A Newly-Detected Reductase from Rauvolfia Closes a Gap in the Biosynthesis of the Antiarrhythmic Alkaloid Ajmaline

• Abstract
• Introduction
• Materials and Methods
• Plant cell cultures
• Standard enzyme assay
• Coupled enzyme assay
• HPLC conditions
• Identification of the enzyme product
• Isolation and enrichment of the reductase
• Anion exchange chromatography on SOURCE 30Q
• Hydroxyapatite chromatography
• Affinity chromatography on 2′,5′-ADP Sepharose 4B
• Ion exchange chromatography on DEAE Sepharose
• Mono Q HR5/5 ion exchange chromatography
• Protein determination
• SDS-PAGE
• Properties of the reductase
• Results and Discussion
• 1,2-Dihydrovomilenine reductase purification
• Properties of the pre-purified 1,2-dihydrovomilenine
  reductase
• Taxonomic distribution of 1,2-dihydrovomilenine reductase
• Acknowledgements
• References

Abstract

A new enzyme, 1,2-dihydrovomilenine reductase (E.C. 1.3.1), has been detected in Rauvolfia cell suspension cultures. The enzyme specifically converts 2β(R)-1,2-dihydrovomilenine through an NADPH-dependent reaction into 17-O-acetylnorajmaline, a close biosynthetic precursor of the antiarrhythmic alkaloid ajmaline from Rauvolfia. A five-step purification procedure using SOURCE 30Q chromatography, hydroxyapatite chromatography, 2′,5′-ADP Sepharose 4B affinity chromatography and ion exchange chromatography on DEAE Sepharose and Mono Q delivered a ∼ 200-fold enriched enzyme in a yield of ∼ 6 %. SDS-PAGE showed an M_r for the enzyme of ∼ 48 kDa. Optimum pH and optimum temperature of the reductase were at pH 6.0 and 37 °C. The enzyme shows a limited distribution in cell cultures expressing ajmaline biosynthesis, and is obviously highly specific for the ajmaline pathway.

Introduction

Among the species of the genus Rauvolfia, the Indian Rauvolfia serpentina has been since the Vedic and Ayurvedic period the most important medicinal plant for a wide range of phytotherapeutic applications. After the isolation of six major alkaloids by Siddiqui and Siddiqui [1], [2] several years elapsed before the therapeutical value of the antiarrhythmic alkaloid ajmaline was established [3] and the use of reserpine as an antihypertensive and neuroleptic drug was clearly defined [4], [5]. Both alkaloids became important drugs on the general pharmaceutical market. During the last two decades, cell systems derived from R. serpentina plants have been exhaustively investigated for alkaloid formation [6], [7]. Based on these results, cell suspension cultures were selected and applied to deliver a whole series of novel enzymes allowing for the first time the elucidation in great detail of the ajmaline biosynthetic pathway starting with the progenitors tryptamine and secologanin [8], (Fig. [1]). The cDNAs encoding strictosidine synthase [9], polyneuridine aldehyde esterase [10], raucaffricine glucosidase [11] and cytochrome P450 reductase were expressed recently in heterologous systems. Except for the P450 reductase, the cloning was accomplished by a ”reverse genetic” approach based on partial polypeptide sequences of the purified enzymes.

Although most of the enzymes of ajmaline biosynthesis are now well known, cytochrome P450 catalysed reactions and the reduction of the late pathway intermediate vomilenine still needed much deeper investigation. Crude enzyme extracts of Rauvolfia cells were shown to reduce the indolenine alkaloid vomilenine to the indoline stage represented by acetylnorajmaline (Fig. [1]). This conversion formally requires two steps of reduction, and neither the sequential order nor the number of reductases involved has been clearly resolved [8].

In this communication we describe the detection, isolation, partial purification and major properties of a novel enzyme catalysing the NADPH-dependent reduction of the vomilenine derivative, 2β(R)-1,2-dihydrovomilenine, into 17-O-acetylnorajmaline. At the enzymatic level, this reaction finally closes a missing link in the late stages of the biosynthesis of ajmaline in Rauvolfia.

Materials and Methods

Plant cell cultures

Rauvolfia serpentina cell suspension cultures and those chosen for taxonomic distribution studies of the 1,2-dihydrovomilenine reductase were grown in Linsmaier-Skoog medium [12] for 10 days in 1 l Erlenmeyer flasks at 24 °C under constant diffuse light (∼ 600 lux) on a rotary shaker (100 rpm). The cell material was harvested by filtration, frozen with liquid nitrogen and stored at -25 °C.

Standard enzyme assay

The standard enzyme assay mixture contained 0.4 mM 2β(R)-1,2-dihydrovomilenine, 2 mM NADPH, 50 mM potassium phosphate buffer (pH 7.0) and an appropriate amount of enzyme in a total volume of 50 μl. The mixture was incubated for 15 min at 30 °C with shaking (550 rpm). The reaction was terminated by adding 75 μl methanol. Then the solution was mixed (vortex) for 5 sec and centrifuged (18,000 × g) for 5 min. The clear supernatant was analysed by HPLC.

Coupled enzyme assay

The coupled assay mixture consisted of KPi buffer at a final concentration of 50 mM (pH 7.0), 0.14 mM vomilenine and vomilenine reductase with a total activity of 13.8 nkat. In addition, the total incubation volume of 200 μl contained 2 mM NADPH and various amounts of 1,2-dihydrovomilenine reductase. The mixture was incubated under the same conditions as for the above mentioned standard assay for 60 min. The reaction was terminated by adding 200 μl methanol. The solution was mixed for 5 sec (vortex). After centrifugation (18,000 × g for 5 min), the clear supernatant was analysed directly by HPLC.

HPLC conditions

For the HPLC assay a LiChrospher 60 RP-select B column (250 × 4 mm, Merck, Darmstadt, Germany) equipped with a select B pre-column (Merck, Darmstadt, Germany) was applied. The solvent system used was: A, acetonitrile, B, 25 mM K₂HPO₄ in 0.45 % H₃PO₄. The analyses were carried out with a gradient of acetonitrile from 28 % to 30 % in 4 min, and to 35 % in 2 min at a flow rate of 1.68 ml/min.

Identification of the enzyme product

A prepurified enzyme fraction (22.04 mg protein) was incubated with 50 mM buffer (KPi, pH 7.0) at 30 °C in the presence of 0.14 mM vomilenine (totally 180 μg) and NADPH (final conc. 2 mM) for 1.5 h. The pH of the mixture was adjusted with ammonia to 9. Then the basic solution was extracted twice with 8.16 ml ethyl acetate and the organic layers combined and evaporated. The residue was chromatographed on TLC plates (Macherey and Nagel, Düren, Germany) with the solvent system chloroform/methanol/ammonia (8.8 : 1.2 : 0.1). The major compound detected under UV light (R_f value 0.52) showed an orange ceric sulphate reaction and yielded the following EI-MS data m/z (rel. int.) = 354 (23.4, M⁺), 339 (5.1), 325 (3.1), 311 (3.1), 293 (3.3), 279 (1.9), 265 (5.1), 249 (3.1), 234 (3.2), 224 (13.1), 208 (5.1), 196 (10.2), 180 (26.3), 168 (100), 156 (11.7), 143 (23.4), 130 (37.2), 117 (19.0), 103 (6.6).

Isolation and enrichment of the reductase

Buffers used for enzyme purification were: buffer A, 10 mM Tris/HCl, pH 7.8; buffer B: 10 mM potassium phosphate, pH 7.0 and buffer C: 10 mM Tris/HCl, pH 7.3; columns used for enzyme purification were obtained from Amersham Pharmacia (Freiburg, Germany) except the hydroxyapatite column which was purchased from Bio-Rad (München, Germany).

All isolation and purification steps were carried out at 4 °C. Plant cell cultures (1.2 kg, fresh weight) were shock frozen in liquid nitrogen. 1.2 l of 100 mM Tris/HCl buffer (pH 8.0) containing 20 mM β-mercaptoethanol (1 ml/g fresh weight) were added to the frozen material and stirred at room temperature till thawed. The cells were ground in an Ultraturrax for 2.5 min. The resulting homogenate was centrifuged at 10,000 × g for 30 min. The supernatant was subjected to ammonium sulphate precipitation. The protein fraction precipitating between 40 % and 60 % saturation was resuspended in buffer A (containing 2.5 mM β-mercaptoethanol) and was dialysed against the same buffer overnight prior to chromatographic purification. The coupled enzyme assay was applied for monitoring reductase activities during the purification procedure.

Anion exchange chromatography on SOURCE 30Q

The dialysed protein solution was centrifuged at 10,000 × g for 30 min. The supernatant was loaded onto a SOURCE 30Q column (50 × 110 mm) which was pre-equilibrated with buffer A. Elution of the enzyme was carried out with a linear gradient of increasing KCl concentration from 0 to 0.2 M in buffer A (500 ml) at a flow rate of 8.0 ml/min.

Hydroxyapatite chromatography

Fractions containing high enzyme activities from SOURCE 30 Q column chromatography were collected and desalted on a Sephadex G-25 column (26 × 370 mm) equilibrated with buffer B. The desalted protein sample was then subjected to a Hydroxyapatite column (16 × 150 mm). Proteins were eluted with a linear gradient of potassium phosphate buffer (pH 7.0, 150 ml) from 10 mM to 0.2 M at a flow rate of 2 ml/min.

Affinity chromatography on 2′,5′-ADP Sepharose 4B

Fractions exhibiting high 1,2-dihydrovomilenine reductase activities were pooled and desalted on a Sephadex G-25 column which was equilibrated with buffer C. Affinity chromatography was carried out on a 2′,5′ ADP Sepharose 4B column (16 × 80 mm) equilibrated with buffer C. Proteins were eluted with buffer C containing 1.0 M NaCl at a flow rate of 1.2 ml/min. Fractions exhibiting high 1,2-dihydrovomilenine reductase activities were pooled and desalted on a Sephadex G-25 column equilibrated with buffer A.

Ion exchange chromatography on DEAE Sepharose

The desalted fraction obtained from affinity chromatography was then applied to a DEAE Sepharose column pre-equilibrated with buffer A. The column was eluted with a linear gradient from 0 to 0.3 M of NaCl in buffer A (60 ml), then eluted with a step gradient from 0.3 M to 0.4 M NaCl and 0.4 M to 0.5 M NaCl in buffer A. The flow rate was 1.2 ml/min. Fractions containing 1,2-dihydrovomilenine reductase activities were collected and desalted as described above.

Mono Q HR5/5 ion exchange chromatography

The desalted and combined fractions of DEAE Sepharose were loaded onto a Mono Q column (HR 5/5) equilibrated with buffer A. Proteins were eluted with a linear gradient of NaCl from 0 to 0.5 M in buffer A (20 ml) at a flow rate of 1.0 ml/min.

Protein determination

Protein concentrations were measured using the method of Bradford [13] with bovine serum albumin as a standard protein.

SDS-PAGE

Denaturing SDS-PAGE was carried out using the discontinuous system described by Laemmli [14] in gels containing 11 % acrylamide. The gels were stained with Coomassie Brilliant Blue solution (0.25 % Coomassie Brilliant Blue R 250, 45 % methanol and 9 % acetic acid in distilled water) and de-stained with a solution of 45 % methanol and 10 % acetic acid in water. Molecular weight standards for SDS-PAGE were the LMW marker mixture purchased from Amersham Pharmacia (Freiburg, Germany).

Properties of the reductase

The pH dependency of the enzyme was determined using the standard enzyme assay and two buffer systems, citrate (50 mM) Na₂HPO₄ (100 mM) between pH 3.7 and pH 7.9 and KH₂PO₄ (100 mM) Na₂HPO₄ (100 mM) between pH 4.6 and pH 8.9.

Temperature optimum of the reaction was measured applying the standard assay. The relative enzyme activity from 20 °C to 75 °C was determined by the HPLC assay. The relative molecular weight of the enzyme was measured after DEAE Sepharose chromatography using an SDS gel calibration curve.

Pre-purified enzyme extracts, prepared by 75 % ammonium sulphate precipitation followed by fractionation with an HiTrap desalting column (5 ml), were obtained from 9 different cell suspension cultures of various families and genera and were used to investigate the taxonomic distribution of the enzyme. The standard enzyme assay and analysis of the incubations by the HPLC assay allowed quantitation of reductase activities compared to R. serpentina cells.

Results and Discussion

The multi-step biosynthetic pathway of the Rauvolfia alkaloid ajmaline consists of several NADPH-dependent reactions. At late stages of this sequence the indolenine alkaloid vomilenine is reduced to the indoline-type 1,2-dihydrovomilenine. This intermediate is further converted to the ajmalan-system (17-O-acetylnorajmaline, Fig. [1]) by 1,2-dihydrovomilenine reductase. Taking advantage of the newly-developed, coupled enzyme assay, vomilenine, which can be simply isolated from ”hairy roots” of Rauvolfia [15], could be used as substrate. In contrast, 1,2-dihydrovomilenine is not available from cell systems and can only be synthesised enzymatically by vomilenine reduction. Application of this enzyme assay therefore allowed the development of an efficient purification protocol for this novel enzyme.

1,2-Dihydrovomilenine reductase purification

A six-step procedure including ammonium sulphate precipitation, followed by five chromatographic steps gave optimum results to highly enrich 1,2-dihydrovomilenine reductase (Figs. [2] and [3], Table [1]). Finally a 200-fold enrichment of the enzyme was achieved with an acceptable recovery of nearly 6 %, when the procedure described in the next paragraph was followed.

Protein fractions from ammonium sulphate precipitation were purified first by anion exchange chromatography on SOURCE 30Q. Because a significant amount of protein did not bind and was eluted with the flow-through (Fig. [2]), the enrichment factor could be doubled with this particular column. An about 5-fold purification was reached. Further purification was achieved on a hydroxyapatite column. 1,2-Dihydrovomilenine reductase was eluted at the beginning of the applied gradient and became well separated from the following eluting protein peaks (Fig. [2]). A 22.5-fold enzyme enrichment resulted from this step with a yield of 21 %. The next purification step included affinity chromatography on 2′,5′-ADP Sepharose which was considered to bind specifically NADPH-dependent enzymes [16]. Enzymes bound to 2′,5′ ADP Sepharose can be eluted with high salt concentrations, e. g., by 1 M NaCl in buffer C (Fig. [2]). After the affinity chromatography, 1,2-dihydrovomilenine reductase became enriched about 65-fold with a recovery of ∼ 13 %. Enzymatically active fractions from 2′,5′-ADP Sepharose chromatography were best purified when applied to an ion exchange column (DEAE Sepharose) (Fig. [2]). An enrichment of ˜ 77-fold of enzyme activity was usually achieved. SDS-PAGE of fractions eluted from DEAE Sepharose indicated, however, that this enzyme preparation was still not homogenous (data not shown). Therefore, protein fractions from DEAE Sepharose were collected and applied to ion exchange chromatography (Mono Q) (Fig. [2]). Coomassie staining of different fractions from Mono Q after SDS-PAGE revealed a protein band correlated in intensity with the relative enzyme activity. Therefore it can be concluded that the protein band assigned by the arrow in Fig. [3] corresponds to the enriched 1,2-dihydrovomilenine reductase. The molecular weight of this particular protein band, presumably the reductase, was estimated to be 48 ± 5 % kDa (Fig. [4]).

Properties of the pre-purified 1,2-dihydrovomilenine
reductase

For further characterisation, the dependence of the enzyme activity on pH and temperature was investigated. The results for the influence of pH on the enzyme activity are shown in Fig. [5]. 1,2-Dihydrovomilenine reductase was stable through a relatively small pH range of 5.8 and pH 6.2 in 50 mM citric acid/100 mM Na₂HPO₄ buffer, with a maximum activity at pH 6.2. In 100 mM KH₂PO₄/100 mM Na₂HPO₄ buffer, the enzyme showed a relatively wide range of optimal pH from half maximum activity at pH 5.2 and pH 7.7 with a maximum at pH 6.0.

When the thermal stability of the enzyme was tested at the pH optimum in citric acid/Na₂HPO₄ buffer, the reductase was stable in a broad range from 30 °C to 50 °C, with a maximum activity around 37 °C. Surprisingly, the enzyme was still active above 55 °C; however, for some enzymes such as the strictosidine glucosidase or the arbutin synthase isolated from the Rauvolfia cell culture used here, high temperature optima of 50 °C were also observed earlier. The product of the reductase reaction was identified as 17-O-acetylnorajmaline by orange staining with ceric ammonium sulphate [17], its R_f-value on TLC, its R_t by HPLC separation and the EI-MS fragmentation. Because vomilenine is not accepted as a substrate by the reductase, first hydrogenation of the indolenine double bond is necessary, which is catalysed by vomilenine reductase. These results clearly define the sequential order of reductions catalyzed by vomilenine reductase and followed by 1,2-dihydrovomilenine reductase. Only two further reactions are involved in ajmaline biosynthesis following these reductions, the deacetylation at the 17-O-acetyl group and N _α-methylation.

Taxonomic distribution of 1,2-dihydrovomilenine reductase

As we have demonstrated for other enzymes of the ajmaline biosynthetic pathway, such as polyneuridine aldehyde esterase [18], vinorine synthase [19], vinorine hydroxylase [20] or 17-O-acetylajmalan esterase [21], their occurrence in cell culture systems is associated with expression of the biosynthesis of ajmaline type alkaloids. All these enzymes exhibit a rather limited substrate acceptance and are taxonomically limited not only to the Apocynaceae family but more specifically to the genus Rauvolfia. Therefore it was interesting to check whether this is also true for the here described reductase. The taxonomic distribution of 1,2-dihydrovomilenine reductase in different plant cell suspension cultures such as R. serpentina, Apocynaceae (100 % relative activity); R. mannii (Stapf), Apocynaceae (18 %); R. mombasiana (Stapf), Apocynaceae (9.5 %); Catharanthus roseus (L.), Apocynaceae (0 %); Nicotiana tabacum (L.), Solanaceae (0 %); Solanum lycopersicum (L.), Solanaceae (0 %); Gossypium hirsutum (L.), Malvaceae (0 %); Lonicera morrowii (L.), Caprifoliaceae (0 %) and Saponaria officinalis (L.), Caryophyllaceae (0 %) indicates clearly that 1,2-dihydrovomilenine reductase is restricted to the genus Rauvolfia. The enzyme, in fact, occupies not only a specific position in ajmaline biosynthesis but also closes a gap at the end of the pathway. Future microsequencing of the enzyme and heterologous expression in Escherichia coli will most probably allow its much more detailed molecular analysis; such experiments are presently in progress.

Acknowledgements

This work was supported by a grant of Deutsche Forschungsgemeinschaft (Bonn, Bad-Godesberg, Germany), by the Fonds der Chemischen Industrie (Frankfurt/Main, Germany) and the BMBF (Bonn, Germany). We are also grateful to Prof. Dr. W. E. Court (Mold, Wales) for linguistic advice.

References

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• 19 Pfitzner A, Polz L, Stöckigt J. Properties of vinorine synthase - the Rauwolfia enzyme involved in the formation of the ajmaline skeleton. Z.  Naturforsch.. 1986;  41c 103-14
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• 21 Polz L, Schübel H, Stöckigt J. Characterization of 2β(R)-17-O-acetylajmalan: acetylesterase - a specific enzyme involved in the biosynthesis of the Rauwolfia alkaloid ajmaline. Z.  Naturforsch.. 1987;  42c 333-42
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1 Shujuan Gao and Gerald von Schumann have contributed equally to this study.

Prof. Dr. J. Stöckigt

Department of Pharmaceutical Biology

Institute of Pharmacy

Johannes Gutenberg-University

Staudinger Weg 5

55099 Mainz, Germany

Email: stoeckig@mail.uni-mainz.de

Phone: +49-(0)6131-39-25751

Fax: +49-(0)6131-39-23752

References

• 1 Siddiqui S, Siddiqui R H. Chemical examination of the roots of Rauwolfia serpentina, Benth.  J Ind Chem Soc. 1931;  8 667-81
  PubMedSearch in Google Scholar
• 2 Siddiqui S, Siddiqui R H. The alkaloids of Rauwolfia serpentina, Benth. Part I. J Ind Chem.  Soc. 1932;  9 539-44
  PubMedSearch in Google Scholar
• 3 Kleinsorge H. Klinische Untersuchungen über die Wirkungsweise des Rauwolfia Alkaloids Ajmalin bei Herzrhythmus-Störungen, insbesondere bei Extrasystolie. Med.  Klin.. 1959;  54 409-12
  PubMedSearch in Google Scholar
• 4 Kähler H J. Rauwolfia alkaloide. In: Kähler HJ, editor Boehringer Mannheim GmbH 1970: 99-105
  Search in Google Scholar
• 5 Kroneberg G. Pharmakologie der Rauwolfia .  Planta Medica. 1957;  5/6 156-65
  PubMedSearch in Google Scholar
• 6 Stöckigt J, Pfitzner A, Firl J. Indole alkaloids from cell suspension cultures of Rauwolfia serpentina Benth.  Plant Cell Reports. 1981;  1 36-9
  CrossrefPubMedSearch in Google Scholar
• 7 Falkenhagen H, Stöckigt J, Kuzovkina I N, Alterman I E, Kolshorn H. Indole alkaloids from ”hairy roots” of Rauwolfia serpentina Can J.  Chem. 1993;  71 1-3
  PubMedSearch in Google Scholar
• 8 Stöckigt J. The Alkaloids. Biosynthesis in Rauwolfia serpentina. Modern aspects of an old medicinal plant. In: Cordell GA, editor Vol. 47 New York; Academic Press 1995: 115-72
  Search in Google Scholar
• 9 Kutchan T M, Dittrich H, Bracher D, Zenk M H. Enzymology and molecular biology of alkaloid biosynthesis.  Tetrahedron. 1991;  47 5945-54
  CrossrefPubMedSearch in Google Scholar
• 10 Dogru E, Warzecha H, Seibel F, Haebel S, Lottspeich F, Stöckigt J. The gene encoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesis in plants in an ortholog of the α/β hydrolase super family. Eur. J.  Biochem.. 2000;  267 1397-406
  PubMedSearch in Google Scholar
• 11 Warzecha H, Gerasimenko I, Kutchan T M, Stöckigt J. Molecular cloning and functional bacterial expression of a plant glucosidase specifically involved in alkaloid biosynthesis.  Phytochemistry. 2000;  54 657-66
  CrossrefPubMedSearch in Google Scholar
• 12 Linsmaier E M, Skoog F. Organic growth factor requirements of tobacco tissue cultures. Physiol.  Plant. 1965;  18 100-27
  PubMedSearch in Google Scholar
• 13 Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal.  Biochem.. 1976;  72 248-54
  CrossrefPubMedSearch in Google Scholar
• 14 Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.  Nature. 1970;  227 680-5
  PubMedSearch in Google Scholar
• 15 Falkenhagen H, Kuzovkina I N, Alterman I E, Nikolaeva L A, Stöckigt J. Alkaloid formation in hairy roots and cell suspensions of Rauwolfia serpentina Benth. Nat. Prod.  Lett.. 1993;  3 107-12
  PubMedSearch in Google Scholar
• 16 Yasukochi Y, Masters B SS. Some properties of a detergent-solubilized NADPH-cytochrome c (cytochrome P-450) reductase purified by biospecific affinity chromatography. J. Biol.  Chem.. 1976;  251 5337-44
  PubMedSearch in Google Scholar
• 17 Court W E, Iwu M M. Chromogenic reactions of Rauvolfia alkaloids after separation by thin-layer chromatography.  Chrom.. 1980;  187 199-207
  PubMedSearch in Google Scholar
• 18 Pfitzner A, Stöckigt J. Polyneuridine aldehyde esterase: An unusually specific enzyme involved in the biosynthesis of sarpagine type alkaloids. J. Chem. Soc. Chem. Commun. 1983: 459-60
  Search in Google Scholar
• 19 Pfitzner A, Polz L, Stöckigt J. Properties of vinorine synthase - the Rauwolfia enzyme involved in the formation of the ajmaline skeleton. Z.  Naturforsch.. 1986;  41c 103-14
  PubMedSearch in Google Scholar
• 20 Falkenhagen H, Stöckigt J. Enzymatic biosynthesis of vomilenine, a key intermediate of the ajmaline pathway, catalyzed by a novel cytochrome P 450 - dependent enzyme from plant cell cultures of Rauwolfia serpentina. Z.  Naturforsch.. 1995;  50c 45-53
  PubMedSearch in Google Scholar
• 21 Polz L, Schübel H, Stöckigt J. Characterization of 2β(R)-17-O-acetylajmalan: acetylesterase - a specific enzyme involved in the biosynthesis of the Rauwolfia alkaloid ajmaline. Z.  Naturforsch.. 1987;  42c 333-42
  PubMedSearch in Google Scholar

1 Shujuan Gao and Gerald von Schumann have contributed equally to this study.

Prof. Dr. J. Stöckigt

Department of Pharmaceutical Biology

Institute of Pharmacy

Johannes Gutenberg-University

Staudinger Weg 5

55099 Mainz, Germany

Email: stoeckig@mail.uni-mainz.de

Phone: +49-(0)6131-39-25751

Fax: +49-(0)6131-39-23752