding a protein of 439 amino acids from L. japonica. The RT-PCR results showed that tissue distribution of HQT was in accordance with the pattern of CGA content. The corresponding cDNAs were expressed in Escherichia coli as fusion proteins. Recombinant HQT exhibited catalysis activity in esterification of quinic acid with caffeoyl-CoA to generate CGA. Purified proteins demonstrated similar kinetic properties as HQT isolated from other species. This work is of significance for further studies concerned with strengthening the efficacy of L. japonica by means of improved CGA content and exploring the biosynthesis of CGA in vitro.

Use of the growing environment as a source of variation to identify the quantitative trait transcripts and modules of co-expressed genes that determine chlorogenic acid accumulation

Developing Coffea arabica seeds accumulate large amounts of chlorogenic acids (CGAs) as a storage form of phenylpropanoid derivatives, making coffee a valuable model to investigate the metabolism of these widespread plant phenolics. However, developmental and environmental regulations of CGA metabolism are poorly understood. In the present work, the expression of selected phenylpropanoid genes, together with CGA isomer profiles, was monitored throughout seed development across a wide set of contrasted natural environments. Although CGA metabolism was controlled by major developmental factors, the mean temperature during seed development had a direct impact on the time-window of CGA biosynthesis, as well as on final CGA isomer composition through subtle transcriptional regulations. We provide evidence that the variability induced by the environment is a useful tool to test whether CGA accumulation is quantitatively modulated at the transcriptional level, hence enabling detection of rate-limiting transcriptional steps [quantitative trait transcripts (QTTs)] for CGA biosynthesis. Variations induced by the environment also enabled a better description of the phenylpropanoid gene transcriptional network throughout seed development, as well as the detection of three temporally distinct modules of quantitatively co-expressed genes. Finally, analysis of metabolite-to-metabolite relationships revealed new biochemical characteristics of the isomerization steps that remain uncharacterized at the gene level.

Production of novel antioxidative phenolic amides through heterologous expression of the plant’s chlorogenic acid biosynthesis genes in yeast

Phenolic esters like chlorogenic acid play an important role in therapeutic properties of many plant extracts. We aimed to produce phenolic esters in baker’s yeast, by expressing tobacco 4CL and globe artichoke HCT. Indeed yeast produced phenolic esters. However, the primary product was identified as N-(E)- p-coumaroyl-3-hydroxyanthranilic acid by NMR. This compound is an amide condensation product of p-coumaric acid, which was supplied to the yeast, with 3-hydroxyanthranilic acid, which was unexpectedly recruited from the yeast metabolism by the HCT enzyme. N-(E)- p-coumaroyl-3-hydroxyanthranilic acid has not been described before, and it shows structural similarity to avenanthramides, a group of inflammation-inhibiting compounds present in oat. When applied to mouse fibroblasts, N-(E)- p-coumaroyl-3-hydroxyanthranilic acid induced a reduction of intracellular reactive oxygen species, indicating a potential therapeutic value for this novel compound.

Isolation and mapping of a C3′H gene (CYP98A49) from globe artichoke, and its expression upon UV-C stress

Globe artichoke represents a natural source of phenolic compounds with dicaffeoylquinic acids along with their biosynthetic precursor chlorogenic acid (5-caffeoylquinic acid) as the predominant molecules. We report the isolation and characterization of a full-length cDNA and promoter of a globe artichoke p-coumaroyl ester 3'-hydroxylase (CYP98A49), which is involved in both chlorogenic acid and lignin biosynthesis. Phylogenetic analyses demonstrated that this gene belongs to the CYP98 family. CYP98A49 was also heterologously expressed in yeast, in order to perform an enzymatic assay with p-coumaroylshikimate and p-coumaroylquinate as substrates. Real Time quantitative PCR analysis revealed that CYP98A49 expression is induced upon exposure to UV-C radiation. A single nucleotide polymorphism in the CYP98A49 gene sequence of two globe artichoke varieties used for genetic mapping allowed the localization of this gene to linkage group 10 within the previously developed maps.

The isolation and mapping of a novel hydroxycinnamoyltransferase in the globe artichoke chlorogenic acid pathway

BackgroundThe leaves of globe artichoke and cultivated cardoon (Cynara cardunculus L.) have significant pharmaceutical properties, which mainly result from their high content of polyphenolic compounds such as monocaffeoylquinic and dicaffeoylquinic acid (DCQ), and a range of flavonoid compounds.ResultsHydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase (HQT) encoding genes have been isolated from both globe artichoke and cultivated cardoon (GenBank accessions DQ915589 and DQ915590, respectively) using CODEHOP and PCR-RACE. A phylogenetic analysis revealed that their sequences belong to one of the major acyltransferase groups (anthranilate N-hydroxycinnamoyl/benzoyltransferase). The heterologous expression of globe artichoke HQT in E. coli showed that this enzyme can catalyze the esterification of quinic acid with caffeoyl-CoA or p-coumaroyl-CoA to generate, respectively, chlorogenic acid (CGA) and p-coumaroyl quinate. Real time PCR experiments demonstrated an increase in the expression level of HQT in UV-C treated leaves, and established a correlation between the synthesis of phenolic acids and protection against damage due to abiotic stress. The HQT gene, together with a gene encoding hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase (HCT) previously isolated from globe artichoke, have been incorporated within the developing globe artichoke linkage maps.ConclusionA novel acyltransferase involved in the biosynthesis of CGA in globe artichoke has been isolated, characterized and mapped. This is a good basis for our effort to understand the genetic basis of phenylpropanoid (PP) biosynthesis in C. cardunculus.

Regulation of polyphenols accumulation by combined overexpression/silencing key enzymes of phyenylpropanoid pathway

There is a growing interest in the metabolic engineering of plant with increased desirable polyphenols such as chlorogenic acid (CGA) and rutin. In this study, the effects of overexpression of both phenylalanine ammonia lyase (AtPAL2), the first enzyme of the phenylpropanoid pathway, and hydroxycinnamoyl-CoA quinate:hydroxycinnamoyl transferase (NtHQT), the last enzyme of CGA biosynthesis, and the overexpression of AtPAL2 together with silencing of NtHQT were investigated in tobacco. Transgenic tobacco plants overexpressing AtPAL2 showed two and five times increases of CGA and rutin levels than the wild-type (WT) plants, respectively. Overexpression of NtHQT further increases the accumulation of CGA in the AtPAL2 plants to about three times than that of the WT level, while silencing of NtHQT in AtPAL2 plants results in about 12 times increase in rutin level than that of the WT plants. Simultaneous overexpression of phenylalanine ammonia lyase (PAL) and overexpression/silencing HQT could be used for the production of functional food with increased polyphenols.

Chlorogenic acid facilitates root hair formation in lettuce seedlings.

Root hairs, which arise from root epidermal cells, are tubular structures that increase the efficiency of water absorption and nutrient uptake. A low pH (pH 4) medium induced root hair formation in lettuce (Lactuca sativa L.) seedlings, and the decapitation of shoots inhibited root hair formation. The addition of shoot extract to the medium restored root hair formation in the decapitated lettuce seedlings. These results suggest that factors essential to the formation of root hairs may be present in the shoot. We purified one factor from the shoot that facilitates root hair formation. This factor was identified as chlorogenic acid (CGA), a common polyphenol in higher plants. The presence of exogenous CGA in the medium induced root hair formation in decapitated lettuce seedlings at pH 4.0 and in intact lettuce seedlings at pH 6.0. The optimum concentration of CGA for root hair formation was identified as 10(-5) M. Decapitation of the shoots reduced the CGA content in the roots to approximately one-third that in intact plants. Application of the CGA biosynthesis inhibitor L-alpha-aminooxy-beta-phenylpropionic acid (AOPP, 10(-6) M) to intact seedlings grown at pH 4.0 reduced both the CGA content of the roots and the total amount of root hairs. The addition of exogenous CGA restored root hair formation in intact seedlings treated with AOPP. These results suggest that CGA is essential for root hair formation in lettuce seedlings.

Modulation of chlorogenic acid biosynthesis in Solanum lycopersicum; consequences for phenolic accumulation and UV-tolerance

Chlorogenic acid (CGA) is one of the most abundant phenolic compounds in tomato (Solanum lycopersicum). Hydroxycinnamoyl CoA quinate transferase (HQT) is the key enzyme catalysing CGA biosynthesis in tomato. We have studied the relationship between phenolic accumulation and UV-susceptibility in transgenic tomato plants with altered HQT expression. Overall, increased CGA accumulation was associated with increased UV-protection. However, the genetic manipulation of HQT expression also resulted in more complex alterations in the profiles of phenolics. Levels of rutin were relatively high in both HQT gene-silenced and HQT-overexpressing plants raised in plant growth tunnels. This suggests plasticity in the flux along different branches of phenylpropanoid metabolism and the existence of regulatory mechanisms that direct the flow of phenolic precursors in response to both metabolic parameters and environmental conditions. These changes in composition of the phenolic pool affected the relative levels of UV-tolerance. We conclude that the capability of the phenolic compounds to protect against potentially harmful UV radiation is determined both by the total levels of phenolics that accumulate in leaves as well as by the specific composition of the phenolic profile.

Biosynthesis of Chlorogenic Acids in Growing and Ripening Fruits of Coffea arabica and Coffea canephora Plants

Chlorogenic acids are major secondary metabolites found in coffee seeds. The accumulation of chlorogenic acids and free quinic acids was studied in Coffea arabica cv. Tall Mokka and Coffea canephora seeds. Growth stages are specified from I to V, corresponding to rapid expansion and pericarp growth (I), endosperm formation (II), mature (green) (III), ripening (pink) (IV), and fully ripened (red) (V) stages. We detected monocaffeoylquinic acids (3CQA, 4CQA and 5CQA), dicaffeoylquinic acids (3,4diCQA, 3,5diCQA and 4,5diCQA) and a monoferuloylquinic acid (5FQA) in whole fruits (stage I), pericarps and seeds. The most abundant chlorogenic acid was 5CQA, which comprised 50-60% of the total of C. arabica and 45-50% of C. canephora seeds. The content of dicaffeoylquinic acid, mainly 3,5-diCQA, was high in C. canephora. A high content of 5FQA was found in seeds of stages III to V, especially in C. canephora. Total chlorogenic acids were accumulated up to 14 mg per fruit in C. arabica and 17 mg in C. canephora, respectively. In contrast, free quinic acid varied from 0.4-2.0 mg (C. arabica) and 0.2-4.0 mg (C. canephora) per fruit during growth. High biosynthetic activity of 5CQA, which was estimated via the incorporation of [U-14C]phenylalanine into chlorogenic acids, was found in young fruits (perisperm and pericarp) in stage I, and in developing seeds (endosperm) in stages II and III. The biosynthetic activity of chlorogenic acids was clearly reduced in ripening and ripe seeds, especially in C canephora. Transcripts of PAL1, C3'H and CCoAMT, three genes related to the chlorogenic acid biosynthesis, were detected in every stagè of growth, although the amounts were significantly less in stage V. Of these genes, CCoAMT, a gene for FQA biosynthesis, was expressed more weakly in stage I. The transcript level of CCoAMT was higher in seeds than in pericarp, but the reverse was found in PAL1. The pattern of expression of genes for the CQA and FQA synthesis is roughly related to the estimated biosynthetic activity, and to the accumulation pattern of chlorogenic acids.

Isolation and functional characterization of a cDNA coding a hydroxycinnamoyltransferase involved in phenylpropanoid biosynthesis in Cynara cardunculus L

BackgroundCynara cardunculus L. is an edible plant of pharmaceutical interest, in particular with respect to the polyphenolic content of its leaves. It includes three taxa: globe artichoke, cultivated cardoon, and wild cardoon. The dominating phenolics are the di-caffeoylquinic acids (such as cynarin), which are largely restricted to Cynara species, along with their precursor, chlorogenic acid (CGA). The scope of this study is to better understand CGA synthesis in this plant.ResultsA gene sequence encoding a hydroxycinnamoyltransferase (HCT) involved in the synthesis of CGA, was identified. Isolation of the gene sequence was achieved by using a PCR strategy with degenerated primers targeted to conserved regions of orthologous HCT sequences available. We have isolated a 717 bp cDNA which shares 84% aminoacid identity and 92% similarity with a tobacco gene responsible for the biosynthesis of CGA from p-coumaroyl-CoA and quinic acid. In silico studies revealed the globe artichoke HCT sequence clustering with one of the main acyltransferase groups (i.e. anthranilate N-hydroxycinnamoyl/benzoyltransferase). Heterologous expression of the full length HCT (GenBank accession DQ104740) cDNA in E. coli demonstrated that the recombinant enzyme efficiently synthesizes both chlorogenic acid and p-coumaroyl quinate from quinic acid and caffeoyl-CoA or p-coumaroyl-CoA, respectively, confirming its identity as a hydroxycinnamoyl-CoA: quinate HCT. Variable levels of HCT expression were shown among wild and cultivated forms of C. cardunculus subspecies. The level of expression was correlated with CGA content.ConclusionThe data support the predicted involvement of the Cynara cardunculus HCT in the biosynthesis of CGA before and/or after the hydroxylation step of hydroxycinnamoyl esters.

Functional characterization of two p-coumaroyl ester 3′-hydroxylase genes from coffee tree: evidence of a candidate for chlorogenic acid biosynthesis

Chlorogenic acid (5-CQA) is one of the major soluble phenolic compounds that is accumulated in coffee green beans. With other hydroxycinnamoyl quinic acids (HQAs), this compound is accumulated in particular in green beans of the cultivated species Coffea canephora. Recent work has indicated that the biosynthesis of 5-CQA can be catalyzed by a cytochrome P450 enzyme, CYP98A3 from Arabidopsis. Two full-length cDNA clones (CYP98A35 and CYP98A36) that encode putative p-coumaroylester 3'-hydroxylases (C3'H) were isolated from C. canephora cDNA libraries. Recombinant protein expression in yeast showed that both metabolized p-coumaroyl shikimate at similar rates, but that only one hydroxylates the chlorogenic acid precursor p-coumaroyl quinate. CYP98A35 appears to be the first C3'H capable of metabolising p-coumaroyl quinate and p-coumaroyl shikimate with the same efficiency. We studied the expression patterns of both genes on 4-month old C. canephora plants and found higher transcript levels in young and in highly vascularized organs for both genes. Gene expression and HQA content seemed to be correlated in these organs. Histolocalization and immunolocalization studies revealed similar tissue localization for caffeoyl quinic acids and p-coumaroylester 3'-hydroxylases. The results indicated that HQA biosynthesis and accumulation occurred mainly in the shoot tip and in the phloem of the vascular bundles. The lack of correlation between gene expression and HQA content observed in some organs is discussed in terms of transport and accumulation mechanisms.

Phenolic compounds in coffee

Phenolic compounds are secondary metabolites generally involved in plant adaptation to environmental stress conditions. Chlorogenic acids (CGA) and related compounds are the main components of the phenolic fraction of green coffee beans, reaching levels up to 14 % (dry matter basis). These compounds have a number of beneficial health properties related to their potent antioxidant activity as well as hepatoprotective, hypoglycemic and antiviral activities. The main groups of CGA found in green coffee beans include caffeoylquinic acids, dicaffeoylquinic acids, feruloylquinic acids, p-coumaroylquinic acids and mixed diesters of caffeic and ferulic acids with quinic acid, each group with at least three isomers. During coffee processing, CGA may be isomerized, hydrolyzed or degraded into low molecular weight compounds. The high temperatures of roasting also produce transformation of part of CGA into quinolactones and, along with other compounds, melanoidins. This review focuses on the chemical characteristics, biosynthesis, and distribution of CGA and related compounds in coffee. The influence of genetic, physiological and environmental factors as well as processing on the chemical composition of coffee beans is discussed. The impact of CGA composition of green coffee on cup quality is also approached. Despite the existence of substantial published information on the total levels of CGA in coffee, more research is needed on the composition of minor phenolic compounds and specific CGA isomers (and related substances) in green and roasted coffee beans, as well as their impact on coffee quality.

Postharvest Variation in Apple (Malus × domestica Borkh.) Flavonoids following Harvest, Storage, and 1-MCP Treatment

The impact of 1-methylcyclopropene (1-MCP) on the synthesis and retention of flavonoid compounds during storage and ripening of red Delicious (Malus x domestica Borkh.) apples was investigated. Numerous anthocyanins, flavonols, flavan-3-ols, and a hydroxycinnamic acid from three different fruit harvest maturities were monitored after a 120 day storage and 1 week shelf life period using high-performance liquid chromatography/diode array detector analysis. The total flavonoid concentration was 5% greater in fruit treated with 1-MCP, whereas chlorogenic acid levels were 24% lower. All compounds analyzed increased in concentration during fruit harvest; however, the anthocyanins generally declined after storage, while chlorogenic acid levels increased. 1-MCP treatment resulted in the retention of anthocyanins in the latter stages of storage but did not affect the flavonols and flavan-3-ols. Chlorogenic acid biosynthesis from early and optimal fruit harvest maturities was greatly inhibited by 1-MCP during storage and the 1 week shelf life period. However, 1-MCP did not affect chlorogenic acid concentrations in late-harvested fruit. Results suggest that 1-MCP may inhibit the activity of phenylalanine ammonia-lyase and subsequent biosynthesis of flavonoid compounds. However, because very little postharvest biosynthesis of flavonoids occurs in apples, 1-MCP treatment may be useful for maintaining some of the intrinsic flavonoid levels of red Delicious apples, if applied at the proper harvest maturity.

Genetic mapping of a caffeoyl-coenzyme A 3-O-methyltransferase gene in coffee trees. Impact on chlorogenic acid content.

Chlorogenic acids (CGA) are involved in the bitterness of coffee due to their decomposition in phenolic compounds during roasting. CGA mainly include caffeoyl-quinic acids (CQA), dicaffeoyl-quinic acids (diCQA) and feruloyl-quinic acids (FQA), while CQA and diCQA constitute CGA sensu stricto (CGA s.s.). In the two cultivated species Coffea canephora and Coffea arabica, CGA s.s. represents 88% and 95% of total CGA, respectively. Among all enzymes involved in CGA biosynthesis, caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is not directly involved in the CGA s.s. pathway, but rather in an upstream branch leading to FQA through feruloyl-CoA. We describe how a partial cDNA corresponding to a CCoAOMT encoding gene was obtained and sequenced. Specific primers were designed and used for studying polymorphism and locating the corresponding gene on a genetic map obtained from an interspecific backcross between Coffea liberica var. Dewevrei and Coffea pseudozanguebariae. Offspring of this backcross were also evaluated for the chlorogenic acid content in their green beans. A 10% decrease was observed in backcross progenies that possess one C. pseudozanguebariae allele of the CCoAOMT gene. This suggests that CGA s.s. accumulation is dependent on the CCoAMT allele present and consequently on the activity of the encoded isoform, whereby CGA accumulation increases as the isoform activity decreases. Possible implications in coffee breeding are discussed.

Duplicate Loci as QTL

The C-glycosyl flavone maysin is an important component of corn earworm (Helicoverpa zea Boddie) (CEW) resistance in maize (Zea mays L.) silks. Chlorogenic acid (CGA), a product of the phenylpropanoid pathway, has also been implicated in CEW antibiosis. The transition from the 9-carbon phenylpropanoid to the 15-carbon flavonoid pathway represents a potentially important regulation point in C-glycosyl flavone biosynthesis. The enzyme chalcone synthase (CHS) catalyzes this reaction and is represented by a set of duplicate loci in maize, colorless2 (c2) and white pollenl (whpl ). Chromosomal regions of the maize genome have been identified as quantitative trait loci (QTL) for maysin synthesis that correspond to these two loci. Our objective was to investigate the role of CHS in flavone and CGA biosynthesis. We tested c2 and whpl as candidate loci using three related F 2 populations segregating for both structural and regulatory loci. Silks were harvested and analyzed from each population for flavonoid and phenylpropanoid content. Phenotypic data was combined with linkage maps generated for each population to conduct statistical and QTL analysis. Our results (i) support earlier findings of the requirement of a functional pericarp color1 (p1) allele for C-glycosyl fiavone biosynthesis, ( ii) demonstrate that variation at the c2 region on chromosome 4 influences both maysin and CGA levels in silks, (iii) reveal a dosage-dependent relationship between c2 and whpl in which the whp1 region acts cooperatively with functional c2 to increase flavones and partially compensates for nonfunctional c2, and (iv) demonstrate that mutations in c2 and whpl shunt intermediates from maysin synthesis to CGA. These results demonstrate that CHS function is a regulatory focal point of substrate flow between the flavone and phenylpropanoid pathways.

Two Loci Exert Major Effects on Chlorogenic Acid Synthesis in Maize Silks

Chlorogenic acid (CGA) in maize (Zea mays L.) silks has been implicated in resistance to corn earworm (Helicoverpa zea Boddie) with a mechanism similar to the flavone, maysin. However, the genetic basis of CGA synthesis is poorly understood. Our goal was to identify candidate loci affecting the biosynthesis of CGA using quantitative trait locus (QTL) analyses on three F 2 populations: (A619 × Mp708)F 2 , (A619 × Mo6)F 2 , and (Mo6 x Mp708)F 2 . Chlorogenic acid and flavone contents in silks were measured, linkage maps generated, and significant loci identified with composite interval mapping (CIM) and ANOVA multiple-effects models for the three populations. Of the QTLs detected, two exerted major effects; one corresponding to the p1 locus, and the other a novel locus we named qtl2. The main effect of the functional allele at the p1 locus was to increase both CGA and flavones, while the positive allele at the qtl2 locus only increased CGA. An epistatic interaction between p1 and qtl2 alleles in the (A619 × Mo6)F 2 population caused an increase in CGA with a concomitant decrease in flavones. The rare ability of Mo6 and Mp708 to synthesize CGA in excess of flavones is primarily a result from the effects at the qtl2 locus.

4-Coumaroyl Coenzyme A 3-Hydroxylase Activity from Cell Cultures ofLithospermum erythrorhizonand Its Relationship to Polyphenol Oxidase

A 4-coumaroyl-CoA 3-hydroxylase activity was purified 4600-fold from cell cultures ofLithospermum erythrorhizon.The enzyme showed a molecular mass of 42,400 ± 1700 Da in gel chromatography and required ascorbate, NADH, or NADPH as cofactors. 4-Coumaroyl-CoA, 4-coumarate,p-cresol, and several other phenolic substances, but not tyrosine, were accepted as substrates for the hydroxylation. Besides hydroxylase activity, the enzyme showed diphenol oxidase activity. Both activities were inhibited by diethyldithiocarbamate or β-mercaptoethanol, although at different concentrations. The enzyme showed striking similarity to a 4-coumaroyl-glucose 3-hydroxylase from sweet potato (Ipomoe batatas) roots, which has reportedly been purified to homogeneity and identified as a specific enzyme of chlorogenic acid biosynthesis. Close examination and comparison to a commercially available polyphenol oxidase, however, suggest that the enzyme activities purified from bothLithospermumand sweet potato are polyphenol oxidases rather than specific enzymes of secondary metabolism.

Correlation between Biosynthesis of Chlorogenic Acid and Flowering in Asparagus Seedlings Induced by Carbamate Compounds

The flowering of Asparagus seedlings induced by carbamate compounds we had developed was triggered when the chemicals were applied in such a way that they were active during the period of shoot differentiation, i.e., 4 to 10 days after seeding. The rate of flowering was closely correlated to the decrease in the chlorogenic acid content of the bud primordium caused by the carbamate treatment. Cytokinins stimulated metabolism in the buds and decreased the inhibitory effect of the carbamates on it. The site of action of the chemicals appears to be somewhere on the metabolic pathway that leads to the synthesis of chlorogenic acid.

Purification and characterization of p-coumaroyl- dglucose hydroxylase of sweet potato ( Ipomoea batatas) roots

p-Coumaroyl- d-glucose hydroxylase in sweet potato ( Ipomoea batatas Lam.) has been purified to apparent electrophoretic homogeneity using a combination of anion- and cation-exchange, hydrophobic and gel filtration chromatography. The purified enzyme was a monomer with a molecular weight of 33,000 and p I of 8.3. The purified enzyme showed not only hydroxylase activity but also polyphenol oxidase activity. l-Ascorbic acid was the best electron donor for the hydroxylation reaction, which had an optimum pH of 7.0. The enzyme hydroxylated p-coumaroyl- d-glucose, p-coumaric acid, and p-cresol but did not act on o-coumaric acid, m-coumaric acid, 4-hydroxy-3-methoxycinnamic acid, p-hydroxy-benzoic acid or l-tyrosine. While the enzyme utilized p-coumaroyl- d-glucose and p-coumaric acid equally at pH 7.0, it hydroxylated only p-coumaroyl- d-glucose at pH 5.5. The enzyme oxidized diphenols such as d,l-(3,4-dihydroxyphenyl) alanine and caffeic acid, but exhibited no clear pH optimum in this reaction characteristic of polyphenol oxidase. Both the hydroxylase and the polyphenol oxidase activities were strongly inhibited by β-mercaptoethanol, diethyldithiocarbamate, KCN, and p-coumaric acid (in concentrations higher than 5 m m). Ammonium sulfate and sodium chloride activated the hydroxylase activity but not the polyphenol oxidase activity of the enzyme. The enzyme activity and l-ascorbic acid contents changed in a manner suggesting their involvements in chlorogenic acid biosynthesis during incubation of sliced sweet potato root tissues.

Isolation of 1-<italic>O-trans-p</italic>-Coumaroyl-rβ-D-glucopyranose from Sweet Potato Roots and Examination of Its Role in Chlorogenic Acid Biosynthesis

Isolation of 1-<italic>O-trans-p</italic>-Coumaroyl-rβ-D-glucopyranose from Sweet Potato Roots and Examination of Its Role in Chlorogenic Acid Biosynthesis