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1.
This communication reviews data on the accumulation and biosynthesis of rosmarinic acid in cell suspension cultures ofColeus blumei. The influence of the medium, mainly the carbohydrate source on growth and rosmarinic acid production in these cell cultures is described. The biosynthetic pathway of rosmarinic acid was elucidated inColeus blumei cell cultures: eight enzymatic activities are involved in the transformation of the precursors phenylalanine and tyrosine to the end product rosmarinic acid.Abbreviations CAH cinnamic acid 4-hydroxylase - 4CL 4-coumarate:CoA ligase - HPPR hydroxyphenylpyruvate reductase - 3-H hydroxycinnamoyl-hydroxyphenyllactate 3-hydroxylase - 3-H hydroxycinnamoyl-hydroxyphenyllactate 3-hydroxylase - PAL phenylalanine ammonia-lyase - RAS rosmarinic acid synthase (hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyl transferase) - TAT tyrosine aminotransferase  相似文献   

2.
Berger A  Meinhard J  Petersen M 《Planta》2006,224(6):1503-1510
Purification of rosmarinic acid synthase (hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyltransferase) from suspension cells of Coleus blumei Benth. (Lamiaceae) by fractionated ammonium sulphate precipitation, hydrophobic interaction chromatography and two affinity chromatography steps led to the identification of peptide sequences, which enabled a PCR-based approach to isolate the full-length cDNA encoding this enzyme. The open reading frame of the cDNA had a length of 1290 base pairs encoding a protein of 430 amino acid residues with a molecular mass of 47,932 Da with typical characteristics of an acyltransferase of the BAHD superfamily. The cDNA was heterologously expressed in Escherichia coli. The enzyme displayed the activity of rosmarinic acid synthase using 4-coumaroyl- and caffeoyl-coenzyme A and 4-hydroxyphenyllactate as well as 3.4-dihydroxyphenyllactate as substrates. Shikimic acid and quinic acid were not able to serve as hydroxycinnamoyl acceptors. This therefore is the first report of the cDNA-cloning of a rosmarinic acid synthase.  相似文献   

3.
Suspension cultures of Coleus blumei accumulate very high amounts of rosmarinic acid, an ester of caffeic acid and 3,4-dihydroxyphenyllactate, in medium with elevated sucrose concentrations. Since the synthesis of this high level of rosmarinic acid occurs in only five days of the culture period, the activities of the enzymes involved in the biosynthesis are very high. Therefore all the enzymes necessary for the formation of rosmarinic acid from the precursors phenylalanine and tyrosine could be isolated from cell cultures of Coleus blumei: phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, hydroxycinnamoyl:CoA ligase, tyrosine aminotransferase, hydroxyphenylpyruvate reductase, rosmarinic acid synthase and two microsomal 3- and 3-hydroxylases. The main characteristics of these enzymes of the proposed biosynthetic pathway of rosmarinic acid will be described.Abbreviations DHPL 3,4-dihydroxyphenyllactate - DHPP 3,4-dihydroxyphenylpyruvate - pHPL 4-hydroxyphenyllactate - pHPP 4-hydroxyphenylpyruvate - RA rosmarinic acid  相似文献   

4.
Agrobacteria mediated Coleus blumei tumour tissues were cultured in vitro on MS medium. Sixteen diversified transformed callus cultures were maintained for several years in the absence of plant growth regulators and antibiotics without affecting the growth rate. Rosmarinic acid was detected spectrophotometrically in all tissue lines but in different quantities. The highest rosmarinic acid accumulation detected was 11% of dry tissue mass. The relation between culture growth and rosmarinic acid production was investigated in three callus lines. The lines showed different rosmarinic acid accumulation in relation to their growth rate; it was either parallel or inversely related to the tissue growth. The effects of certain medium constituents on the callus growth and rosmarinic acid accumulation were examined in four tumour cell lines. Addition of 4% or 5% sucrose stimulated rosmarinic acid synthesis and decreased callus growth. Nitrogen reduction to one half or one quarter of initial concentration did not affect rosmarinic acid synthesis and decreased callus growth in three lines, while it increased rosmarinic acid accumulation and callus growth in one line. Addition of 0.1 mg/l Phe stimulated rosmarinic acid production in two lines but had little effect on the rosmarinic acid level in others. Rosmarinic acid production was significantly improved on modified macronutrients, where the Ac2 line produced 16.5 mg of rosmarinic acid per tube (0.2 g of dry wt) after being in culture for 35 days.  相似文献   

5.
Pyrroline-5-carboxylate reductase (P5CR) catalyzes the reduction of Delta1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)(+). The enzymatic cycle between P5C and proline is very important in many physiological and pathological processes. Human P5CR was over-expressed in Escherichia coli and purified to homogeneity by chromatography. Enzymatic assays of the wild-type protein were carried out using 3,4-dehydro-L-proline as substrate and NAD(+) as cofactor. The homopolymer was characterized by cross-linking and size exclusion gel filtration chromatography. Human P5CR was crystallized by the hanging-drop vapor-diffusion method at 37 degrees C. Diffraction data were obtained to a resolution of 2.8A and were suitable for high resolution X-ray structure determination.  相似文献   

6.
7.
Maike Petersen 《Planta》1993,191(1):18-22
Rosmarinic acid synthase from cell cultures of Coleus blumei Benth. was purified to apparent homogeneity by fractionated ammonium sulfate precipitation (60–80% saturation), hydrophobic interaction chromatography, affinity chromatography and gel filtration. This purification procedure resulted in a 225-fold-enriched specific enzyme activity with a yield of 9%. The protein preparation was apparently pure according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis. The apparent molecular mass determined by gel filtration and SDS-PAGE was 77 kDa, indicating that rosmarinic acid synthase is a monomeric enzyme.Abbreviations DTT dithiothreitol - HIC hydrophobic interaction chromatography - RA rosmarinic acid - RAS rosmarinic acid synthase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis The financial support of the Deutsche Forschungsgemeinschaft is gratefully acknowledged. Two-dimensional gel electrophoresis was done with the help of Dr. Guy Bauw, University of Gent, Belgium.  相似文献   

8.
Rosmarinic acid   总被引:25,自引:0,他引:25  
Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. It is commonly found in species of the Boraginaceae and the subfamily Nepetoideae of the Lamiaceae. However, it is also found in species of other higher plant families and in some fern and hornwort species. Rosmarinic acid has a number of interesting biological activities, e.g. antiviral, antibacterial, antiinflammatory and antioxidant. The presence of rosmarinic acid in medicinal plants, herbs and spices has beneficial and health promoting effects. In plants, rosmarinic acid is supposed to act as a preformed constitutively accumulated defence compound. The biosynthesis of rosmarinic acid starts with the amino acids L-phenylalanine and L-tyrosine. All eight enzymes involved in the biosynthesis are known and characterised and cDNAs of several of the involved genes have been isolated. Plant cell cultures, e.g. from Coleus blumei or Salvia officinalis, accumulate rosmarinic acid in amounts much higher than in the plant itself (up to 36% of the cell dry weight). For this reason a biotechnological production of rosmarinic acid with plant cell cultures has been proposed.  相似文献   

9.
Fungal metabolism of biphenyl.   总被引:9,自引:0,他引:9       下载免费PDF全文
gamma-Glutamyl phosphate reductase, the second enzyme of proline biosynthesis, catalyses the formation of l-glutamic acid 5-semialdehyde from gamma-glutamyl phosphate with NAD(P)H as cofactor. It was purified 150-fold from crude extracts of Pseudomonas aeruginosa PAO 1 by DEAE-cellulose chromatography and hydroxyapatite adsorption chromatography. The partially purified preparation, when assayed in the reverse of the biosynthetic direction, utilized l-1-pyrroline-5-carboxylic acid as substrate and reduced NAD(P)(+). The apparent K(m) values were: NAD(+), 0.36mm; NADP(+), 0.31mm; l-1-pyrroline-5-carboxylic acid, 4mm with NADP(+) and 8mm with NAD(+); P(i), 28mm. 3-(Phosphonoacetylamido)-l-alanine, a structural analogue of gamma-glutamyl phosphate, inhibited this enzyme competitively (K(i)=7mm). 1-Pyrroline-5-carboxylate reductase (EC 1.5.1.2), the third enzyme of proline biosynthesis, was purified 56-fold by (NH(4))(2)SO(4) fractionation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography. It reduced l-1-pyrroline-5-carboxylate with NAD(P)H as a cofactor to l-proline. NADH (K(m)=0.05mm) was a better substrate than NADPH (K(m)=0.02mm). The apparent K(m) values for l-1-pyrroline-5-carboxylate were 0.12mm with NADPH and 0.09mm with NADH. The 3-acetylpyridine analogue of NAD(+) at 2mm caused 95% inhibition of the enzyme, which was also inhibited by thio-NAD(P)(+), heavy-metal ions and thiol-blocking reagents. In cells of strain PAO 1 grown on a proline-medium the activity of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase was about 40% lower than in cells grown on a glutamate medium. No repressive effect of proline on 1-pyrroline-5-carboxylate reductase was observed.  相似文献   

10.
R. Wagner  K. G. Wagner 《Planta》1985,165(4):532-537
The enzyme activities of the pyridine-nucleotide cycle, which transform nicotinic acid mononucleotide (NaMN) into NAD, have been characterized. The investigations were based on the extraction of protein, its purification on disposable gel-filtration columns, and determination of the enzymatic activities by high-performance liquid chromatography techniques. The latter technique avoided the synthesis and use of radioactive precursors. The NaMN-adenylyltransferase which converts NaMN into NaAD (nicotinic acid adenine dinucleotide) and NAD-synthetase which converts NaAD into NAD were characterized by their kinetic parameters and their specific activities in different tobacco tissues. This is the first report on NAD-synthetase from tissue of a higher plant. It was found that NAD-synthetase accepted both glutamine and asparagine for the amide transfer. Adenylyltransfer also occured with nicotinamide mononucleotide (NMN) which was transformed to NAD, whereas the glutamine-dependent amidation was only observed with NaAD. Thus, an additional route for the synthesis of NAD (NaMNNMNNAD) obviously does not exist. A comparison of the enzyme activities in tobacco tissues with different capacities for the synthesis of nicotine showed that, in contrast to quinolinic acid phosphoribosyltransferase whose activity was strictly correlated with the nicotine content, only NaMN-adenylyltransferase showed a smooth correlation, whereas NAD-synthetase was not affected at all.Abbreviations HPLC high-performance liquid chromatography - QA quinolinic acid - NaMN nicotinic acid mononucleotide - NaAD nicotinic acid adenine dinucleotide - NMN nicotinamide mononucleotide  相似文献   

11.
12.
Suspension cultures of Coleus blumei were characterized with respect to growth and rosmarinic acid formation in media with different sugars and various sugar concentrations. Sucrose is the sugar with the highest stimulating effect on growth and rosmarinic acid accumulation, followed by glucose and fructose. The sugar alcohol mannitol cannot be metabolized by the plant cells. Sucrose is cleaved into glucose and fructose by the Coleus cells. Sucrose concentrations from 1 to 5% have an increasing positive effect on growth and rosmarinic acid synthesis in the cell cultures with a maximum rosmarinic acid content of 12% of the dry weight in medium with 5% sucrose; in medium with 6% sucrose rosmarinic acid accumulation obviously did not reach its highest level in the culture period of 14 days. A very high yield of rosmarinic acid (2 mg ml-1 suspension) could also be achieved by maintaining a sucrose concentration of 2% during the whole culture period. The start of rosmarinic acid synthesis by the cell cultures seems to be regulated by the growth limitation when a nutrient, e.g. phosphate is depleted from the medium. The rate of rosmarinic acid accumulation is related to the amount of carbon left in the medium when growth ceases.Abbreviations RA rosmarinic acid  相似文献   

13.
A. Razzaque  B. E. Ellis 《Planta》1977,137(3):287-291
Cell suspension cultures of Coleus blumei Benth. have been found to accumulate 8–11% of their dry weight as rosmarinic acid (-O-caffeoyl-3,4-dihydroxyphenyl-lactic acid). Actively-growing tissue converts >20% of exogenously supplied phenylalanine and tyrosine to the caffeoyl ester and this high rate of synthesis coincides with an increase in phenylalanine ammonia-lyase specific activity. Administration to the cultures of known phenylpropanoid precursors of rosmarinic acid failed to enhance the latter's production and in some cases inhibited it.Abbreviations RA rosmarinic acid (-O-caffeoyl-3,4-dihydroxyphenyllactic acid - DOPA dihydroxyphenylalanine - PAL phenylalanine ammonialyase - DOPL dihydroxyphenyl-lactic acid  相似文献   

14.
Vogelsang K  Schneider B  Petersen M 《Planta》2006,223(2):369-373
Cell suspension cultures of the hornwort Anthoceros agrestis Paton (Anthocerotaceae) were cultivated and characterized in CB-media containing 2 and 4% sucrose. The suspension cells accumulated rosmarinic acid up to 5.1% of the cell dry weight as well as caffeoyl-4'-hydroxyphenyllactate. Moreover, a more hydrophilic compound was detected which was isolated and identified as rosmarinic acid 3'-O-beta-D-glucoside, a new rosmarinic acid derivative. This new rosmarinic acid derivative was found up to 1.0% of the cell dry weight in suspension cells of A. agrestis.  相似文献   

15.
Biogenesis of rosmarinic acid in Mentha   总被引:1,自引:0,他引:1       下载免费PDF全文
The biogenesis of rosmarinic acid (alpha-O-caffeoyl-3,4-dihydroxyphenyl-lactic acid), the second most common ester of caffeic acid in the plant kingdom, was studied in Mentha arvense and Mentha piperita. Administration of (14)C-labelled compounds showed that, whereas the caffeoyl moiety was formed from phenylalanine via cinnamic acid and p-coumaric acid, the 3,4-dihydroxyphenyl-lactic acid moiety was formed from tyrosine and 3,4-dihydroxyphenylalanine. Time-course studies and the use of labelled rosmarinic acid showed that endogenous rosmarinic acid had a low turnover rate. The caffeoyl moiety did not appear to contribute to the formation of insoluble polymers, as has been suggested for chlorogenic acid in other plants.  相似文献   

16.
A biosynthetic pathway for rosmarinic acid is proposed. This pathway is deduced from studies of the enzymes detectable in preparations from suspension cells of Coleus blumei. Phenylalanine is transformed to 4-coumaroyl-CoA by the enzymes of the general phenylpropanoid pathway: phenylalanine ammonia-lyase (EC 4.3.1.5), cinnamic acid 4-hydroxylase (EC 1.14.13.11) and hydroxycinnamic acid:CoA ligase (EC 6.2.1.12). Tyrosine is metabolized to 4-hydroxyphenyllactate by tyrosine aminotransferase (EC 2.6.1.5) and hydroxyphenylpyruvate reductase. The ester can be formed from 4-coumaroyl-CoA and 4-hydroxyphenyllactate by the catalytic activity of rosmarinic acid synthase with concomitant release of CoA. Microsomal hydroxylase activities introduce the hydroxyl groups at positions 3 and 3 of the aromatic rings of the ester 4-coumaroyl-4-hydroxyphenyllactate giving rise to rosmarinic acid.Abbreviations Caf-pHPL caffeoyl-4-hydroxyphenyllactate - DHPL 3,4-dihydroxyphenyllactic acid - pC-DHPL 4-coumaryl-3,4-dihydroxyphenyllactate - pC-pHPL 4-coumaryl-4-hydroxyphenyllactate - pHPL 4-hydroxyphenyllactic acid - RA rosmarinic acid The financial support of the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie is gratefully acknowledged.  相似文献   

17.
R. Wagner  F. Feth  K. G. Wagner 《Planta》1986,167(2):226-232
In order to elucidate the NAD-recycling pathway the following enzyme activities have been characterized in different tobacco tissues and in tomato root: NAD pyrophosphatase, nicotinamide mononucleotide (NMN)/nicotinic acid mononucleotide (NaMN) glycohydrolases, nicotinamidase and nicotinic acid phosphoribosyltransferase. The investigations were performed with protein extracts purified by gel filtration and enzymatic activities were determined by high-performance liquid chromatography methods. The kinetic parameters of the different enzymes from tobacco root and their specificity are reported. The data are in favor of the so-called pyridine-nucleotide cycle VI (NADNMNnicotinamidenicotinic acidNaMNnicotinic acid adenine dinucleotideNAD). In the nicotine-producing tobacco root a further direct route leading from NaMN to nicotinic acid is proposed. These data are reconciled with the assumption that it is nicotinic acid which is provided by the pyridine-nucleotide cycle for the synthesis of nicotine.Abbreviations HPLC high-performance liquid chromatography - Na nicotinic acid - NaAD nicotinic acid adenine dinucleotide - NaMN nicotinic acid mononucleotide - NMN nicotinamide mononucleotide - PRPP 5-phosphoribosyl-1-pyrophosphate This contribution is dedicated to Professor Augustin Betz on the occasion of his 65th birthday  相似文献   

18.
Prephenate aminotransferase (PAT) from rosmarinic acid-producing cell cultures of Anchusa officinalis has been purified to apparent electrophoretic homogeneity using a combination of high-performance anion-exchange, chromatofocusing, and gel filtration chromatography. The purified enzyme has a native molecular weight of 220,000 and subunit molecular weights of 44,000 and 57,000, indicating a possible alpha 2 beta 2 subunit structure. The purified PAT displays high affinity for prephenate (Km = 80 microM) but could also utilize other aromatic alpha-keto acids at less than 20% the rate with prephenate. L-Aspartate (Km = 80 microM) is about three times as effective as L-glutamate as amino-donor substrate. Anchusa PAT is not subject to feedback inhibition from L-phenylalanine or tyrosine, but its activity is affected by a rosmarinic acid metabolite, 3,4-dihydroxyphenyllactic acid.  相似文献   

19.
B. E. Ellis  S. Remmen  G. Goeree 《Planta》1979,147(2):163-167
Phenylalanine ammonia-lyase from an over-producer strain of Coleus blumei Benth. cell cultures accumulating high levels of rosmarinic acid (RA) has been shown to possess no special feed-back sensitivity to RA or its precursors. No tyrosine-3-hydroxylase activity could be detected in culture extracts and no specific inhibitors of tyrosine incorporation into RA were found. L--aminooxy--phenyl propionic acid, however, was effective in specifically blocking phenylalanine incorporation. This block also led to an accumulation of label from tyrosine in 4-hydroxyphenyllactic acid rather than in 3,4-dihydroxyphenylalanine (DOPA) or 3,4-dihydroxyphenyllactic acid. These observations require a re-evaluation of the possible role of DOPA as a major biogenic precursor to RA.Abbreviations AOPP -aminooxy--phenylpropionic acid - DOPA 3,4-dihydroxyphenylalanine - RA rosmarinic acid (-O-caffeoyl-3,4-dihydroxyphenyllactic acid) - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)  相似文献   

20.
A unique enzyme that catalyzes the formation of 4-hydroxyaniline from 4-aminobenzoic acid was found in the homogenate of Agaricus bisporus. The enzyme was prepared from the homogenate by (NH4)2SO4 fractionation, gel filtration and ion-exchange chromatography. The products formed from 4-aminobenzoic acid by the enzyme were shown to be 4-hydroxyaniline and CO2. The reaction required FAD, NAD(P)H and O2. These results indicate that the enzyme is a new FAD-dependent monooxygenase.  相似文献   

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