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Characterization of rosmarinic acid synthase from cell cultures of Coleus blumei
003l-9422/9I s3.00+ 0.00 Q 1991Pcrgamon Press plc
Phyrochemistry.Vol. 30, No. 9, pp. 2877-2881.1991 Printed in Great Britain.
CHARACTERIZATION OF ROSMARINIC ACID SYNTHASE CELL CULTURES OF COLEUS BLUMEZ
FROM
MAIKE S. PETERSEN Institut fiir Entwicklungs- und Molekularbiologie der Pflanzen, D-4000
Diisseldorf
Heinrich-Heine-Universitit 1, Germany
Diisseldorf.
Universiti%sstrasse
1,
(Received in reoised form 12 February 199 1)
Key Word Index-Coleus blumei; Lamiaceae; rosmarinic acid biosynthesis; acyltransferase; rosmarinic acid; hydroxycinnamic acid esters; hydroxyphenyllactic
acid.
Abstract-Rosmarinic acid synthase (RAS) catalyses the transesterilication reaction of CoA-activated cinnamic acids, e.g. 4coumaroyl- or caffeoyl-CoA, and hydroxyphenyllactic acids, e.g. 4-hydroxy- or 3,4-dihydroxyphenyllactic acid, during the biosynthesis of rosmarinic acid. The enzyme isolated from suspension cultures of Coleus blumei is soluble and has an optimal pH of 7.g7.5. Michaelis-Menten kinetics were observed with respect to the substrates 4coumaroyl- and catTeoyl-CoA with Km-values of 20 and 33 PM, respectively. Only R( +)-stereoisomers of the hydroxyphenyllactic acid are accepted by RAS. For 4-hydroxy- and 3+dihydroxyphenyllactic acid the &-values were 0.17 and 0.37 mM, respectively. The RAS reaction was inhibited by 4-hydroxymercuribenzoate, the S( -)stereoisomer of 3,4-dihydroxyphenyllactic acid, hydroxyphenylpyruvates and rosmarinic acid, whereas cinnamic acids were not inhibitory. Coenzyme A showed a noncompetitive inhibition. The reverse reaction of RAS, the splitting of rosmarinic acid with help of coenzyme A into caffeoyl-CoA and 3,4_dihydroxyphenyllactate, was readily detected. The kinetic data for this reaction are &,-values of 310 pM for coenzyme A and 15 PM for rosmarinic acid.
Rosmarinic acid (RA), an ester of caffeic acid and 3,4dihydroxyphenyllactic acid (Fig. l), is one of the most abundant caffeic acid esters occurring in plants. It is mainly found in species of the families Boraginaceae and Lamiaceae, but also occurs in Blechnum ferns [I]. Investigations on the biosynthesis of rosmarinic acid were started by Ellis and Towers [2], who identified the amino acids phenylalanine and tyrosine as the precursors for rosmarinic acid in plants of Mentha. In these experiments the radioactively labelled phenylalanine was only incorporated into the caffeic acid moiety of rosmarinic acid, whereas tyrosine and DOPA were the only precursors for the 3,4_dihydroxyphenyllactic acid part. Rosmarinic acid is also produced in suspension cultures of several species of the Lamiaceae and Boraginaceae, e.g. Anchusa ojicinalis [3], Orthosiphon aristatus [S], and Coleus blumei [S, 63. In suspension cultures of Coleus blumei the rosmarinic acid content can be influenced by the composition of the culture medium, particularly the sucrose concentration [6,7J Rosmarinic acid contents of up to 19% of the cell dry weight were found in Coleus blumei suspension cultures grown in medium with 4% sucrose [7]. Investigation of the enzymes involved in the biosynthesis of rosmarinic acid have been conducted in cell cultures of Coleus blumei and Anchusa oficinalis. Phenylalanine is transformed by the enzymes of the general phenylpropanoid pathway to 4-coumaroyl- and/or caffeoyl-CoA, and the activities of phenylalanine ammonia lyase, cinnamic acid 4-hydroxylase and hydroxycinnamoyl CoA-ligase could be detected in enzyme preparations
from suspension cells of Coleus blumei [S]. Incorporation of tyrosine into RA is initiated by tyrosine aminotransferase [9, 101. The resulting 4-hydroxyphenylpyruvate (pHPP) is reduced by a hydroxyphenylpyruvate reductase to 4-hydroxyphenyllactic acid (pHPL) [7, 113. This enzyme also accepts 3,4_dihydroxyphenylpyruvate (DHPP) as substrate, and forms 3,4_dihydroxyphenyllactic acid (DHPL). The transesterification reaction of caffeoyl- or 4coumaroyl-CoA and Chydroxy- or 3,4dihydroxyphenyllactic acid is catalysed by rosmarinic acid synthase (RAS), as previously reported [7J We now report a more extensive characterization of this enzyme.
RESULTS
Rosmarinic acid content of the cells and actioity of rosmarinic acid synthase during the growth cycle of the culture
The rosmarinic acid content of the cells and the activity of RAS were monitored during a growth cycle of the suspension cultures of Coleus blumei in medium with 2% (CB,) and 4% (CB,) sucrose. Rosmarinic acid accumulation was very low in medium with 2% sucrose, but was strongly enhanced in CB,-medium (Fig. 2). The rosmarinic acid content of the cells increased from day 4 to 10 of the culture period and reached a maximum level at more than 13% of the cell dry weight at day 10. The specific RAS activity from cells grown in CB,-medium increased from day 3 to 9 of the culture period and reached a maximum level of about 210 nkat g- ’ protein (Fig. 3). The enzyme activities in cells from medium with 2%
2877
2878
M. S. PETERSEN
Fig.
1. Reaction
of rosmarinic
acid synthase
R1=R2=OH R’=OH, R’=H,
2
4
6
caffeoyl-CoA
+ DHPL
4-coumaroyl-CoA
R’=OH
8
substrates
caffeoyl-CoA
R’=H
R’=R2=H
0
with different
4-coumaroyl-CoA
10
12
Characteristics
of rosmarinic
acid synthase
RAS is a soluble enzyme and can be precipitated by ammonium sulphate between 60 and 80% saturation. This precipitation step results in a four-fold increase of the specific activity of the enzyme. The addition of Polyclar AT, an adsorbent of phenolic compounds, during the enzyme preparation is necessary to remove rosmarinic acid from the enzyme extract, because this compound inhibits the RAS reaction (see below). The pH-optimum of RAS is at pH 7.K7.5 in 0.1 M potassium phosphate buffer. Half maximal activities are achieved at pH 6.5 and 8.5. In 0.1 M Tris-HCI buffer the pH-optimum differs slightly and the enzyme activities are lower than in potassium phosphate buffer. The stability of rosmarinic acid was tested in both buffer systems (0.1 M potassium phosphate and 0.1 M Tri-HCI) at overlapping pH intervals from pH 5.5-10 under the conditions of the enzyme assay. Under these conditions (10 min at 30”) rosmarinic acid was stable. The temperature optimum for
reaction
+ CoA
-+ Ccoumaroyl-DHPL
+ CoA
+ pHPL
+ 4-coumaroyl-pHPL
+ CoA
0
2
4
products:
acid + CoA
+ DHPL
14
sucrose were substantially lower, with a maximum acitivity of about 90 nkat g- ’ protein. The higher rosmarinic acid content in cells from CB,-medium is thus correlated with the elevated RAS activity.
+ rosmarinic
+ pH PL -+ calTeoyl-pHPL
Idays 1 Fig. 2. Growth ( W. 0) and RA accumulation (3.0) of suspenL sion cultures of Coleus hlumei during a culture period of 14 days in medium with 2% (m, 0) and 4% (0, 0) sucrose.
and the corresponding
6
8
10
12
14
[days 1 Fig. 3. Activity during a culture and 4% (0, 0) CoA ( W, 0)
of RAS from cell cultures of Coleus hlumei period of 14 days in medium with 2% ( n , II) sucrose. The enzyme was tested with catTeoyland 4-coumaroyl-CoA (3, 3) as substrates.
the RAS reaction was at 30”, when caffeoyl-CoA is used as a substrate, and at 40” with 4-coumaroyLCoA. RAS preparations could be stored at - 20” for several months without severe loss in enzyme activity. RAS activity was also stable for 5 hr at 30”. DTT and ascorbic acid were routinely added to the RAS assay. Ascorbic acid at 0.5 mM enhanced the yield of rosmarinic acid by 50%. This may be. due to the antioxidative activity of ascorbic acid, which protects the substrates and reaction products from oxidation. The RAS activity was also stimulated more than six-fold at a concentration of 10 mM of DTT. The RAS reaction in a standard assay was linear with
protein concentrations up to 15Opg of the ammonium sulphate precipitated protein, and with time up to 10 min. Phenolases and esterases were proven not to be active in standard assays for RAS. Substrates
RAS catalyses the transfer of cinnamic acids from the corresponding CoA-thioester to the aliphatic hydroxyl group of a hydroxyphenyllactic acid. If the substrates are
Rosmarinic acid synthase caffeoyl-CoA and 3,4dihydroxyphenyllactic acid, the reaction products are rosmarinic acid and coenzyme A. However, our enzyme preparations also accepted substrates other than caffeoyl-CoA and 3,4dihydroxyphenyllactic acid, in which case other rosmarinic acid-like esters were formed (Fig. 1). To our knowledge, most of these structures have not been identified in Go, although caffeoyl-4-hydroxyphenyllactate was found in traces in methanolic extracts from suspension cells of our Coleus cultures and has been reported as a natural constituent of Lycopus [ 123. Among the tyrosine-derived substrates, 4-hydroxyphenyllactate and 3,4-dihydroxyphenyllactate were accepted as substrates, as was 3-methoxy+hydroxyphenyllactate. 3,4-Dimethoxyphenyllactate and phenyllactate did not support a reaction, suggesting that the 4-hydroxyl-group might be necessary for RAS activity. For the substrates Chydroxyphenyllactate and 3,4dihydroxyphenyllactate, Michaelis-Menten kinetics were observed with saturation concentrations of 1 mM for pHPL and DHPL and apparent &-values of 0.17 and 0.37 mM, respectively. No substrate inhibition occurred with either compound. Hydroxyphenyllactic acids exist in two different stereoisomer forms. RAS accepted only the R( +)-stereoisomer of 3,4-dihydroxyphenyllactic acid, while the S( - )-stereoisomer inhibited the reaction. Unfortunately, only racemic 4hydroxyphenyllactate was available for testing. If, as for 3,4-dihydroxyphenyllactate, the S( - )-stereoisomer of 4-hydroxyphenyllactate also inhibits the reaction, the Km-value for 4-hydroxyphenyllactate could possibly be lower than the indicated concentration. The activity of RAS was routinely determined with caffeoyl- and 4-coumaroyl-CoA as substrates, but cinnamoyl-CoA was also accepted. With sinapoyl-CoA and feruloyl-CoA obviously esters were also formed as reaction products, but in very low concentrations. These reaction products, unfortunately, were not stable enough to allow a further characterization. The RAS reaction was saturated at 1OOpM caffeoylCoA and displayed an apparent K, value of 33 FM. With 4-coumaroyl-CoA, a strong substrate inhibition occurred and a saturation concentration could not be determined. The apparent K,-value for 4coumaroyl-CoA was ca 20 PM. Competition experiments with calfeoyl- and 4-coumaroyl-CoA applied simultaneously at different concentrations showed that the reaction with caffeoyl-CoA was much more strongly inhibited by 4-coumaroyl-CoA than was the reaction with 4-coumaroyl-CoA by caffeoyl-CoA. The mode of inhibition was competitive. 4-CoumaroylCoA, therefore, seems to be the preferred substrate for RAS.
Inhibition experiments
RAS is not inhibited by cinnamic, 4-coumaric and caffeic acid up to concentrations of 1 mM, whereas 3,4dihydroxy- and 4-hydroxyphenylpyruvate and the wrong stereoisomer of 3,4-dihydroxyphenyllactate reduced the rate of rosmarinic acid synthesis. 4-Hydroxymercuribenzoate completely blocked RAS activity at concentrations higher than 0.5 mM. Coenzyme A showed a prominent inhibitory effect. Kinetic studies of the inhibition mode revealed a non-competitive inhibition (Fig. 4). The inhibi-
2879
0
0.02
004
006
006
01
[s1 Fig. 4. Inhibition of RAS by 0 mM (0). 0.5 mM (C), 1 mM (Cl), 2 mM (m) and 5 mM (A) coenzyme A at different concentrations of caffeoyl-CoA. The inhibition mode was determined with help of Lineweaver-Burk diagrams and shows a non-competitive inhibition with inhibition constants K, = K,, = 1.7 mM.
tion constants Ki and Ki, were determined to be at 1.7 mM. A strong product inhibition occurred when rosmarinic acid was added to RAS tests with 4-coumaroyl-CoA as the substrate. At concentrations higher than 0.2 mM rosmarinic acid, the inhibition mode seemed to be an uncompetitive one. Reverse reaction of rosmarinic acid synthase RAS also catalysed the splitting of rosmarinic acid into caffeoyl-CoA and 3,4-dihydroxyphenyllactic acid, when coenzyme A was supplied. Caffeic acid did not appear among the products, showing that esterases were not responsible for the splitting of rosmarinic acid. This reverse reaction also showed Michaelis-Menten kinetics with apparent K, values of 15 PM for rosmarinic acid and 310pM for coenzyme A and was saturated at a rosmarinic acid concentration of 50 pM. For coenzyme A, saturation was only achieved at a concentration of ca 1 mM. Under standard reaction conditions, the influence of the reverse reaction of RAS can be neglected, although the high affinity of RAS for rosmarinic acid may account for the strong inhibitory effect of rosmarinic acid on the RAS reaction. DISCUSSION
Rosmarinic acid synthase catalyses a key step in rosmarinic acid biosynthesis. As in other biosynthetic pathways leading to caffeic acid esters, an activated cinnamic acid is necessary as precursor. In the case of rosmarinic acid biosynthesis, this is a CoA-thioester, as also described for the biosynthesis of flavonoids [13], chlorogenie acid [14, IS] and other cinnamic acid conjugates. Cinnamic acid glucose esters, as another possibility of an activated precursor, or the unactivated cinnamic acid itself did not lead to a synthesis of rosmarinic acid with enzyme preparations from cells of Coleus blumei. Cinnamic acid glucose esters have been described to serve as activated precursors in the biosynthesis of sinapoylmalate [ 163, sinapine [ 173, acylated betacyanins [ 181 and, in some species, of chlorogenic acid [ 193.
M. S.
2880
PETERSEN
The activity of RAS is positively correlated with the accumulation of rosmarinic acid during the culture cycle of suspension cultures of Coleus blumei. In medium with 4% sucrose, the RAS activity is substantially higher than in medium with 2% sucrose. Therefore, the synthetic rate for rosmarinic acid may be regulated by the enzyme activity of RAS. With the discovery of RAS and hydroxyphenylpyruvate reductase [73 the biosynthetic pathway of rosmarinic acid is nearly clarified. The cinnamic acid precursor is formed from phenylalanine by the well known enzymes of the general phenylpropanoid pathway, phenylalanine ammonia lyase, cinnamic acid 4-hydroxylase and hydroxycinnamoyl CoA-ligase, which could all be detected in enzyme preparations from suspension cells of Coleus blumei [8]. Tyrosine aminotransferase [9, IO] and hydroxyphenylpyruvate reductase [7] are involved in the synthesis of the tyrosine-derived precursor hydroxyphenyllactic acid. In the biosynthetic pathway for rosmarinic acid, the point at which both 3-hydroxyl-groups are introduced into the aromatic rings of the precursors or the ester itself is unclear. Experiments by Ellis et al. [203 showed that inhibition of PAL by the specific inhibitor aaminooxy+phenylpropionic acid resulted in an accumulation of 4-hydroxyphenyllactic acid in suspension
cultures of Coleus blumei. Therefore, the 3-hydroxylation might occur at a relatively late stage of the biosynthesis. Furthermore, RAS showed highest affinities for the monohydroxylated substrates 4-coumaroyl-CoA and 4hydroxyphenyllactic acid, and the V&K,,, values indicate that these might be the preferred substrates for RAS. In this model, an ester of 4-coumaric acid and 4-hydroxyphenyllactic acid might normally be the direct reaction product of RAS. If this is the case. the name rosmarinic acid synthase is not strictly correct for this enzyme, instead, 4coumaroyl-coenzyme A: 4-hydroxyphenyllactic acid 4-coumaroyl transferase would be the correct nomenclature. This hypothesis is further sustained by the detection of membrane-bound hydroxylating activities which introduce the 3-hydroxyl groups into the aromatic rings of less hydroxylated rosmarinic acid-like esters (Fig. l), forming rosmarinic acid (Petersen, unpublished results). This indicates that, perhaps, in rosmarinic acid biosynthesis the hydroxylation pattern of the aromatic rings is only established after the completion of the Cskeleton, as proposed for flavonoid [21-243 and chlorogenie acid biosynthesis [25, 261.
EXPERIMENTAL Cell cultures. Suspension
cultures of Coleus blumei Benth. were from a callus culture, which was a gift from Dr B. Ulbrich (Nattermann & Cie., Cologne, Germany). The cultures were maintained as described previously [73. Chemicab. Cinnamic acid CoAesters and 3+dihydroxyphenyllactic actd were prepd as described previously [7]. S( -)-3,4Dihydroxyphenyllactic acid was a gift from Dr N. Rao (Forschungszentrum Jiilich GmbH, Germany). Enzyme preparation. RAS was prepd from ‘I-day-old suspension cells of Coleus blumei, which were grown in medium with 4% sucrose. The cells were filtered under suction and homogenized with l/10 weight of Polyclar AT and I ml of 0.1 M K-Pi huger pH 7 with 1 mM DTTg- ’ fr. wt of the cells by an Ultra-Turrax (Janke & Kunkel, Staufen i. Br.. Germany) for 150 sec. The homogenate was filtered through a nylon screen (pore established
size 100 pm) and centrifuged for 15min at 48 000 y. The supernatant was used for a fractionated (NH&SO,-pptn with satd (NH&SO, soln which was adjusted to pH 7 with NH,OH. Most of the RAS activity was pptd between 60 and 80% satn of (NH&SO, and was sedimented by centrifugation at 48000 q for 20 min. The pellet was redissolved in butler and desalted on a Sephadex G25 column (Pharmacia). The desalted protein was stored at -20’ until it was used for enzyme assays. Protem determination. Protein concns were measured spectrophotometrically according to ref. L27] using bovine serum albumin as a standard. Assayfor rosmarinic acid synthase. The assay for RAS contained in a total vol. of 250 ~1 0.1 M K-Pi buffer pH 7:2.5 pmol DTT, 125 nmol ascorbic acid, 50 nmol caffeoyl-CoA or 4-coumaroyl-CoA, IOOnmol pHPL or DHPL and ca 5Opg of the (NH&SO,-pptd and desalted protein. The assay was incubated at 30” for 5 min and stopped by addition of20 ~1 of6 M HCI. The assays were extracted x 3 with 0.5 ml EtOAc each. After evapn of the EtOAc under vacuum the residue was redissolved in 300 ~1 MeOH-Hz0 (1: 1) adjusted to pH 3 with H,PO,. The reaction products were identified and quantified by HPLC-analysis on Shandon Hypersil ODS-column (290 x 4.6 mm, parttcle size 5 pm) with MeOH-H,O (I : 1) acidified with 100 ~1 H,PO, I- ’ as an eluent at a flow rate of 1.5 ml min-‘. The products were detected spectrophotometrically at 333 nm. The values for reaction products other than RA can only be given in relative umts, because standards are not available and the molar extinction coefficients arc not known. The reverse reactton of RAS was measured in an assay with a total vol. of 250~1 0.1 M K-Pi butfer pH7 containing 2.5pmol DIT, 125 nmol ascorbic actd, 5Onmol RA. 25Onmol CoA and 20~1 of the (NH,)$O,-pptd and desalted protem. The assay was incubated at M” for 10 min and stopped by addition of 20~1 of 6M HCI. Extraction and HPLC-analysis were performed as above, but with a linear elution gradient during IOmm from 20% MeOH IO 50% MeOH in H,O acidified with lOO$ H~PO,I~~’ at a flow rate of 1.5 mlmin-’ and detection at 280 nm. ldentifcation of the reaction productst Esters other than RA were synthesized enzymatically by supplying RAS with other cinnamic acid CoA-esters and/or hydroxyphenyllactic acids as substrates. For identification they were first purified by semiprep. HPLC on a Shandon Hypersil ODScolumn (290 x 8 mm, particle size 5 pm) with MeOH-H,O (1: I) acidified with 3 ml HOAc I- ’ as an eluent. After evapn of the eluent, the esters were cleaved enzymatically with 4mg Rhozyme HP-150 (Pollock and Pool Ltd. Reading, U.K.) in 200 ~1 NH,OAc buffer pH 6 at 30” for 4 hr. The reaction was stopped by addition of 50 ~1 6 M HCI and the assays were extracted x 3 with 0.5 ml EtOAc each. After cvapn of the solvent the residues were dissolved in MeOH-H,O (1.1) adjusted IO pH 3 with H,PO, and analysed by HPLC as described for the enzyme assay, but with detection at 280 nm. The products of the enzymatic ester cleavage were identified using cinnamic, Ccoumaric, caffeic, ferulic, and sinapic acid and pHPL and DHPL as authentic standards. Acknowledgement-
The
Forschungsgemeinschaft
financial support by the is gratefully acknowledged.
Dcutsche
REFERENCES 1. De-Eknamkul,
Vol. 4 Medicinal and Aromafic I (Bajaj, Y. P. S.. ed.), p. 310. Springer, Berlin.
Agriculture Hunts
W. and Ellis, B. E. (1983) in Biotechnology
and Forestry
in
Rosmarinic acid synthase 2. Ellis, B. E. and Towers, G. H. N. (1970) B&hem J. lla 291. 3. De-Eknamkul, W. and Ellis, B. E. (1984) Planto Med. 51,346. 4. Sumaryono, W., Proksch, P., Wray, V., Witte, L. and Hartmann, T. (1989) Planta Med. 55, 110. 5. Razraque, A. and Ellis, B. E. (1977) Planto 137, 287. 6. Zenk, M. H., El-Shagi, H. and Ulbrich, B. (1977) Noturwissenschajien 64, 585. 7. Petersen, M. and Alfermann, A. W. (1988) Z. Norurjorsch. 4% 501. 8. Karwatzki, B., Petersen, M. and Alfermann, A. W. (1989) Planta Med. 55, 663. 9. De-Eknamkul, W. and Ellis, B. E. (1987) Phytochemistry 26, 1941. 10. De-Eknamkul, W. and Ellis, B. E. (1987) Arch. Biochem. Biophys. 257, 430. 11. Hausler, E., Petersen, M. and Alfermann, A. W. (1990) PIanta Med. 56, 619620. 12. Gumbinger, H. G., Vahlensieck, U. and Winterhoff, H. (1990) Poster at the symposium on ‘Biology and Chemistry of Active Natural Substances’, Bonn. 13. Kreuzaler, F. and Hahlbrock, K. (1972) FEBS Letters 2& 69.
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14. Stiickigt, J. and Zenk, M. H. (1975) FEBS Letters 42, 131. 15. Rhodes, M. J. C. and Woohorton, L. S. C. (1976) Phytochemistry IS, 947. 16. Tkotz, N. and Strack. D. (1980) Z. Naturforsch. 3Sc, 835. 17. Griiwe, W. and Strack, D. (1986) Z. Naturjorsch. 41c, 28. 18. Bokem, M. and Strack, D. (1988) Planta 174, 101. 19. Villegas, R. J. A. and Kojima, M. (1986) J. Biol. Chem. Ml, 8729. 20. Ellis, B. E., Remmen, S. and Goeree, G. (1979) Planta 147, 163. 21. Stotz, G. and Forkmann. G. (1982) Z. Naturforsch, 37~. 19. 22. Forkmann, G., Heller, W. and Grisebach, H. (1980) Z. Naturforsch. 3Sc. 691. 23. Hagmann, M. L., Heller, W. and Grisebach, H. (1983) Eur. J. Biochem. 134,547. 24. Larson, R. L. and Bussard, J. B. (1986) Plant Physiol. 80,483. 25. Heller, W. and Kiihnl, T. (1985) Arch. Biochem Biophys. 241, 453. 26. Kuhnl, T., Koch, U., Heller, W. and Wellmann, E. (1987) Arch. Biochem. Biophys. 258, 226. 27. Bradford, M. M. (1976) Anal. B&hem. 72, 248.
Phyrochemistry.Vol. 30, No. 9, pp. 2877-2881.1991 Printed in Great Britain.
CHARACTERIZATION OF ROSMARINIC ACID SYNTHASE CELL CULTURES OF COLEUS BLUMEZ
FROM
MAIKE S. PETERSEN Institut fiir Entwicklungs- und Molekularbiologie der Pflanzen, D-4000
Diisseldorf
Heinrich-Heine-Universitit 1, Germany
Diisseldorf.
Universiti%sstrasse
1,
(Received in reoised form 12 February 199 1)
Key Word Index-Coleus blumei; Lamiaceae; rosmarinic acid biosynthesis; acyltransferase; rosmarinic acid; hydroxycinnamic acid esters; hydroxyphenyllactic
acid.
Abstract-Rosmarinic acid synthase (RAS) catalyses the transesterilication reaction of CoA-activated cinnamic acids, e.g. 4coumaroyl- or caffeoyl-CoA, and hydroxyphenyllactic acids, e.g. 4-hydroxy- or 3,4-dihydroxyphenyllactic acid, during the biosynthesis of rosmarinic acid. The enzyme isolated from suspension cultures of Coleus blumei is soluble and has an optimal pH of 7.g7.5. Michaelis-Menten kinetics were observed with respect to the substrates 4coumaroyl- and catTeoyl-CoA with Km-values of 20 and 33 PM, respectively. Only R( +)-stereoisomers of the hydroxyphenyllactic acid are accepted by RAS. For 4-hydroxy- and 3+dihydroxyphenyllactic acid the &-values were 0.17 and 0.37 mM, respectively. The RAS reaction was inhibited by 4-hydroxymercuribenzoate, the S( -)stereoisomer of 3,4-dihydroxyphenyllactic acid, hydroxyphenylpyruvates and rosmarinic acid, whereas cinnamic acids were not inhibitory. Coenzyme A showed a noncompetitive inhibition. The reverse reaction of RAS, the splitting of rosmarinic acid with help of coenzyme A into caffeoyl-CoA and 3,4_dihydroxyphenyllactate, was readily detected. The kinetic data for this reaction are &,-values of 310 pM for coenzyme A and 15 PM for rosmarinic acid.
Rosmarinic acid (RA), an ester of caffeic acid and 3,4dihydroxyphenyllactic acid (Fig. l), is one of the most abundant caffeic acid esters occurring in plants. It is mainly found in species of the families Boraginaceae and Lamiaceae, but also occurs in Blechnum ferns [I]. Investigations on the biosynthesis of rosmarinic acid were started by Ellis and Towers [2], who identified the amino acids phenylalanine and tyrosine as the precursors for rosmarinic acid in plants of Mentha. In these experiments the radioactively labelled phenylalanine was only incorporated into the caffeic acid moiety of rosmarinic acid, whereas tyrosine and DOPA were the only precursors for the 3,4_dihydroxyphenyllactic acid part. Rosmarinic acid is also produced in suspension cultures of several species of the Lamiaceae and Boraginaceae, e.g. Anchusa ojicinalis [3], Orthosiphon aristatus [S], and Coleus blumei [S, 63. In suspension cultures of Coleus blumei the rosmarinic acid content can be influenced by the composition of the culture medium, particularly the sucrose concentration [6,7J Rosmarinic acid contents of up to 19% of the cell dry weight were found in Coleus blumei suspension cultures grown in medium with 4% sucrose [7]. Investigation of the enzymes involved in the biosynthesis of rosmarinic acid have been conducted in cell cultures of Coleus blumei and Anchusa oficinalis. Phenylalanine is transformed by the enzymes of the general phenylpropanoid pathway to 4-coumaroyl- and/or caffeoyl-CoA, and the activities of phenylalanine ammonia lyase, cinnamic acid 4-hydroxylase and hydroxycinnamoyl CoA-ligase could be detected in enzyme preparations
from suspension cells of Coleus blumei [S]. Incorporation of tyrosine into RA is initiated by tyrosine aminotransferase [9, 101. The resulting 4-hydroxyphenylpyruvate (pHPP) is reduced by a hydroxyphenylpyruvate reductase to 4-hydroxyphenyllactic acid (pHPL) [7, 113. This enzyme also accepts 3,4_dihydroxyphenylpyruvate (DHPP) as substrate, and forms 3,4_dihydroxyphenyllactic acid (DHPL). The transesterification reaction of caffeoyl- or 4coumaroyl-CoA and Chydroxy- or 3,4dihydroxyphenyllactic acid is catalysed by rosmarinic acid synthase (RAS), as previously reported [7J We now report a more extensive characterization of this enzyme.
RESULTS
Rosmarinic acid content of the cells and actioity of rosmarinic acid synthase during the growth cycle of the culture
The rosmarinic acid content of the cells and the activity of RAS were monitored during a growth cycle of the suspension cultures of Coleus blumei in medium with 2% (CB,) and 4% (CB,) sucrose. Rosmarinic acid accumulation was very low in medium with 2% sucrose, but was strongly enhanced in CB,-medium (Fig. 2). The rosmarinic acid content of the cells increased from day 4 to 10 of the culture period and reached a maximum level at more than 13% of the cell dry weight at day 10. The specific RAS activity from cells grown in CB,-medium increased from day 3 to 9 of the culture period and reached a maximum level of about 210 nkat g- ’ protein (Fig. 3). The enzyme activities in cells from medium with 2%
2877
2878
M. S. PETERSEN
Fig.
1. Reaction
of rosmarinic
acid synthase
R1=R2=OH R’=OH, R’=H,
2
4
6
caffeoyl-CoA
+ DHPL
4-coumaroyl-CoA
R’=OH
8
substrates
caffeoyl-CoA
R’=H
R’=R2=H
0
with different
4-coumaroyl-CoA
10
12
Characteristics
of rosmarinic
acid synthase
RAS is a soluble enzyme and can be precipitated by ammonium sulphate between 60 and 80% saturation. This precipitation step results in a four-fold increase of the specific activity of the enzyme. The addition of Polyclar AT, an adsorbent of phenolic compounds, during the enzyme preparation is necessary to remove rosmarinic acid from the enzyme extract, because this compound inhibits the RAS reaction (see below). The pH-optimum of RAS is at pH 7.K7.5 in 0.1 M potassium phosphate buffer. Half maximal activities are achieved at pH 6.5 and 8.5. In 0.1 M Tris-HCI buffer the pH-optimum differs slightly and the enzyme activities are lower than in potassium phosphate buffer. The stability of rosmarinic acid was tested in both buffer systems (0.1 M potassium phosphate and 0.1 M Tri-HCI) at overlapping pH intervals from pH 5.5-10 under the conditions of the enzyme assay. Under these conditions (10 min at 30”) rosmarinic acid was stable. The temperature optimum for
reaction
+ CoA
-+ Ccoumaroyl-DHPL
+ CoA
+ pHPL
+ 4-coumaroyl-pHPL
+ CoA
0
2
4
products:
acid + CoA
+ DHPL
14
sucrose were substantially lower, with a maximum acitivity of about 90 nkat g- ’ protein. The higher rosmarinic acid content in cells from CB,-medium is thus correlated with the elevated RAS activity.
+ rosmarinic
+ pH PL -+ calTeoyl-pHPL
Idays 1 Fig. 2. Growth ( W. 0) and RA accumulation (3.0) of suspenL sion cultures of Coleus hlumei during a culture period of 14 days in medium with 2% (m, 0) and 4% (0, 0) sucrose.
and the corresponding
6
8
10
12
14
[days 1 Fig. 3. Activity during a culture and 4% (0, 0) CoA ( W, 0)
of RAS from cell cultures of Coleus hlumei period of 14 days in medium with 2% ( n , II) sucrose. The enzyme was tested with catTeoyland 4-coumaroyl-CoA (3, 3) as substrates.
the RAS reaction was at 30”, when caffeoyl-CoA is used as a substrate, and at 40” with 4-coumaroyLCoA. RAS preparations could be stored at - 20” for several months without severe loss in enzyme activity. RAS activity was also stable for 5 hr at 30”. DTT and ascorbic acid were routinely added to the RAS assay. Ascorbic acid at 0.5 mM enhanced the yield of rosmarinic acid by 50%. This may be. due to the antioxidative activity of ascorbic acid, which protects the substrates and reaction products from oxidation. The RAS activity was also stimulated more than six-fold at a concentration of 10 mM of DTT. The RAS reaction in a standard assay was linear with
protein concentrations up to 15Opg of the ammonium sulphate precipitated protein, and with time up to 10 min. Phenolases and esterases were proven not to be active in standard assays for RAS. Substrates
RAS catalyses the transfer of cinnamic acids from the corresponding CoA-thioester to the aliphatic hydroxyl group of a hydroxyphenyllactic acid. If the substrates are
Rosmarinic acid synthase caffeoyl-CoA and 3,4dihydroxyphenyllactic acid, the reaction products are rosmarinic acid and coenzyme A. However, our enzyme preparations also accepted substrates other than caffeoyl-CoA and 3,4dihydroxyphenyllactic acid, in which case other rosmarinic acid-like esters were formed (Fig. 1). To our knowledge, most of these structures have not been identified in Go, although caffeoyl-4-hydroxyphenyllactate was found in traces in methanolic extracts from suspension cells of our Coleus cultures and has been reported as a natural constituent of Lycopus [ 123. Among the tyrosine-derived substrates, 4-hydroxyphenyllactate and 3,4-dihydroxyphenyllactate were accepted as substrates, as was 3-methoxy+hydroxyphenyllactate. 3,4-Dimethoxyphenyllactate and phenyllactate did not support a reaction, suggesting that the 4-hydroxyl-group might be necessary for RAS activity. For the substrates Chydroxyphenyllactate and 3,4dihydroxyphenyllactate, Michaelis-Menten kinetics were observed with saturation concentrations of 1 mM for pHPL and DHPL and apparent &-values of 0.17 and 0.37 mM, respectively. No substrate inhibition occurred with either compound. Hydroxyphenyllactic acids exist in two different stereoisomer forms. RAS accepted only the R( +)-stereoisomer of 3,4-dihydroxyphenyllactic acid, while the S( - )-stereoisomer inhibited the reaction. Unfortunately, only racemic 4hydroxyphenyllactate was available for testing. If, as for 3,4-dihydroxyphenyllactate, the S( - )-stereoisomer of 4-hydroxyphenyllactate also inhibits the reaction, the Km-value for 4-hydroxyphenyllactate could possibly be lower than the indicated concentration. The activity of RAS was routinely determined with caffeoyl- and 4-coumaroyl-CoA as substrates, but cinnamoyl-CoA was also accepted. With sinapoyl-CoA and feruloyl-CoA obviously esters were also formed as reaction products, but in very low concentrations. These reaction products, unfortunately, were not stable enough to allow a further characterization. The RAS reaction was saturated at 1OOpM caffeoylCoA and displayed an apparent K, value of 33 FM. With 4-coumaroyl-CoA, a strong substrate inhibition occurred and a saturation concentration could not be determined. The apparent K,-value for 4coumaroyl-CoA was ca 20 PM. Competition experiments with calfeoyl- and 4-coumaroyl-CoA applied simultaneously at different concentrations showed that the reaction with caffeoyl-CoA was much more strongly inhibited by 4-coumaroyl-CoA than was the reaction with 4-coumaroyl-CoA by caffeoyl-CoA. The mode of inhibition was competitive. 4-CoumaroylCoA, therefore, seems to be the preferred substrate for RAS.
Inhibition experiments
RAS is not inhibited by cinnamic, 4-coumaric and caffeic acid up to concentrations of 1 mM, whereas 3,4dihydroxy- and 4-hydroxyphenylpyruvate and the wrong stereoisomer of 3,4-dihydroxyphenyllactate reduced the rate of rosmarinic acid synthesis. 4-Hydroxymercuribenzoate completely blocked RAS activity at concentrations higher than 0.5 mM. Coenzyme A showed a prominent inhibitory effect. Kinetic studies of the inhibition mode revealed a non-competitive inhibition (Fig. 4). The inhibi-
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0
0.02
004
006
006
01
[s1 Fig. 4. Inhibition of RAS by 0 mM (0). 0.5 mM (C), 1 mM (Cl), 2 mM (m) and 5 mM (A) coenzyme A at different concentrations of caffeoyl-CoA. The inhibition mode was determined with help of Lineweaver-Burk diagrams and shows a non-competitive inhibition with inhibition constants K, = K,, = 1.7 mM.
tion constants Ki and Ki, were determined to be at 1.7 mM. A strong product inhibition occurred when rosmarinic acid was added to RAS tests with 4-coumaroyl-CoA as the substrate. At concentrations higher than 0.2 mM rosmarinic acid, the inhibition mode seemed to be an uncompetitive one. Reverse reaction of rosmarinic acid synthase RAS also catalysed the splitting of rosmarinic acid into caffeoyl-CoA and 3,4-dihydroxyphenyllactic acid, when coenzyme A was supplied. Caffeic acid did not appear among the products, showing that esterases were not responsible for the splitting of rosmarinic acid. This reverse reaction also showed Michaelis-Menten kinetics with apparent K, values of 15 PM for rosmarinic acid and 310pM for coenzyme A and was saturated at a rosmarinic acid concentration of 50 pM. For coenzyme A, saturation was only achieved at a concentration of ca 1 mM. Under standard reaction conditions, the influence of the reverse reaction of RAS can be neglected, although the high affinity of RAS for rosmarinic acid may account for the strong inhibitory effect of rosmarinic acid on the RAS reaction. DISCUSSION
Rosmarinic acid synthase catalyses a key step in rosmarinic acid biosynthesis. As in other biosynthetic pathways leading to caffeic acid esters, an activated cinnamic acid is necessary as precursor. In the case of rosmarinic acid biosynthesis, this is a CoA-thioester, as also described for the biosynthesis of flavonoids [13], chlorogenie acid [14, IS] and other cinnamic acid conjugates. Cinnamic acid glucose esters, as another possibility of an activated precursor, or the unactivated cinnamic acid itself did not lead to a synthesis of rosmarinic acid with enzyme preparations from cells of Coleus blumei. Cinnamic acid glucose esters have been described to serve as activated precursors in the biosynthesis of sinapoylmalate [ 163, sinapine [ 173, acylated betacyanins [ 181 and, in some species, of chlorogenic acid [ 193.
M. S.
2880
PETERSEN
The activity of RAS is positively correlated with the accumulation of rosmarinic acid during the culture cycle of suspension cultures of Coleus blumei. In medium with 4% sucrose, the RAS activity is substantially higher than in medium with 2% sucrose. Therefore, the synthetic rate for rosmarinic acid may be regulated by the enzyme activity of RAS. With the discovery of RAS and hydroxyphenylpyruvate reductase [73 the biosynthetic pathway of rosmarinic acid is nearly clarified. The cinnamic acid precursor is formed from phenylalanine by the well known enzymes of the general phenylpropanoid pathway, phenylalanine ammonia lyase, cinnamic acid 4-hydroxylase and hydroxycinnamoyl CoA-ligase, which could all be detected in enzyme preparations from suspension cells of Coleus blumei [8]. Tyrosine aminotransferase [9, IO] and hydroxyphenylpyruvate reductase [7] are involved in the synthesis of the tyrosine-derived precursor hydroxyphenyllactic acid. In the biosynthetic pathway for rosmarinic acid, the point at which both 3-hydroxyl-groups are introduced into the aromatic rings of the precursors or the ester itself is unclear. Experiments by Ellis et al. [203 showed that inhibition of PAL by the specific inhibitor aaminooxy+phenylpropionic acid resulted in an accumulation of 4-hydroxyphenyllactic acid in suspension
cultures of Coleus blumei. Therefore, the 3-hydroxylation might occur at a relatively late stage of the biosynthesis. Furthermore, RAS showed highest affinities for the monohydroxylated substrates 4-coumaroyl-CoA and 4hydroxyphenyllactic acid, and the V&K,,, values indicate that these might be the preferred substrates for RAS. In this model, an ester of 4-coumaric acid and 4-hydroxyphenyllactic acid might normally be the direct reaction product of RAS. If this is the case. the name rosmarinic acid synthase is not strictly correct for this enzyme, instead, 4coumaroyl-coenzyme A: 4-hydroxyphenyllactic acid 4-coumaroyl transferase would be the correct nomenclature. This hypothesis is further sustained by the detection of membrane-bound hydroxylating activities which introduce the 3-hydroxyl groups into the aromatic rings of less hydroxylated rosmarinic acid-like esters (Fig. l), forming rosmarinic acid (Petersen, unpublished results). This indicates that, perhaps, in rosmarinic acid biosynthesis the hydroxylation pattern of the aromatic rings is only established after the completion of the Cskeleton, as proposed for flavonoid [21-243 and chlorogenie acid biosynthesis [25, 261.
EXPERIMENTAL Cell cultures. Suspension
cultures of Coleus blumei Benth. were from a callus culture, which was a gift from Dr B. Ulbrich (Nattermann & Cie., Cologne, Germany). The cultures were maintained as described previously [73. Chemicab. Cinnamic acid CoAesters and 3+dihydroxyphenyllactic actd were prepd as described previously [7]. S( -)-3,4Dihydroxyphenyllactic acid was a gift from Dr N. Rao (Forschungszentrum Jiilich GmbH, Germany). Enzyme preparation. RAS was prepd from ‘I-day-old suspension cells of Coleus blumei, which were grown in medium with 4% sucrose. The cells were filtered under suction and homogenized with l/10 weight of Polyclar AT and I ml of 0.1 M K-Pi huger pH 7 with 1 mM DTTg- ’ fr. wt of the cells by an Ultra-Turrax (Janke & Kunkel, Staufen i. Br.. Germany) for 150 sec. The homogenate was filtered through a nylon screen (pore established
size 100 pm) and centrifuged for 15min at 48 000 y. The supernatant was used for a fractionated (NH&SO,-pptn with satd (NH&SO, soln which was adjusted to pH 7 with NH,OH. Most of the RAS activity was pptd between 60 and 80% satn of (NH&SO, and was sedimented by centrifugation at 48000 q for 20 min. The pellet was redissolved in butler and desalted on a Sephadex G25 column (Pharmacia). The desalted protein was stored at -20’ until it was used for enzyme assays. Protem determination. Protein concns were measured spectrophotometrically according to ref. L27] using bovine serum albumin as a standard. Assayfor rosmarinic acid synthase. The assay for RAS contained in a total vol. of 250 ~1 0.1 M K-Pi buffer pH 7:2.5 pmol DTT, 125 nmol ascorbic acid, 50 nmol caffeoyl-CoA or 4-coumaroyl-CoA, IOOnmol pHPL or DHPL and ca 5Opg of the (NH&SO,-pptd and desalted protein. The assay was incubated at 30” for 5 min and stopped by addition of20 ~1 of6 M HCI. The assays were extracted x 3 with 0.5 ml EtOAc each. After evapn of the EtOAc under vacuum the residue was redissolved in 300 ~1 MeOH-Hz0 (1: 1) adjusted to pH 3 with H,PO,. The reaction products were identified and quantified by HPLC-analysis on Shandon Hypersil ODS-column (290 x 4.6 mm, parttcle size 5 pm) with MeOH-H,O (I : 1) acidified with 100 ~1 H,PO, I- ’ as an eluent at a flow rate of 1.5 ml min-‘. The products were detected spectrophotometrically at 333 nm. The values for reaction products other than RA can only be given in relative umts, because standards are not available and the molar extinction coefficients arc not known. The reverse reactton of RAS was measured in an assay with a total vol. of 250~1 0.1 M K-Pi butfer pH7 containing 2.5pmol DIT, 125 nmol ascorbic actd, 5Onmol RA. 25Onmol CoA and 20~1 of the (NH,)$O,-pptd and desalted protem. The assay was incubated at M” for 10 min and stopped by addition of 20~1 of 6M HCI. Extraction and HPLC-analysis were performed as above, but with a linear elution gradient during IOmm from 20% MeOH IO 50% MeOH in H,O acidified with lOO$ H~PO,I~~’ at a flow rate of 1.5 mlmin-’ and detection at 280 nm. ldentifcation of the reaction productst Esters other than RA were synthesized enzymatically by supplying RAS with other cinnamic acid CoA-esters and/or hydroxyphenyllactic acids as substrates. For identification they were first purified by semiprep. HPLC on a Shandon Hypersil ODScolumn (290 x 8 mm, particle size 5 pm) with MeOH-H,O (1: I) acidified with 3 ml HOAc I- ’ as an eluent. After evapn of the eluent, the esters were cleaved enzymatically with 4mg Rhozyme HP-150 (Pollock and Pool Ltd. Reading, U.K.) in 200 ~1 NH,OAc buffer pH 6 at 30” for 4 hr. The reaction was stopped by addition of 50 ~1 6 M HCI and the assays were extracted x 3 with 0.5 ml EtOAc each. After cvapn of the solvent the residues were dissolved in MeOH-H,O (1.1) adjusted IO pH 3 with H,PO, and analysed by HPLC as described for the enzyme assay, but with detection at 280 nm. The products of the enzymatic ester cleavage were identified using cinnamic, Ccoumaric, caffeic, ferulic, and sinapic acid and pHPL and DHPL as authentic standards. Acknowledgement-
The
Forschungsgemeinschaft
financial support by the is gratefully acknowledged.
Dcutsche
REFERENCES 1. De-Eknamkul,
Vol. 4 Medicinal and Aromafic I (Bajaj, Y. P. S.. ed.), p. 310. Springer, Berlin.
Agriculture Hunts
W. and Ellis, B. E. (1983) in Biotechnology
and Forestry
in
Rosmarinic acid synthase 2. Ellis, B. E. and Towers, G. H. N. (1970) B&hem J. lla 291. 3. De-Eknamkul, W. and Ellis, B. E. (1984) Planto Med. 51,346. 4. Sumaryono, W., Proksch, P., Wray, V., Witte, L. and Hartmann, T. (1989) Planta Med. 55, 110. 5. Razraque, A. and Ellis, B. E. (1977) Planto 137, 287. 6. Zenk, M. H., El-Shagi, H. and Ulbrich, B. (1977) Noturwissenschajien 64, 585. 7. Petersen, M. and Alfermann, A. W. (1988) Z. Norurjorsch. 4% 501. 8. Karwatzki, B., Petersen, M. and Alfermann, A. W. (1989) Planta Med. 55, 663. 9. De-Eknamkul, W. and Ellis, B. E. (1987) Phytochemistry 26, 1941. 10. De-Eknamkul, W. and Ellis, B. E. (1987) Arch. Biochem. Biophys. 257, 430. 11. Hausler, E., Petersen, M. and Alfermann, A. W. (1990) PIanta Med. 56, 619620. 12. Gumbinger, H. G., Vahlensieck, U. and Winterhoff, H. (1990) Poster at the symposium on ‘Biology and Chemistry of Active Natural Substances’, Bonn. 13. Kreuzaler, F. and Hahlbrock, K. (1972) FEBS Letters 2& 69.
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14. Stiickigt, J. and Zenk, M. H. (1975) FEBS Letters 42, 131. 15. Rhodes, M. J. C. and Woohorton, L. S. C. (1976) Phytochemistry IS, 947. 16. Tkotz, N. and Strack. D. (1980) Z. Naturforsch. 3Sc, 835. 17. Griiwe, W. and Strack, D. (1986) Z. Naturjorsch. 41c, 28. 18. Bokem, M. and Strack, D. (1988) Planta 174, 101. 19. Villegas, R. J. A. and Kojima, M. (1986) J. Biol. Chem. Ml, 8729. 20. Ellis, B. E., Remmen, S. and Goeree, G. (1979) Planta 147, 163. 21. Stotz, G. and Forkmann. G. (1982) Z. Naturforsch, 37~. 19. 22. Forkmann, G., Heller, W. and Grisebach, H. (1980) Z. Naturforsch. 3Sc. 691. 23. Hagmann, M. L., Heller, W. and Grisebach, H. (1983) Eur. J. Biochem. 134,547. 24. Larson, R. L. and Bussard, J. B. (1986) Plant Physiol. 80,483. 25. Heller, W. and Kiihnl, T. (1985) Arch. Biochem Biophys. 241, 453. 26. Kuhnl, T., Koch, U., Heller, W. and Wellmann, E. (1987) Arch. Biochem. Biophys. 258, 226. 27. Bradford, M. M. (1976) Anal. B&hem. 72, 248.