Deoxysarpagine hydroxylase--a novel enzyme closing a short side pathway of alkaloid biosynthesis in Rauvolfia.

Article abstract of DOI:10.1016/S0968-0896(02)00126-8

Microsomal preparations from cell suspension cultures of the Indian plant Rauvolfia serpentina catalyze the hydroxylation of deoxysarpagine under formation of sarpagine. The newly discovered enzyme is dependent on NADPH and oxygen. It can be inhibited by typical cytochrome P450 inhibitors such as cytochrome c, ketoconazole, metyrapone, tetcyclacis and carbon monoxide. The CO-effect is reversible with light (450 nm). The data indicate that deoxysarpagine hydroxylase is a novel cytochrome P450-dependent monooxygenase. A pH optimum of 8.0 and a temperature optimum of 35 degrees C were determined. K(m) values were 25 microM for NADPH and 7.4 microM for deoxysarpagine. Deoxysarpagine hydroxylase activity was stable in presence of 20% sucrose at -25 degrees C for >3 months. The analysis of presence of the hydroxylase in nine cell cultures of seven different families indicates a very limited taxonomic distribution of this enzyme.

Full text of DOI:10.1016/S0968-0896(02)00126-8

Bioorganic & Medicinal Chemistry 10 (2002) 2479–2483
Deoxysarpagine Hydroxylase — A Novel Enzyme Closing a Short
Side Pathway of Alkaloid Biosynthesis in Rauvolfia
Bingwu Yu, Martin Ruppert and Joachim Stockigt*
Johannes Gutenberg-University, Institute of Pharmacy, Department of Pharmaceutical Biology,
Staudinger Weg 5, 55099 Mainz, Germany
Received 12 February 2002; accepted 2 April 2002
Abstract—Microsomal preparations from cell suspension cultures of the Indian plant Rauvolfia serpentina catalyze the hydroxyla-
tion of deoxysarpagine under formation of sarpagine. The newly discovered enzyme is dependent on NADPH and oxygen. It can be
inhibited by typical cytochrome P450inhibitors such as cytochrome c, ketoconazole, metyrapone, tetcyclacis and carbon monoxide.
The CO-effect is reversible with light (450nm). The data indicate that deoxysarpagine hydroxylase is a novel cytochrome P450-
dependent monooxygenase. A pH optimum of 8.0and a temperature optimum of 35 ꢀC were determined. Km values were 25 mM for
NADPH and 7.4 mM for deoxysarpagine. Deoxysarpagine hydroxylase activity was stable in presence of 20% sucrose at À25 ꢀC for
>3 months. The analysis of presence of the hydroxylase in nine cell cultures of seven different families indicates a very limited
taxonomic distribution of this enzyme. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
out labeling7 clearly demonstrated that the final bio-
synthetic step in sarpagine biosynthesis must be the
hydroxylation of deoxysarpagine at carbon position 10
(Scheme 1). Although these results marked an impor-
tant step in understanding the sarpagine formation, they
did not allow a final conclusion concerning the type of
reaction, the mechanism of reaction or the nature of the
involved enzyme.
The biosynthesis of the monoterpenoid indole alkaloid
ajmaline, which has been in use as an antiarrhythmic
drug for about four decades,1À3 has been previously
elucidated at the enzymatic level in much detail.4,5 Cell
suspension cultures of the Indian medicinal plant Rau-
volfia serpentina Benth. ex Kurz are an efficient source
for the isolation of the particular enzymes, because the
cells also produce a great variety of indole alkaloids of
different types and especially of the Sarpagan and
Ajmalan types.6 Although the biosynthetic pathway
leading to ajmaline is now well known, side routes
emerging directly from this pathway still need further
exploitation. Such knowledge might become very
important in the near future for the metabolic regula-
tion of the cellular alkaloid formation in Rauvolfia,
which is presently unknown.
In this communication, we describe the in vitro bio-
synthesis of sarpagine from 10-deoxysarpagine which is
catalyzed by a novel NADPH- and cytochrome P450-
dependent Rauvolfia enzyme named deoxysarpagine
hydroxylase (DOSH).
Results and Discussion
The alkaloid sarpagine is a typical Rauvolfia constituent
and has been isolated as one of the first examples of this
alkaloid group a long time ago.8 Its biological activities
were not pronounced very much and the alkaloid did
not become a commercially applied drug. Sarpagine
deserves, however, attention in the light of biosynthesis,
especially what the enzyme was concerned introducing
oxygen into the aromatic ring. Therefore, we con-
centrated on searching for the enzyme which would
hydroxylate 10-deoxysarpagine leading to sarpagine.
Hydroxylation reactions at carbon atoms are usually
Despite several not deeply investigated examples of such
side sequences, like the biosynthesis of the alkaloid
groups tetraphyllicines, of the rauglucines and of the
raumaclines, the formation of the typical Rauvolfia
alkaloid sarpagine, also a side product of ajmaline bio-
synthesis, is in principle well known. Recently published
in vivo NMR experiments at 800 MHz performed with-
*Corresponding author. Tel.: +49-6131-39-25751; fax: +49-6131-39-
23752; e-mail: stoeckig@mail.uni-mainz.de
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00126-8

2480
B. Yu et al. / Bioorg. Med. Chem. 10 (2002) 2479–2483
catalyzed by either cytochrome P450-dependent
enzymes9 or 2-oxoglutarate-utilizing dioxygenases.10,11
The presumptive substrate, 10-deoxysarpagine, was
easily separated by HPLC from the putative enzyme
product sarpagine, and thus it was possible to establish
a simple and fast HPLC-based enzyme assay. Micro-
somal fractions of Rauvolfia cells prepared by a stan-
dard method12 were incubated with 10-deoxysarpagine
in the presence of NADPH. Based on HPLC analysis
this incubation mixture showed the cell-free synthesis of
sarpagine. In detail, when a crude cell-free extract or a
microsomal preparation of R. serpentina cultured cells
was incubated with 0.1 mM 10-deoxysarpagine and
NADPH at pH 8.0for 4 h, a new peak at Rt 7.0min
was detected by HPLC. The isolated compound showed
an UV spectrum identical to that of sarpagine (data not
shown). It also had the same behavior pattern on TLC.
In addition, EI–MS analysis revealed the same frag-
mentation pattern, indicating that the enzyme product
indeed was sarpagine. Since no conversion was observed
with cell-free extracts or microsomal preparations which
had been denatured by heating, the sarpagine formation
was clearly enzyme-catalyzed. We named this enzyme
deoxysarpagine hydroxylase (DOSH).
As the cell-free conversion was not high yielding, we
screened for optimum enzyme activity during the whole
period of cell growth (Fig. 1). The maximum enzyme
activity was detected on the third day of cell growth
which is much earlier than observed for other enzymes
in ajmaline biosynthesis4 and then decreased. Enzymatic
sarpagine synthesis in the microsomal fraction obtained
from these cells was more than 10times higher com-
pared to that in the crude cell-free extract, suggesting
that the enzyme activity was membrane-associated. The
transformation catalyzed by the hydroxylase showed an
optimum at pH 8.0in Tris–HCl buffer. This pH is
higher than reported for most other enzymes in ajmaline
biosynthesis (Scheme 1) despite two exceptions, viz.
vinorine synthase (pH 8.5) and vinorine hydroxylase
(pH 8.3)5,13,14 which suggests different subcellular
enzyme localization. The optimum temperature for
DOSH reaction is 35 ꢀC. Under these conditions the
catalyzed reaction was distinctly dependent on NADPH
and oxygen. NADPH could not be replaced by NADH,
indicating a remarkable cofactor specificity of DOSH.
The appropriate Michaelis–Menten kinetics showed an
apparent Km value for NADPH of 25 mM (Fig. 2)
demonstrating a high affinity of the enzyme towards the
cofactor. The low Km value of 7.4 mM for 10-deoxy-
sarpagine (Fig. 3) let us conclude that 10-deoxy-
sarpagine is most probably the natural substrate of the
isolated hydroxylase. Because of the membrane-associ-
ation and the necessity for NADPH for the enzymatic
activity, we suggested that the reaction is cytochrome
P450-dependent. Since NADP+ is a typical competitive
inhibitor of this class of enzymes,15 we measured the
hydroxylase activity at different NADPH/NADP+
ratios. Indeed, the results showed a distinct inhibition of
the hydroxylation in presence of the oxidised cofactor.
A Lineweaver–Burk plot for three different NADP+
concentrations proved the competitive inhibition of the
enzyme by NADP+ (Fig. 4).
Scheme 1. Major steps of the biosynthetic pathways of sarpagan- and
ajmalan-type alkaloids in cell suspension culture of Rauvolfia serpen-
tina. The reaction catalyzed by the deoxysarpagine hydroxylase
(hydroxylation to sarpagine at carbon position 10) is boxed (DOSH,
deoxysarpagine hydroxylase; PNAE, polyneuridine aldehyde esterase;
SG, strictosidine glucosidase; SS, strictosidine synthase; VH, vinorine
hydroxylase; VS, vinorine synthase).
Figure 1. Changes of deoxysarpagine hydroxlase activity of R. ser-
pentina cell cultures during a cultivation period of 8 days,.

B. Yu et al. / Bioorg. Med. Chem. 10 (2002) 2479–2483
2481
Cytochrome c is another typical P450inhibitor. 16 When
10 mM cytochrome c was included to the reaction mix-
ture, a 45.7% inhibition was observed, whereas nearly
complete inhibition was found at 20 mM cytochrome c
(Table 1). This result strongly suggested the cytochrome
changed from N2/O2 to CO/O2 in light. In the dark the
conversion was reduced from ꢁ70to ꢁ20%. Under the
same conditions light at 450nm can in principle reverse
the reaction, but this was not observed at 700 nm. Thus,
the hydroxylase was finally confirmed as a member of
the class of cytochrome P450enzymes and most prob-
ably was a new member. When a selection of cell sus-
pension cultures of different plant families and genera
was tested for hydroxylase activity it turned out that
exclusively R. serpentina cells contained DOSH indicat-
ing a specific occurrence of the enzyme in this particular
cell system producing sarpagine (Table 3).
P450character of the hydroxylase. We also tested a
17,18
series of typical cytochrome P450inhibitors
and all
showed significant inhibition of the reaction, which
indicated that DOSH belongs to the class of cytochrome
P450monooxygenases. The strongest inhibitor is BAS
110W which resulted in complete inhibition at 1 mM
and 83.3% inhibition at 0.1 mM concentration. Further
studies showed that the hydroxylase could also be
inhibited by carbon monoxide. In particular, the rever-
sal of the inhibition by light with a maximum at 450nm
Sarpagine is one of the major indole alkaloids in root
bark of Rauvolfia species8 but occurring also in other
plant parts,21 Rauvolfia cell cultures6 and Rauvolfia
hybride systems.22 It might be a late alkaloid in the
biosynthesis of alkaloids of this particular plant or cell
systems. The enzymatic formation of sarpagine descri-
bed here is obviously a reaction terminating one of the
side routes of alkaloid metabolism emerging from
ajmaline biosynthesis.
is regarded as the most reliable proof for a cytochrome
19,20
P450containing enzyme.
In fact, the activity of
deoxysarpagine hydroxylase was reduced 30% in dark
when compared to that in light in the presence of a
nitrogen/oxygen mixture, indicating that light can
increase the hydroxylase activity (Table 2). The enzyme
activity decreased to ꢁ30% when the gas mixture was
Table 1. Influence of typical cytochrome P450inhibitors on 1-0
deoxysarpagine hydroxylase activity of cultivated Rauvolfia cellsa
Inhibitor
Concentration (mM)
Rel. inhibition (%)
Ancymidole
1.048.8
0.1
1.0
0.1
1.083.6
0.1
0.02
0.01
1.069.2
0.1
1.0
0.1
45.5
100.0
83.3
BAS 110W
BAS 111W
Cytochrome c
Ketoconazole
LAB 150978
Metyrapone
Tetcyclacis
35.2
90.8
45.7
26.3
74.1
35.7
1.083.1
0.1
1.045.3
0.1
57.8
30.1
Figure 3. The effect of 10-deoxysarpagine concentration on deoxy-
sarpagine hydroxylase activity (standard incubation mixture, Km=7.4
mM, Vmax=0.55 nkat). The insert shows the double-reciprocal plot.
aStandard incubation mixture; 100% enzyme activity=0.45 nkat.
Figure 4. Competitive inhibition of deoxysarpagine hydroxylase by
NADP+ in presence of 0.75, 1.25, 2.5 mM NADPH (Lineweaver–
Figure 2. The effect of NADPH concentration on deoxysarpagine
hydroxylase activity (standard incubation mixture, Km=25 mM,
Vmax=0.36 nkat). The insert shows the double-reciprocal plot.
Burk plot: *, without NADP+; &, 0.5 mM NADP+
NADP+; &, 1.5 mM NADP+).
;
ꢂ
, 1.0mM

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B. Yu et al. / Bioorg. Med. Chem. 10 (2002) 2479–2483
Table 2. Effect of light and carbon monoxide on deoxysarpagine
hydroxylase activity assayed with microsomes isolated from 3 days old
R. serpentina cell culturesa
Table 3. Taxonomic distribution of deoxysarpagine hydroxylase in
various cell suspension culturesa
Cell culture
Family
Sarpagine synthase
(rel. act.%)
Gas mixture
Illumination
Deoxysarpagine
hydroxylase activity (%)
Rauvolfia serpentina
Agrostis tenuis.
Apocynaceae
Poaceae
Apocynaceae
Euphorbiaceae
Caprifoliaceae
Solanaceae
Rubiaceae
Apocynaceae
Caryophyllaceae
100
b
N2/O2 9:1
N2/O2 9:1
CO/O2 9:1
CO/O2 9:1
CO/O2 9:1
CO/O2 9:1
Light
Dark
Dark
100
b
b
b
b
b
b
b
72.1
19.8
29.2
23.4
19.1
Catharanthus roseus
Euphorbia peplus
Lonicera morrowii
Nicotiana tabacum
Ophiorrhiza pumila
Rhazya stricta
Light
Blue (450nm)
Red (700 nm)
aStandard incubation mixture in a special apparatus for varying illu-
mination and different gas mixtures as described previously;13 100%
enzyme activity=0.25 nkat.
Saponaria officinalis
aStandard growth conditions and harvest on day 3.
bNo detectable activity in HPLC, detection limit <1% rel. act., 100%
activity=0.45 nkat.
The obtained results extend our knowledge on enzy-
matic Rauvolfia alkaloid biosynthesis. They also allow
us to study the appropriate cDNA coding for the sar-
pagine synthesizing enzyme after its purification, Cyto-
chrome P450enzymes are also an interesting class of
enzymes from the point of view of organic synthesis.
They hydroxylate a variety of compounds in a regio-
and stereoselective manner which is difficult to achieve
by synthetic methods. For that reason this type of
enzyme might become very important in future times
when a high yielding heterologous expression becomes
available. Therefore, the purification of the novel
DOSH is now under further investigation.
organic layers were combined, concentrated, and chro-
matographed on TLC (silica gel 60F 254; Merck Darm-
stadt, Germany) with solvent system CHCl3/MeOH/
NH3 90:10:0.1. The Rf value of 10-deoxysarpagine (Rf
0.54) was identical with that of a reference compound.
After elution the product was measured by EI–MS
(Finnigan MAT 44S quadrupole instrument) at 70eV.
Fragmentation pattern were identical to a reference
sample of 10-deoxysarpagine.
Enzyme preparations
All enzyme isolations were carried out at 4 ꢀC. The used
buffers were: Buffer A: 100 mM Tris–HCl, pH 8.0, 10
mM KCl, 20% sucrose and 10 mM b-mercaptoethanol.
Buffer B: buffer A with additional 0.1 mM EDTA. The
deep-frozen cell material (200 g) of R. serpentina was
pulverized in the presence of liquid nitrogen, homo-
genized by stirring for 0.5 h in 200 mL buffer A and fil-
tered through four layers of cheese-cloth. This crude
protein extract was centrifuged at 13,000g for 30min.
From the supernatant the microsomes were prepared by
Mg2+ precipitation as described.12 The microsomal
pellets were washed twice with 40mL buffer B, then
suspended in 16 mL buffer B and homogenized with a
Potter homogenisator (8.0Â0.5 cm, Xinghua Company,
Shanghai, China). The obtained suspension was used as
microsomal fraction and was employed directly for
enzyme assays or stored at À25 ꢀC. Protein concentra-
tions were determined according to a method described
by Bradford.25
Materials and Methods
Plant cell material and culture method
Cell suspension cultures of R. serpentina were grown as
previously described23 under constant illumination on
gyratory shakers (100 rpm) in 1-L Erlenmeyer flasks
containing 400 mL LS-medium (Table 3).24 For enzyme
preparation 3 days old cultured cells were harvested by
suction filtration. The cell material was frozen with
liquid nitrogen and stored at À25 ꢀC until required for
isolation of microsomes.
Biochemicals
NADPH, NADP+ and NADH were purchased from
AppliChem (Darmstadt, Germany). Ancymidole, cyto-
chrome c, ketoconazole, metyrapone and sarpagine
were obtained from Sigma-Aldrich (Taufkirchen, Ger-
many). Tetcyclacis, LAB 150978, BAS 110W and BAS
111W were kindly provided by BASF (Ludwigshafen,
Germany). All other commercially available materials
were of highest purity. Vellosimine was isolated from R.
serpentina hairy root cultures by a method which was
earlier described for the alkaloid vinorine.14
Enzyme assay
Deoxysarpagine hydroxylase was assayed in an incu-
bation mixture comprising 10 mL 10-deoxysarpagine (1
mM), 100 mL NADPH (1.2 mM) and 200 mL micro-
somes (1 mg protein) in buffer B (total volume 500 mL).
Incubation was performed for 4 h at 35 ꢀC with shaking
(400 rpm) in open reaction vials. The reaction was
initiated by the addition of NADPH to the incubation
mixture and terminated by adding 500 mL of MeOH.
The mixture was subsequently centrifuged at 14,000g
for 5 min and 40 mL of the supernatant was assayed by
RP-HPLC.
Subsequently, 10-deoxysarpagine was prepared from
vellosimine: vellosimine (1 mg) was dissolved in the
mixture of 0.6 mL MeOH and 0.6 mL citrate–buffer
(1 M, pH 5.0). An excess of NaBH4 was added and the
reaction lasted for 1 h and then the reaction mixture was
extracted with EtOAc three times (2 mL each). The

B. Yu et al. / Bioorg. Med. Chem. 10 (2002) 2479–2483
2483
HPLC analysis
were determined by Lineweaver–Burk plots (Figs. 2 and
3).
The amount of sarpagine was determined by RP-HPLC
using a Merck LiChrosolv RP select B column [250 Â 4
mm, Merck (Darmstadt, Germany)]; gradient 68% 20
Taxonomic distribution of the hydroxylase
mM (NH4)2CO3/32% MeCN for
5
min, 40%
For the determination of the taxonomic distribution of
DOSH the isolation of microsomes from cell suspension
cultures of different plant families and genera and incu-
bation conditions were as described above.
(NH4)2CO3/60% MeCN for 7 min, monitoring the
absorption at 278 nm. The quantities of sarpagine were
calculated from the peak area at 278 nm recorded using
a Chromato-integrator (Hitachi, Japan). Retention
times of 10-deoxysarpagine and sarpagine were 11 and 7
min, respectively.
Acknowledgements
Supported by the Deutsche Forschungsgemeinschaft
(Bonn, Bad-Godesberg), Fonds der Chemischen Indus-
trie (Frankfurt/M.) and by the Bundesministerium fur
Bildung und Forschung (Bonn) is highly acknowledged.
We thank Dr. W. Rademacher from BASF (Ludwig-
shafen) for providing us with a range of cytochrome
P450inhibitors. Dr. Bing-Wu Yu thanks the Deutscher
Akademischer Austauschdienst (Bonn) for a scholar-
ship. We also appreciate the linguistic help of Prof. Dr.
W. E. Court (Mold, Wales).
Identification of the reaction product
The identification of the product was carried out on the
basis of UV and MS-measurement. In an open 100 mL
flask 200 mL 10-deoxysarpagine (1.0 mM), 2 mL
NADPH (1.2 mM), 20mL microsomes (20mg protein)
and 20mL buffer B were incubated while shaking (100
rpm) at 35 ꢀC for 4 h. The appropriate control experi-
ment was performed without substrate. MeOH (20mL)
was added to the mixture to terminate the reaction.
Then the reaction solution was three times extracted
with EtOAc (each 30mL). The organic fractions were
combined and evaporated. The residue was dissolved in
References and Notes
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+
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pH-Optimum determination and oxygen dependence
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Oxygen dependence experiments were performed as
described previously.13 The determination of pH opti-
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above, but using the following two buffer solutions: 100
mM potassium phosphate buffer (pH 5.0–7.5), 100 mM
Tris–HCl buffer (pH 7.0–9.0); each with 10 mM KCl,
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Inhibition experiments
The P450inhibitors, ancymidole, cytochrome c, keto-
conazole, metyrapone, tetcyclacis, LAB 150978, BAS
110W and BAS 111W were tested. Metyrapone and
cytochrome c were dissolved in H2O and others were
dissolved in Buffer B. The concentrations of inhibitors
were adjusted to 1 and 0.1 mM in the reaction solution
except for cytochrome c (20and 10 mM). The inhibition
experiments were carried out using the standard assay
described above.
20. Lau, S. M.; Harder, P. A.; O’Keefe, D. P. Biochemistry
1993, 32, 1945.
21. Court, W. E. Planta Med. 1983, 48, 228.
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Determination of kinetic parameters
The apparent Michaelis constant (Km) and maximal
reaction velocity (Vmax) for NADPH and sarpagine