Heterologous expression of a
Rauvolfia
cDNA encoding strictosidine
glucosidase, a biosynthetic key to over 2000 monoterpenoid indole
alkaloids
Irina Gerasimenko, Yuri Sheludko, Xueyan Ma and Joachim Sto¨ ckigt
Lehrstuhl fu
¨
r Pharmazeutische Biologie, Institut fu
¨
r Pharmazie, Johannes Gutenberg-Universita
¨
t Mainz, Germany
Strictosidine glucosidase (SG) is an enzyme that catalyses the
second step in the biosynthesis of various classes of mono-
terpenoid indole alkaloids. Based on the comparison of
cDNA sequences of SG from Catharanthus roseus and
raucaffricine glucosidase (RG) from Rauvolfia serpentina,
primers for RT-PCR were designed and the cDNA encoding
SG was cloned from R. serpentina cell suspension cultures.
The active enzyme was expressed in Escherichia coli and
purified to homogeneity. Analysis of its deduced amino-acid
sequence assigned the SG from R. serpentina to family 1 of
glycosyl hydrolases. In contrast to the SG from C. roseus,
the enzyme from R. serpentina ispredictedtolackan
uncleavable N-terminal signal sequence, which is believed to
direct proteins to the endoplasmic reticulum. The tempera-
ture and pH optimum, enzyme kinetic parameters and
substrate specificity of the heterologously expressed SG were
studied and compared to those of the C. roseus enzyme,
revealing some differences between the two glucosidases.

terpenoid indole alkaloids is the glucoalkaloid strictosidine
[7–10]. It is formed by condensation of tryptamine, the
decarboxylation product of tryptophan, and the monoter-
pene secologanin catalysed by the enzyme strictosidine
synthase (SS) [11]. The biosynthetic pathways leading to
different classes of indole alkaloids branch off somewhere
downstream of strictosidine. The first point where this
divergence may take place is the deglucosylation of strict-
osidine catalysed by strictosidine glucosidase (SG). The
unstable aglycone formed in this reaction is further conver-
ted through unknown intermediates to different indole
alkaloids exhibiting structurally most diverse carbon skel-
etons (Fig. 1). About 2000 of these secondary metabolites
are known to occur in higher plants. Many of them are
important because of various pharmacological and thera-
peutic applications such as the cytostatic vincaleucoblastine
and vincristine used in cancer chemotherapy, the toxin
strychnine, the vasodilative yohimbine, the neuroleptic
reserpine, the antihypertensive ajmalicine and the anti-
arrhythmic ajmaline.
The complex chemical structure of ajmaline, an alkaloid
from the Indian medicinal plant Rauvolfia serpentina Benth.
ex Kurz, consists of a hexacyclic carbon skeleton bearing
nine chiral carbon centres. About 10 enzymes participate in
its formation [5]. The cloning and heterologous expression
has already been achieved for a number of enzymes of this
pathway, such as SS [12,13], polyneuridine aldehyde esterase
(PNAE) [14], the cytochrome P450 reductase (M. Ruppert
&J.Sto
¨

leading from strictosidine to ajmaline. In the present article,
we report on cloning and heterologous expression in
Escherichia coli of the cDNA from R.serpentina cell
suspension cultures coding for SG [17]. An analogous
enzyme was characterized from cell suspension cultures of
Catharanthus roseus [18,19] and recently it has been cloned
from the same source and heterologously expressed in yeast
[20]. In our study, we compare the primary structure,
general properties, enzyme kinetics and substrate specificity
of both glucosidases. The unstable intermediates and the
end products formed during in vitro deglucosylation of
strictosidine and its Nb-methylated derivative (dolichanto-
side) are also investigated.
MATERIALS AND METHODS
Plant material
Cell suspensions were cultivated in 1-L conical flasks
containing 300 mL liquid Linsmaier and Skoog (LS)
medium [21] at 100 r.p.m. in diffuse light (600 lux).
Cloning of SG cDNA
Total RNA from 6-day-old R. serpentina cell suspension
cultures was isolated using peqGOLD RNAPure solution
(PEQLAB, Erlangen, Germany) according to the manu-
facturer’s manual. OligoT primer (T
15
-NNN) and RLM
reverse transcriptase (Promega, Mannheim, Germany) were
used for first strand cDNA synthesis. PCR was carried out
in Genius thermocycler (Techne, Burkhardtsdorf,
Germany) with Taq DNA polymerase from Gibco (Karls-
ruhe, Germany) under the following conditions: 94 °Cfor

Sequence analysis
The deduced amino-acid sequence was scanned for the
occurrence of conserved patterns using the
PROSITE
[23]
database. For prediction of transmembrane helices the
servers
HMMTOP
[24],
TMHMM
[25] and
SOSUI
(Tokyo
University of Agriculture & Technology) were used. The
subcellular localization was predicted by
PSORT
server [26].
Expression and purification of SG
The restriction enzymes were purchased from New England
Biolabs (Schwalbach/Taunus, Germany); the T4 DNA
ligase was from Promega. The full-length SG cDNA was
inserted in the NcoIandPstI sites of the pSE280 vector
(Invitrogen, Karlsruhe, Germany) and expressed in E. coli
strain TOP10 (Invitrogen) growing in liquid Luria–Bertani
medium supplemented with 50 mgÆL
)1
ampicillin at 37 °C.
A control bacterial culture contained the vector pSE280
without an insert. To obtain a crude enzyme preparation,
100 mL of an overnight grown E. coli culture was centri-

EDTA; 0.5
M
NaCl; 0.1% Triton X-100).
After cracking the cells in a French press, the crude extract
was centrifuged (15 000 g, 30 min) and loaded onto gravity
flow column (diameter 3 cm) packed with chitin beads
(20 mL) and pre-equilibrated with 200 mL of column buffer
(20 m
M
Tris/HCl, pH 8.0; 1 m
M
EDTA; 0.5
M
NaCl). After
washing with 150 mL of cell break buffer followed by
150 mL of column buffer, the column was flashed with
50 mL of cleavage buffer (20 m
M
Tris/HCl, pH 8.0; 1 m
M
EDTA; 0.5
M
NaCl; 50 m
M
dithiothreitol). The flow was
stopped and the column kept for 23 h at 4 °C for cleavage of
Fig. 1. The key role of strictosidine in the
biosynthesis of different classes of monoterpe-
noid indole alkaloids.
Ó FEBS 2002 Cloning of strictosidine glucosidase from Rauvolfia (Eur. J. Biochem. 269) 2205

5 min) the supernatant was analyzed by HPLC on CC 250/
4 Nucleosil 100–5 C18 column (Macherey-Nagel, Du
¨
ren,
Germany) using the following solvent system: acetonitrile/
39 m
M
NaH
2
PO
4
(pH 2.5), gradient 15 : 85 fi 25 : 75
within 1 min, fi 40 : 60 within 6.5 min, fi 40 : 60
for 2.5 min, fi 85 : 15 within 0.5 min, fi 85 : 15 for
4.5 min, fi 15 : 85 within 0.5 min, fi 15 : 85 for 4.5 min;
1.2 mLÆmin
)1
flow rate, detection at 250 nm. For substrate
specificity studies an alternative strictosidine glucosidase
activity assay was used based on quantitative determination
of released glucose. The reaction mixture (total volume
100 lL, 0.1
M
citrate/phosphate buffer, pH 5.0) containing
putative substrates (400 nmol in 20 lLMeOH)and0.13 lg
strictosidine glucosidase (45.5 pkat with strictosidine) was
incubated at 30 °C overnight (16 h). The reaction was
terminated with 200 lLMeOH,and100 lL of the resulting
mixture were added to 1 mL of the Glucose Trinder
Reagent (Sigma, Deisenhofen, Germany). The D

of 5a-carboxystrictosidine, 100 l
M
)250 l
M
of 19,20-dihydrostrictosidine, and 25 l
M
)250 l
M
of
Nb-methylstrictosidine. The pH optimum was determined
by incubation of 20 nmol of strictosidine with 26 ng of SG
for 30 min at 30 °C in different buffers: 0.1
M
citrate/
phosphate (pH 3.8–7.0), 0.1
M
KP
i
(pH 5.8–8.0), and 0.1
M
Tris/HCl (pH 7.0–9.0). The temperature optimum was
determined by incubation of 12.5 nmol of strictosidine with
13 ng of SG in 0.1
M
citrate/phosphate buffer (pH 5.0) for
30 min at different temperatures (13–65 °C). Inhibition by
0.25 m
M
cathenamine, 0.25 m
M

254
plates, 20 · 20 cm (Merck, Darmstadt,
Germany) were used with the solvent systems SS1/petro-
leum ether/acetone/diethylamine (7 : 2 : 1) or SS2/CHCl
3
/
MeOH (8 : 2). Substances were detected by measuring the
A
254
and colours after spraying with ceric ammonium sulfate
reagent (CAS). EI-MS measurements were carried out with
a quadrupole instrument (Finnigan MAT 44S) at 70 eV.
HR-EI-, HR-FAB-, and FD-MS spectra were recorded on
JEOL JMS-700 mass spectrometer.
1
H-NMR spectra were
measured using AMX 400 and DRX 600 instruments
(Bruker, Karlsruhe, Germany) with CDCl
3
and pyridine-
d
5
as solvents. The COSY, NOESY, HSQC and HMBC
experiments were performed on the DRX 600 instrument.
Preparation of substrates
Strictosidine was prepared according to the published
procedure [28] or isolated from Rauvolfia serpen-
tina · Rhazya stricta somatic hybrid cell subcultures
RxR17K as reported [29]. Dolichantoside was prepared
from strictosidine by methylation using NaBH

,calc.forC
28
H
29
O
6
N
3
, 503.2056).
Identification of intermediate under reducing
conditions
(a) Strictosidine (225 nmol) was incubated in 0.1
M
citrate/
phosphate buffer (pH 5.0) (total volume 1.5 mL) with
132 lg crude transgenic E. coli protein in presence of
450 nmol NaBH
3
CN for 15 min at 30 °C. The reaction
mixture was extracted with ethyl acetate. The organic phase
was evaporated and the residue analyzed by 2D-TLC with
2206 I. Gerasimenko et al. (Eur. J. Biochem. 269) Ó FEBS 2002
solvent system SS1. The product located at Rf 0.51 was
identified as tetrahydroalstonine (12) by comparison of its
EI-MS data with those of an authentic sample.
(b) Strictosidine (0.45 lmol) was incubated in 0.1
M
citrate/phosphate buffer (pH 5.0) (total vol. 1.5 mL) with
132 lg crude transgenic E. coli protein in presence of
900 lmol NaBH

(rel.int.%)382(7,M
+
), 381 (10), 367 (7), 213 (10), 199 (8),
185 (100), 171 (15), 156 (18), 144 (17). HR-EI-MS: m/z
382.1884 (M
+
,calc.forC
22
H
26
O
4
N
2
, 382.1893), 367.1681
(M
+
-CH
3
,calc.forC
21
H
23
O
4
N
2
, 367.1658).
1
HNMR

3
), 52.9
(t, C-5), 60.8 (d, C-3), 76.7 (d, C-19), 78.2 (d, C-21), 108.7
(s, C-7), 111.6 (d, C-12), 112.1 (s, C-16), 118.3 (d, C-9), 119.9
(d, C-10), 122.0 (d, C-11), 128.0 (s, C-8), 137.6 (s, C-13),
138.3 (s, C-2), 154.7 (d, C-17), 168.1 (s, CO
2
CH
3
). Import-
ant NOE correlations: H-3–H-14a; H-15–H-19; H-21–H-
12, H
3
-18, H-19, H-20.
RESULTS AND DISCUSSION
Cloning of cDNA encoding strictosidine glucosidase
Primers for PCR were designed on the basis of comparison
of cDNA sequences of strictosidine glucosidase (SG) from
C. roseus [20] and raucaffricine glucosidase (RG) from
R. serpentina [16], two enzymes expected to have the highest
homology to the SG from R. serpentina.RT-PCRexperi-
ments yielded a 1311-bp long DNA fragment with a high
homology of 79.9% to C. roseus SG. After successful
amplification of cDNA ends containing start and stop
codons, the full-length cDNA was generated by PCR with
primers for 3¢ and 5¢ ends including the necessary restriction
sites. As the 5¢ RACE PCR products contained an in-frame
stop codon 12 bp upstream of the start codon, the obtained
ORF of 1599 bp was full-length (Fig. 2). The encoded
protein of 532 amino acids has a calculated molecular mass

SG from C. roseus,theR. serpentina enzyme lacks an
uncleavable N-terminal signal sequence that would direct
the protein to the endoplasmic reticulum (ER) and form a
transmembrane segment, as predicted using
PSORT
software
[26]. The length and peak value of the central hydrophobic
region and the net charge of the N-terminal basically
charged region were considered to predict the presence of
signal sequence and the absence of consensus pattern
around the cleavage sites suggests that the putative signal
sequence of C. roseus SG is uncleavable [26]. The SG from
C. roseus was indicated to be localized in the ER by sucrose
gradient analysis and in vivo enzyme activity staining studies
[20], although earlier ultracentrifugation experiments
showed that the C. roseus SG occurs in at least two forms,
one soluble and one membrane-associated [36].
To prove whether the cDNA cloned from R. serpentina
indeed encoded the SG, it was expressed in E. coli. Crude
extracts of the bacteria transformed with pSE280 vector
containing SG cDNA showed high strictosidine glucosidase
activity (2.4 pkatÆlg
)1
total protein), whereas for control
cultures bearing the same vector without insert no SG activity
could be detected. These results allow us to conclude that the
cloned cDNA indeed encodes SG from R. serpentina.
Properties of heterologously expressed SG
To achieve simple and efficient purification of the enzyme,
SGwasexpressedinfusionwiththeinteintag[37]and

1m
M
serpentine, although at a significant lower degree
(Table 1), indicating a close relationship of both enzymes.
The K
m
value for strictosidine was 0.12 m
M
, which corres-
Fig. 2. cDNA Sequence and deduced amino
acid sequence of SG from R. serpentina. Motifs
conserved in members of glycosyl hydrolases
family 1 are shaded, the putative catalytic
glutamate residues are marked. A, proton
donor. B, nucleophile.
2208 I. Gerasimenko et al. (Eur. J. Biochem. 269) Ó FEBS 2002
ponds well to the data of two SG enzymes characterized
from C. roseus cell cultures [18], although the K
m
value
determined for C. roseus SG recently [19] is much lower
(Table 1). The stable end product of in vitro strictosidine
deglucosylation, cathenamine, as well as the final product of
the indole alkaloid biosynthetic pathway in R. serpentima,
ajmaline, did not inhibit the enzymatic reaction at 0.25 m
M
concentration.
Size-exclusion chromatography on Superdex-75 column
revealed that the purified heterologously expressed SG from
R. serpentina has a molecular mass > 450 kDa, as it has

(I) £ 20 l
M
ND 0.12 m
M
0.1 m
M
(II)
V
max
0.23 n
M
Æmin
)1
(I) 180–230 pkatÆmg
)1
ND 347 pkatÆlg
)1
0.12 n
M
Æmin
)1
(II)
pH optimum 6.0–6.4 6.0–8.5 ND 5.0–5.2
Temperature optimum 30 °CND ND50°C
Inhibition by ND ND
1m
M
Cu
2+
50% 8.8%

2-naphthyl-b-
D
-glucoside, cinnamic acid glucoside, con-
iferin, esculin, fluorescein-glucoside, isatinoxim-glucoside,
prunasin, rhapontin, rutin, sinigrin and zeatin-glucoside).
Thus the SG from R. serpentina has a high degree of
substrate specificity, as it has been also observed for the
C. roseus SG [18,19].
Products of enzymatic deglucosylation of strictosidine
With sufficient expression of SG in E. coli, pure R. serpen-
tina enzyme activities became available for the first time to
investigate the mechanism of strictosidine conversion
in more detail (Fig. 5). Similar experiments have been
previously carried out with rather crude enzyme extracts
from C. roseus cell suspensions [42]. To gain more detailed
insight into the mechanism of strictosidine conversion, we
carried out a series of experiments. Incubation of strictosi-
dine with heterologously expressed SG led to the formation
of cathenamine (8) exhibiting identical EI-MS and
1
H
NMR data (not shown) with those previously reported [43].
As it cannot be excluded that unstable intermediates formed
after strictosidine deglucosylation may change their struc-
ture during EI-MS measurement, milder ionization tech-
niques were applied. But the FD-MS and HR-FAB-MS
spectra confirmed that the main deglucosylation product
represents cathenamine (8). We therefore concluded that the
in vitro deglucosylation of strictosidine by SG from
R. serpentina results in the same product as the reaction

shown).
Deglucosylation of dolichantoside
To retard the intramolecular condensation of the C-21
aldehyde and Nb amino groups leading to ring closure, we
modified the structure of strictosidine. Nb-Methylstrictosi-
dine (dolichantoside) (2) was found to be the only substrate
with substituted b-nitrogen that was converted by the
enzyme at sufficient rate (Table 2). Its incubation with SG
resulted in the formation of several products. EI-MS
screening revealed that the most unpolar of them had a
molecular mass of 382, corresponding to the putative
Nb-methyldialdehyde (6). HR-EI-MS measurement
confirmed the elemental composition C
22
H
26
O
4
N
2
.But
the
1
H-NMR spectrum showed no signals which would
correspond to the expected aldehyde protons, as well as to
the vinyl side chain. Absence of a signal from Na-H
suggested that one of the aldehyde groups of (6)hasreacted
with the Na amino group. In addition, chemical shifts of Nb
methyl protons (d 2.39), H-3 (d 3.87) and protons at C-5 (d
2.60 and 3.52) indicated a tertiary b nitrogen. Presence of a

As reported recently, the enzymatic deglucosylation of
dolichantoside by a crude enzyme preparation from
Strychnos mellodora resulted in the formation of a
quaternary alkaloid, Nb-methyl-21-hydroxy-mayumbine,
as a major product, in which the condensation of C-21
aldehyde and Nb amino groups occurred [45]. The pattern
of conversion products was the same after incubation of
dolichantoside with SG from C. roseus (as crude enzyme
preparation) and a less specific glucosidase from sweet
almonds [45]. The 3-isocorreantine A identified in this
study was, however, not detected in these experiments,
although when treated with an unspecific b-glucosidase,
3-isodolichantoside gave correantine A and its 21-epimer
[44]. Our detection of 3-isocorreantine suggests that the
dialdehyde (6) is released from the enzyme and converts
immediately to (9). Bearing in mind that the reduction
of 18,19-double bond in strictosidine can influence its
binding the SG (which is demonstrated by a higher K
m
value, Table 2), the bond rotation necessary for the
reaction between C-21 and Na is not likely to occur in
the enzyme–substrate complex. The described experiments
indicate that the ring D closure is a fast and spontaneous
reaction.
CONCLUSIONS
It has been suggested that SG may play a role in the
divergence of indole alkaloid biosynthetic pathways [20].
This present study demonstrates that the in vitro conversion
of strictosidine by SGs from two different plants, C. roseus
and R. serpentina, occurs by the same mechanism. It results

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