Molecular cloning and functional bacterial expression of a plant glucosidase specifically involved in alkaloid biosynthesis

Article abstract of DOI:10.1016/S0031-9422(00)00175-8

Monoterpenoid indole alkaloids are a vast and structurally complex group of plant secondary compounds. In contrast to other groups of plant products which produce many glycosides, indole alkaloids rarely occur as glucosides. Plants of Rauvolfia serpentina accumulate ajmaline as a major alkaloid, whereas cell suspension cultures of Rauvolfia mainly accumulate the glucoalkaloid raucaffricine at levels of 1.6 g/l. Cell cultures do contain a specific glucosidase, known as raucaffricine-O-β-D-glucosidase (RG), which catalyzes the in vitro formation of vomilenine, a direct intermediate in ajmaline biosynthesis. Here, we describe the molecular cloning and functional expression of this enzyme in Escherichia coli. RG shows up to 60% amino acid identity with other glucosidases of plant origin and it shares several sequence motifs with family 1 glucosidases which have been characterized. The best substrate specificity for recombinant RG was raucaffricine (K(M) 1.3 mM, V(max) 0.5 nkat/μg protein) and only a few closely related structural derivatives were also hydrolyzed. Moreover, an early intermediate of ajmaline biosynthesis, strictosidine, is a substrate for recombinant RG (K(M) 1.8 mM, V(max) 2.6 pkat/μg protein) which was not observed for the low amounts of enzyme isolated from Rauvolfia cells. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(00)00175-8

Phytochemistry 54 (2000) 657±666
www.elsevier.com/locate/phytochem
Molecular cloning and functional bacterial expression of a plant
glucosidase speci®cally involved in alkaloid biosynthesisp
Heribert Warzechaa, 1, Irina Gerasimenkoa, Toni M. Kutchanb, Joachim Stockigta,*
aLehrstuhl fur Pharmazeutische Biologie, Institut fur Pharmazie, Johannes Gutenberg-Universitat Mainz, Staudinger Weg 5, 55099 Mainz, Germany
È
È
È
bInstitut fur P¯anzenbiochemie, Weinberg 3, 06120 Halle, Germany
È
Received 3 January 2000; received in revised form 15 May 2000
Abstract
Monoterpenoid indole alkaloids are a vast and structurally complex group of plant secondary compounds. In contrast to
other groups of plant products which produce many glycosides, indole alkaloids rarely occur as glucosides. Plants of Rauvol®a
serpentina accumulate ajmaline as a major alkaloid, whereas cell suspension cultures of Rauvol®a mainly accumulate the
glucoalkaloid rauca€ricine at levels of 1.6 g/l. Cell cultures do contain a speci®c glucosidase, known as rauca€ricine-O-b-D-
glucosidase (RG), which catalyzes the in vitro formation of vomilenine, a direct intermediate in ajmaline biosynthesis. Here, we
describe the molecular cloning and functional expression of this enzyme in Escherichia coli. RG shows up to 60% amino acid
identity with other glucosidases of plant origin and it shares several sequence motifs with family 1 glucosidases which have been
characterized. The best substrate speci®city for recombinant RG was rauca€ricine ꢀKM 1.3 mM, Vmax 0.5 nkat/mg protein) and
only a few closely related structural derivatives were also hydrolyzed. Moreover, an early intermediate of ajmaline biosynthesis,
strictosidine, is a substrate for recombinant RG ꢀKM 1.8 mM, Vmax 2.6 pkat/mg protein) which was not observed for the low
amounts of enzyme isolated from Rauvol®a cells. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Rauvol®a serpentina; Apocynaceae; Plant cell suspension culture; Rauca€ricine-O-b-D-glucosidase; Heterologous expression; Substrate
speci®city
1. Introduction
known that the cyanogenic glucosides have important
defensive roles against predation or fungal attack
(Poulton, 1990) whereas glucosylated auxins act as
inactive storage forms of these plant hormones (Cohen
and Bandurski, 1982; Brzobohaty et al., 1993).
Glucosides are widely distributed throughout the
plant kingdom. An overwhelming number of plant sec-
ondary products is reported to occur in glucosylated
form, which increases their water solubility and allows
their high vacuolar accumulation. In addition to their
enhanced solubility, glucosides may have other import-
ant features for plant metabolism, but the functions of
only a few compounds have been elucidated. It is well
Alkaloids can also occur in the glucosylated form,
although this is not necessary for enhanced solubility
since alkaloids will form ion pairs with appropriate or-
ganic acids in the plant cell. Of the 2000 monoterpe-
noid indole alkaloids that have been characterized, a
few are used in medicine as cytostatics (vincaleucoblas-
tine and vincristine), neuroleptics (reserpine) or as
vasodilatives (yohimbine) and only about 40 of these
are glucoalkaloids (Ruyter et al., 1988). In the biosyn-
thesis and metabolism of alkaloids of the ajmalan-
group, which is characteristic for the traditional ayur-
vedic medicinal plant Rauvol®a, two glucosides are
involved. The ®rst is the biosynthetic intermediate
p The cDNA sequence reported herein was deposited in GenBank
under accession number AF 149311.
* Corresponding author. Tel.: +49-6131-395751; fax: +49-6131-
3923752.
E-mail address: stoeckig@mail.uni-mainz.de (J. Stockigt).
1 Present address: Boyce Thompson Institute for Plant Research,
Tower Road, Ithaca, NY 14853-1801, USA.
0031-9422/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00175-8

658
H. Warzecha et al. / Phytochemistry 54 (2000) 657±666
strictosidine (Fig. 1), formed from tryptamine and
secologanin by the enzyme strictosidine synthase
(Stockigt and Zenk, 1977). Strictosidine occupies a
central position in the synthesis of all monoterpenoid
indole alkaloids in plant genera such as Catharanthus,
Rauvol®a, Strychnos, Cinchona, etc. (Kutchan, 1998).
In this case, the glucose moiety functions as a protect-
ing group to stabilize the molecule. After deglucosyla-
tion of strictosidine by strictosidine glucosidase, the
highly reactive and unstable aglycone is directed into
di€erent biosynthetic pathways that are species depen-
dent and result in the production of various indole
and quinoline alkaloids (Rue€er et al., 1978). This
enzyme which was ®rst described 20 years ago
(Hemscheidt and Zenk, 1980) was recently cloned
from Catharanthus roseus (Geerlings et al., 2000). In
Rauvol®a, strictosidine is converted by about 10 di€er-
ent soluble and membrane associated enzymes to the
structurally complex alkaloid ajmaline, harbouring a
six ring system with a total of nine chiral carbon
atoms. Ajmaline is an important plant-derived phar-
maceutical which is commercially isolated from Rau-
vol®a roots and has been used in the therapy of heart
disorders for the past four decades.
Several side reactions occur in cell cultures of Rau-
vol®a serpentina in addition to the major pathway
leading to ajmaline. It has been suggested that these
side reactions might exhaust the intermediates from
the ajmaline biosynthesis (Stockigt, 1995) and may
explain the low ajmaline accumulation rates of cell cul-
tures compared with the intact plant. Greater accumu-
lation in the cultivated cells has important
consequences for the biotechnological production of
ajmaline.
Cell cultures accumulate rauca€ricine, a glucoside of
vomilenine which is a direct biosynthetic precursor of
ajmaline (Fig. 1). Vomilenine glucosyl transferase
(VGT) forms rauca€ricine from vomilenine, resulting
in cell cultures that accumulate this glucoside up to
levels of 1.6 g/l and represents one of the most abun-
dant indole alkaloids to accumulate in a cell culture
(Ruyter and Stockigt, 1991). In fact, on a dry weight
basis rauca€ricine formation in the cultivated plant
cells might exceed that of intact plants by 67-fold. This
side product of ajmaline biosynthesis is deglucosylated
by another highly speci®c enzyme from the cultured
Rauvol®a cells Ð rauca€ricine glucosidase (RG, EC
3.2.1.125). Therefore, rauca€ricine biosynthesis and
the re-utilization of rauca€ricine for the ajmaline bio-
synthetic pathway could be the crucial and rate limit-
ing steps in the formation of the antiarrhythmic drug
ajmaline. For that reason, both enzymes, VGT and
RG, are of an interest.
In this article, we describe the cloning of the RG
cDNA, the functional expression of the encoded pro-
tein in Escherichia coli, and its characterization.
2. Results and discussion
2.1. Cloning of partial and full length RG cDNA
While rauca€ricine could not be deglucosylated by
any commercially available glucosidase, RG was
shown to accept only rauca€ricine and structurally clo-
sely related derivatives as a substrate (Schubel et al.,
1986). In order to study this enzyme that is present in
Rauvol®a cell cultures more thoroughly, we puri®ed
the enzyme and sequenced internal peptides (Warzecha
et al., 1999).
Fig. 1. Schematic representation of ajmaline biosynthesis in Rauvol®a
serpentina. Double arrows are indicating several enzymatic reactions.
Enzymes are: STR1, strictosidine synthase; SG, strictosidine glucosi-
dase; VGT, vomilenine glucosyl transferase; RG, rauca€ricine gluco-
sidase.
Two partial amino acid sequences of RG, the pep-

H. Warzecha et al. / Phytochemistry 54 (2000) 657±666
659
tide fragments PF 37 and PF 47 showed extensive
homology with other glucosidases. As recently
described, the sequences of several glucosidases posses-
sing homology with the peptide fragment PF 37 are
near the COOH-terminus of the corresponding
enzyme, while PF 47 homologues are located near the
NH2-terminus. Both are ¯anking a region of about
320 amino acids. To amplify a partial cDNA of RG,
two degenerate oligonucleotides were synthesized,
using one as a sense primer (RG 47s) and the other as
an antisense primer (RG 37as). A third degenerate oli-
gonucleotide (RG 47int.s) was derived from a con-
served region in other glucosidases, which is located
four amino acids downstream of the RG 47-site, and
was used for nested PCR to verify that the ampli®ed
nucleic acid is indeed a part of a glucosidase cDNA.
(up to 60% identity, Table 1), the majority of which
were plant-derived. The sequence indicates that RG
belongs to family 1 of glucosyl hydrolases (Henrissat,
1991; Henrissat and Davies, 1997). In fact, RG shares
several important motifs that are responsible for the
catalytic activity of family 1 glucosidases. The ®rst
motif is the ``Ile/Val-Thr-Glu-Asn-Gly'' sequence
between residues 418 and 422 found in one of the pep-
tides after microsequencing of RG. The glutamic acid
of this motif was previously identi®ed to be the active
site nucleophile (Withers et al., 1990; Trimbur et al.,
1992). The second residue necessary for cleavage of
glucosides might be the aspartic acid in the sequence
``Asp-X-X-Arg-X-X-Tyr'' between residues 439 and
445 (Trimbur et al., 1992) or the glutamic acid in the
``Asn-Glu-Pro/Ile'' sequence between residues 185 and
187 (Keresztessy et al., 1994). RG exhibits each of
these motifs (in addition to several other conserved
regions) and, therefore, belongs to this family of gluco-
sidases.
The comparison of amino acid sequences of RG and
of the recently heterologously expressed strictosidine
glucosidase (SG) from C. roseus (Geerlings et al.,
2000) is of special interest because of these enzymes'
common involvement in indole alkaloid biosynthesis.
The two glucosidases demonstrate relatively a high
degree of amino acid and nucleotide identity (52 and
67%, respectively), although the homology of RG to
other glucosidases was even more pronounced (see
Table 1). Moreover, the analysis of amino acid
sequence of SG did not reveal the ``Ile/Val-Thr-Glu-
Asn-Gly'' and ``Asp-X-X-Arg-X-X-Tyr'' motifs.
RG seems to be not only a new member of family 1
of glucosidases with a unique substrate speci®city, but
also an excellent example to study substrate speci®city
of the enzyme, e.g. by mutagenesis. For such investi-
gations, heterologous expression of the active enzyme
would be a prerequisite.
Since RG of the R. serpentina cell suspension shows
its optimum activity after 7 days of growth, RNA was
obtained from 6-day-old cells and was used as a tem-
plate for RT-PCR. With the above selected RG 47s
and RG 37as, a PCR product of approximately 970 bp
in length was ampli®ed. A second PCR with this tem-
plate using RG 37as and RG 47int.s as primers
resulted in an expected smaller fragment of 940 bp in
length. The former PCR product was subcloned into
pGEM-T, sequenced and the cDNA was then used as
a probe for screening of R. serpentina cDNA library.
Several clones were isolated which were identical in
sequence, but not in length. The longest cDNA con-
tained an ORF of 1623 nucleotides, a 144 nucleotide
5'-¯anking region and a 229 bp 3'-¯anking region with
a poly-A tail. The protein encoded by this sequence
consists of 540 amino acids with a calculated molecu-
lar weight of 60931 Da (Fig. 2). This value agrees well
with the molecular mass of 61 kDa for the native
enzyme from plant cell cultures (Warzecha et al.,
1999). Additionally, all the six peptide sequences
obtained by microsequencing have been found in the
deduced amino acid sequence of the cDNA (Fig. 2),
con®rming that this sequence encodes RG.
2.3. Heterologous expression
Due to the lack of information about the NH2-ter-
minus of native RG, two possible in-frame ATG
codons may function as translation start sites at
nucleotides 145 or 226 in this cDNA (Fig. 2). The
1623 bp clone was subcloned into the expression vector
pSE280 by the use of the naturally occurring NcoI-
restriction site (named pSE-RG1), whereas the shorter
1542 bp clone (using ATG at position 226) was gener-
ated by PCR (named pSE-RG2). Both constructs were
expressed in E. coli. While the clone pSE-RG1 showed
high glucosidase activity in a crude protein extract, no
deglucosylation of rauca€ricine could be measured in
the enzyme extract from pSE-RG2. This result
strongly suggested that the ®rst ATG is the translation
2.2. Sequence similarities of RG with other plant
glucosidases
Several glucosidases showed great similarity to RG

660
H. Warzecha et al. / Phytochemistry 54 (2000) 657±666
Fig. 2. Nucleotide sequence of the RG cDNA clone and its predicted amino acid sequence. Recognition sequences for EcoRI, located at 661 and
1668; NcoI, at 143 and 703; are in bold letters. Putative start codons are underlined. Boxed areas represent the peptide sequences obtained from
microsequenzing of RG.

H. Warzecha et al. / Phytochemistry 54 (2000) 657±666
661
start signal and that the amino acids between positions
1 and 21 are necessary for enzyme activity.
In the crude protein extract of the E. coli strain con-
taining pSE-RG1, an enzyme activity of 0.7 nkat/mg
protein was measured in the soluble fraction. Although
the pSE280 vector has an IPTG-inducible promoter,
RG activity could be detected without IPTG-induc-
tion. In addition, no pronounced increase of RG ac-
tivity in crude extracts was noted after induction with
IPTG. However, in contrast to the rather dicult and
inecient multistep puri®cation of RG from plant cell
cultures, the recombinant enzyme from E. coli only
required anion exchange (SOURCE 30Q), hydro-
phobic interaction (SOURCE 15 PHE) and size exclu-
sion (Superdex 75 HR) chromatography to yield
homogenous enzyme (Fig. 3).
The puri®ed enzyme catalyzed the deglucosylation
of rauca€ricine and the aglycone was identi®ed using
mass spectrometry and 1H-NMR spectroscopy. This
proved that the puri®ed protein was indeed RG.
2.4. Substrate speci®city of heterologously expressed RG
In contrast with the inherent low quantities of RG
produced in plant cells, the higher amounts of pure
enzyme now available from E. coli have only recently
allowed for an in-depth investigation of the enzyme's
substrate speci®city. Several glucosides, mainly of
plant origin, were tested as putative substrates for RG
using the pure enzyme (Table 2). Two di€erent assays
were used, one based on the detection of glucose
released and the other based on the HPLC resolution
of substrate and the aglycone formed after incubation
with the enzyme. The results obtained indicate that
both the glucosidase from Rauvol®a cells and the het-
erologously expressed enzyme from E. coli exhibit very
similar substrate speci®city. Besides the compounds
listed in the Table 2, the heterologously expressed RG
did not accept several di€erent glucosides as sub-
strates, e.g. indoxyl-b-D-glucoside, strictosidine lactam,
Fig. 3. SDS-PAGE of heterologously expressed rauca€ricine glucosi-
dase puri®ed from E. coli. Proteins were separated on a 11% gel and
coomassie stained. M, marker; 1, fraction with RG activity after
Superdex 75 column.
vincoside lactam, loganin, secologanin, arbutin, iso-
quercitrin, vanillin-b-D-glucoside, esculin, ¯uorescein-
b-D-glucoside, 4-nitrophenyl-b-D-glucoside, 6-bromo-2-
naphthyl-b-D-glucoside, sinigrin, ipecoside or rutin.
Unexpectedly, however, RG deglucosylated strictosi-
dine, as well as its derivate 5a-carboxystrictosidine.
Table 1
Homologies of RG with other plant glucosidasesa
C. roseus SGD
P. serotina AH1
P. avium PH
P. serotina PH
T. repens CG
T. repens nCG
C. speciosus FG
R. serpentina RG
C. roseus SGD
P. serotina AH1
P. avium PH
52
55
49
57
49
72
54
49
72
88
60
52
66
65
62
52
49
59
57
55
61
46
44
50
51
50
50
47
P. serotina PH
T. repens CG
T. repens nCG
a Enzymes identi®ed by their GenBank accession numbers: RG, rauca€ricine glucosidase (AF149311); SGD, Catharanthus roseus strictosidine
glucosidase (AF112888); CG, Trifolium repens cyanogenic glucosidase (X56733); nCG, T. repens noncyanogenic glucosidase (X56734); AH1, Pru-
nus serotina amygdalin hydrolase isoform AH1 (U26025); PH, P. serotina prunasin hydrolase (U50201) or Prunus avium b-glucosidase (U39228);
FG, Costus speciosus furostanol glycosid 26-O-b-glucosidase (D83177).

662
H. Warzecha et al. / Phytochemistry 54 (2000) 657±666
This strictosidine glucosidase activity was not found in
the partially puri®ed plant enzyme (Schubel at al.,
1986). This may be due to the higher amount of RG
that was expressed in the E. coli used in this study.
Moreover, 5a-carboxystrictosidine was accepted as a
substrate for RG, although this strictosidine derivative
plays no role in the biosynthesis of Rauvol®a of the
ajmalan±sarpagan group (Stockigt, 1979). The 3a(S )
stereochemistry was determined to be essential for RG
activity since the 3b(R ) epimer 5a-carboxyvincoside
was not deglucosylated. Although the data about sub-
strate speci®city of heterologously expressed SG (Geer-
lings et al., 2000) were not reported, the same
substrate preference (strictosidine compared to vinco-
side) was observed for SG from C. roseus cell suspen-
sion cultures (Hemscheidt and Zenk, 1980; Luijendijk
et al., 1998).
A comparison of KM and Vmax values for rauca€ri-
cine ꢀKM 1.3 mM, Vmax 0.5 nkat/mg) and for strictosi-
dine ꢀKM 1.8 mM, Vmax 2.6 pkat/mg) shows
rauca€ricine to be the preferred substrate. Though
similar KM values suggest comparable binding capacity
for both substrates, the much lower Vmax value for
strictosidine indicates that this glucoside is hydrolyzed
with signi®cantly lower eciency. Unfortunately there
are no data reported about the enzyme kinetics of het-
erologously expressed SG (Geerlings et al., 2000). The
KM values for strictosidine determined with SG puri-
®ed from C. roseus cells were much lower (0.2 and 0.1
mM for two isolated SG enzymes reported by
Hemscheidt and Zenk, 1980, and R20 mM reported by
Luijendijk et al., 1998), therefore, demonstrating that
this enzyme has a much higher anity for strictosidine
than the heterologously expressed RG. The question
whether C. roseus SG can also catalyze the deglucosy-
lation of rauca€ricine remains unanswered, since the
reported studies on its substrate speci®city do not
include this alkaloid.
Most likely, the hydrolysis of strictosidine is due to
secondary activity of RG. This indicates that strictosi-
dine must be cleaved by a di€erent glucosidase in Rau-
vol®a cells which has not yet been identi®ed. The
results described here are, therefore, also of special
interest for future work to be performed on the im-
portant enzyme strictosidine glucosidase, which pro-
vides the biosynthetic intermediate for the 2000
naturally occurring monoterpenoid indole alkaloids. In
this respect, determination of the gene copy number of
RG or of related genes coding for appropriate isoen-
zymes would be very informative.
2.5. Gene copy number of RG
For Southern blotting, genomic DNA of R. serpen-
tina cell suspension culture was hybridized to a 1000
bp EcoRI fragment of RG cDNA. Two bands were
observed for the BamHI and PvuII digested DNA (6.4
and 8 kb or 1.8 and 3.4 kb, respectively) while EcoRI
digested DNA showed six bands (between 1.7 and 3.4
kb, Fig. 4). Since RG cDNA has two EcoRI restriction
sites 1000 bp apart, a hybridization signal correspond-
ing to this size was expected to be found in the EcoRI
digested DNA. The sole detection of larger fragment
signals could be explained, in principle, by the presence
of one or more introns in the RG gene. An intron
inside the region between the EcoRI sites would
increase the size of the fragment. Moreover, an intron
showing another EcoRI restriction site would cause
the occurence of two bands of di€erent size, according
to the position of the site and the size of the intron. In
this case, the results of the Southern blot indicate the
presence of introns in the RG gene. As shown for
genes from maize and in several Brassicaceae, the
plant glucosidase genes contain up to 11 introns (Esen
and Bandaranayake, 1998; Rask et al., 2000). At this
Table 2
Substrate speci®city of heterologous expressed rauca€ricine glucosidasea
Substrate tested with rauca€ricine glucosidase
Relative activity
(%), determined by HPLC
Relative activity
(%), determined by glucose assay
Rauca€ricine (2 mM)
100 (49.8 pkat)b
n.d.
100 (40.75 pkat)b
77.8
21-Glucopyranosyl-hydroxysarpagan-17-al (2 mM)
21-Glucopyranosyl-hydroxysarpagan-17-ol (2 mM)
1,2-Dihydrorauca€ricine (2 mM)
1-Methyl-1,2-dihydrorauca€ricine (2 mM)
Rauca€ricine (4 mM)
84.9
82.7
62.8
70.9
47.4
100 (104.0 pkat)c
40.5
100 (77.3 pkat)c
4.2
Acetylrauglucine (4 mM)
Strictosidine (4 mM)
n.d.
6.5
7.1
12.8
5a-Carboxystrictosidine (4 mM)
23.1
a Activity of RG against rauca€ricine was set to 100%. Activity was tested by HPLC assay and/or glucose testing. n.d., not determined.
b The incubation mixture contained 0.02 mg protein.
c The incubation mixture contained 0.08 mg protein.

H. Warzecha et al. / Phytochemistry 54 (2000) 657±666
663
the native enzyme from the Rauvol®a cell suspension
culture. The molecular weight of 66:60025% deter-
mined for the active enzyme by gel chromatography
(Schubel et al., 1986) and of 61 kDa by gel electro-
phoresis (Warzecha et al., 1999) is in agreement with
the calculated weight of the complete amino acid
sequence. While the identity of the enzyme was
unequivocally con®rmed by the identi®cation of the
enzyme product vomilenine, the former's ability to
hydrolyze strictosidine was here detected for the ®rst
time. This is perhaps a result of the greater availability
of RG from heterologous expression of the protein in
E. coli. This shows the importance of re-investigating
every recombinant enzyme for determining its func-
tion, especially when quantities available from plant
material are very low and, therefore, limit extensive
biochemical characterizations. Although the activity of
recombinant enzymes can usually be detected in crude
protein extracts, characterization should be carried out
with puri®ed enzymes only. Several microorganisms
possess the ability to convert plant secondary products
like strictosidine (Shen et al., 1998) and it can not be
excluded that host enzymes in crude extracts may be
responsible for product formation. This fact could dis-
tort statements about substrate speci®city and the
identity of products. Moreover, the structure of the
formed products must be con®rmed by spectroscopic
methods such as MS and NMR to unequivocally ver-
ify the functional identity of the enzyme.
The present results do not allow us to conclude
whether RG also catalyses the SG activity in Rauvol®a
and, therefore, other glucosidase clones from this plant
are now being isolated, expressed and characterized
(Gerasimenko and Stockigt, in preparation). The iso-
lation and identi®cation of RG will also allow the inhi-
bition or overexpression of this side reaction in
cultivated Rauvol®a cells and the investigation of the
signi®cance of this particular enzyme for the entire al-
kaloid metabolism in Rauvol®a.
3. Experimental
3.1. Plant cell cultures
Fig. 4. Southern blot with DNA of R. serpentina cell suspension cul-
ture. Genomic DNA (10 mg per lane) was digested with restriction
enzymes and resolved by agarose gel electrophoresis. 32P-labled 1000
bp RG cDNA fragment obtained from EcoRI digestion was used as
hybridization probe. While in the cDNA two recognition sites for
EcoRI occur, none of the other restriction endonucleases should
hydrolyze within the RG reading frame.
Cell suspension cultures of R. serpentina BENTH.
ex KURZ were cultivated in 1 l conical ¯asks contain-
ing 400 ml LS medium (Linsmaier and Skoog, 1965)
on a gyratory shaker (100 rpm) at 24228C in di€use
light (600 lx). After 7 days, half of the cell suspension
was routinely added to 200 ml of fresh LS medium
under aseptic conditions.
stage it could not be concluded if there is more than
one copy of the RG gene in the Rauvol®a genome.
In conclusion, RG obtained by heterologous ex-
pression in E. coli shows similar properties to those of
3.2. Isolation and analysis of nucleic acids
Total RNA was isolated from plant cell suspension

664
H. Warzecha et al. / Phytochemistry 54 (2000) 657±666
cultures as described in (Ausubel et al., 1997).
Poly(A)+ RNA was puri®ed from total RNA using
Oligotex2 beads (Qiagen, Hilden, Germany). DNA
was obtained from cell cultures using the DNeasy Kit
(Qiagen). Cell suspensions were harvested, immediately
frozen with liquid nitrogen and freeze dried. The lyo-
philized cells were then pulverized and processed
according to the manufacturers instructions. For
Southern hybridization analyses, genomic DNA was
digested with restriction endonucleases, separated by
electrophoresis in a 1% agarose gel in TAE bu€er
(Sambrook et al., 1989), partially hydrolyzed in 0.2 N
HCl for 15 min, 30 min in 1.5 M NaCl/0.5 M NaOH,
neutralized 2 Â 30 min in 1.5 M NaCl/0.5 M Tris±HCl
(pH 7.2) and 1 mM EDTA. After transferring the
nucleic acids by capillary blotting (Sambrook et al.,
1989) to a nylon membrane (Hybond N+, Amersham±
Pharmacia) and UV crosslinking the DNA to the ®lter,
prehybridization and hybridization were carried out
overnight (5Â SSPE [0.19 M NaCl, 10 mM NaPi, 1
mM EDTA], 5Â Denhardt's, 0.1% SDS, 200 mg/ml
salmon sperm, 50% formamide) at 428C. The ®lters
were washed two times with 2Â SSC (0.15 M NaCl, 30
mM Na-citrate) (Sambrook et al., 1989) 0.1% SDS at
428C and then several times with 0.2Â SSC, 0.1% SDS
at increasing temperatures (50±658C). The hybridizing
DNA was identi®ed by autoradiography on Biomax
®lms (Kodak). DNA probes were 32P-labeled with the
Random Priming System (Life Technologies, Eggen-
stein).
matics Institute) was used. Multiple sequence align-
ment and calculation of homologies were performed
using MegAlign (LASERGENE).
3.4. Heterologous expression
For heterologous expression of RG, the vector
pSE280 (Invitrogen) was restriction digested with NcoI
and XhoI. Due to a second internal NcoI site in ad-
dition to that at the translation start (position 143), a
partial digestion of pBlueRG (XhoI digested) with
NcoI was carried out. After electrophoretic separation,
the fragment of approximately 1850 bp was puri®ed
and cloned into pSE280 (XhoI/NcoI digested). The
plasmids (pSE280RG) were expressed in TOP 10 E.
coli cells. For use of the second ATG codon as a
translation start signal, the cDNA was ampli®ed
with two extended deoxy-oligonucleotides as PCR
primers (RG2for: ACTAGCCATGGGAACGGGCT-
CTTC; RG2rev: CGATCTCGAGCTACTTTCTTAA-
TCTCTTG) and the product was cloned into pSE280.
E. coli TOP 10 was cultivated at 378C in conical ¯asks
(LB medium containing 1% trypton, 0.5% yeast
extract, 1% NaCl). For induction of RG expression,
isopropyl-b-D-thiogalactopyranoside was added to the
cells (OD 0.5) to a ®nal concentration of 1 mM. After
induction, the cells were cultivated for an additional 4
h at 378C in a gyratory shaker (200 rpm).
3.5. Protein puri®cation
A cDNA library was constructed of mRNA from 6-
day-old cell suspension culture using the l ZAP II sys-
tem (Stratagene). Screening was performed according
to the manufacturer's instructions.
To produce a small scale cell lysate of E. coli (up to
20 ml culture), the cells were pelleted, resuspended in
bu€er A (50 mM NaH2PO4, pH 8, 0.3 M NaCl) and
sonicated 6 Â 10 s with 200 W on ice. For larger
volumes, the cells were disrupted by the use of a
French press. After centrifugation, the supernatant
was used for enzyme assays or further puri®cation
steps.
For ammonium sulfate precipitation of proteins,
(NH4)2SO4 powder was added to the supernatant over
a period of 1 h (48C, continously stirring) up to a salt
concentration of 30%. After centrifugation (30 min,
10,000 Â g, 48C) (NH4)2SO4 was added to the super-
natant to a ®nal concentration of 75%. The solution
was centrifuged again, and the precipitated protein
was dissolved in 100 ml bu€er B (20 mM Tris±HCl,
pH 7.5, 10 mM b-mercaptoethanol (b-ME)). After
another centrifugation, the supernatant was dialyzed
against 10 l bu€er B overnight.
3.3. PCR and cloning
Partial cDNA clones encoding RG were generated
by PCR using cDNA produced by reverse transcrip-
tion of total RNA isolated from 6-day-old cell suspen-
sion cultures. DNA ampli®cation was performed under
the following conditions: 5 min at 948C, 3 cycles of
948C for 30 s, 408C for 2 min and 728C for 1 min;
then 30 cycles of 948C for 30 s, 508C for 2 min, 728C
for 1 min. The last step was followed by an additional
elongation step at 728C for 5 min. The ampli®ed DNA
was separated by agarose gel electrophoresis. The
band of approximately desired size was eluted from
the gel (NucleoSpin Extraction, Macherey & Nagel,
Duren, Germany) and cloned into the pGEM-T vector
(Promega, Mannheim, Germany) for further analysis.
pBluescript containing full length clones of RG cDNA
(pBlueRG) were isolated from the l ZAP library and
were used for sequencing (Genterprise, Mainz).
For anion exchange chromatography, the dialyzed
protein was applied to a SOURCE 30Q XK 50/30 col-
umn (Pharmacia) equilibrated with bu€er C (20 mM
Tris±HCl, pH 8.0; 10 mM b-ME). After washing the
column with 0.5 column volumes (CV, 240 ml) of buf-
fer C, the proteins were eluted with a linear KCl gradi-
For the alignment of the amino acid sequence of
RG the Fasta 3 program at EBI (European Bioinfor-

H. Warzecha et al. / Phytochemistry 54 (2000) 657±666
665
ent (6 CV, 0±0.5 M KCl) prepared from bu€ers C and
D (20 mM Tris±HCl, pH 8.0, 10 mM b-ME, 1 M
KCl) at a ¯ow rate of 20 ml/min. RG activity
appeared at KCl concentrations of 0.23±0.26 M. Frac-
tions containing enzyme activity were pooled and pre-
pared for the next puri®cation step by adding
(NH4)2SO4 to a resulting concentration of 1 M.
The combined fractions were resolved by hydro-
phobic interaction chromatography on a SOURCE 15
PHE XK 16/20 column (Pharmacia) which had been
equilibrated with bu€er E (20 mM Tris±HCl, pH 8.0,
10 mM b-ME; 1 M (NH4)2SO4). After washing the
column with 0.8 CV (CV 30 ml) of bu€er E, proteins
were eluted with a linear (NH4)2SO4 gradient (10 CV,
1±0 M (NH4)2SO4) prepared from bu€ers E and C at
a ¯ow rate of 10 ml/min. RG activity eluted at
(NH4)2SO4 concentrations of 0.59±0.51 M. Fractions
containing active RG were pooled and concentrated
using Centriprep 10 and Microcon 30 concentrators
(Amicon, Witten).
hydrolyzed rauca€ricine after 12 h of incubation under
the conditions described.
KM and Vmax values for rauca€ricine were deter-
mined in presence of 0.25 mg protein using both the
HPLC and the glucose release assay. KM and Vmax
values for strictosidine were determined in presence of
4 mg protein using the glucose release assay.
3.7. Other methods
Protein electrophoresis, staining, and quanti®cation
were performed, as described recently (Warzecha et al.,
1999).
Acknowledgements
This work was supported by a grant of the Deutsche
Forschungsgemeinschaft (DFG, Bonn) and by the
Fonds der Chemischen Industrie (Frankfurt/Main).
We thank Dipl. Biol. Yuri Sheludko for providing
samples of alkaloids and Mrs Laura Shevy (Boyce
Thompson Institute for Plant Research, Tower Road,
Ithaca, New York) for her kind help in correcting the
English version.
Concentrated fractions were applied to a Superdex
75 HR 10/30 column (Pharmacia) for size exclusion
chromatography (CV 30 ml). The proteins were eluted
with 20 mM Tris±HCl bu€er, pH 7.8, containing 100
mM KCl and 10 mM b-ME at a ¯ow rate of 30 ml/h
collecting 0.5 ml fractions.
3.6. Analysis of substrates and products
References
Glucosidase activity was analyzed with the HPLC
assay, as described recently (Warzecha et al., 1999).
For the identi®cation of vomilenine, the product of the
enzymatic reaction was dissolved in methanol and ana-
lyzed by GC±MS. For 1H-NMR investigations, the
product was extracted with CH2Cl2 and, after drying,
dissolved in DMSO-d6 MS and 1H-NMR data were
identical with those of a vomilenine standard.
For the testing of glucosidase activity with a broad
range of substrates, the glucose liberated was measured
using Glucose reagent (Trinder) (Sigma). This system
is based on a coupled enzyme system which consists of
glucose oxidase and peroxidase. Unless otherwise sta-
ted the incubation mixture (total volume 100 ml) con-
tained 4 mM of the corresponding substrate, 10 ml of
citrate±NaOH bu€er (pH 5.0), and 0.08 mg of RG. In-
cubations were carried out at 288C with agitation. The
reaction was terminated by addition of 200 ml metha-
nol. Of this mixture, 200 ml were added to 1 ml of Glu-
cose reagent (0.5 mM 4-aminoantipyrine; 20 mM p-
hydroxybenzene sulfonate; 15.000 U/l glucose oxidase;
10,000 U/l peroxidase; pH 7.0) and after 18 min, the
absorbance at 505 nm was recorded. Parallel incu-
bations without RG were treated identically. The
detection limit of this assay was 0.5 pkat (20 nM glu-
cose liberated in 12 h). It was shown that RG still
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman,
J.G., Smith, J.A., Struhl, K., 1997. Phenol/SDS method for plant
RNA preparation. In: Short Protocols in Molecular Biology, 3rd
ed. Wiley, New York.
Brzobohaty, B., Moore, I., Kristo€ersen, P., Bako, L., Campos, N.,
Schell, J., Palme, K., 1993. Release of an active cytokinin by a b-
glucosidase localized in the maize root meristem. Science 262,
1051±1054.
Cohen, J.D., Bandurski, R.S., 1982. Chemistry and physiology of the
bound auxins. Annu. Rev. Plant Physiol. 33, 403±430.
Esen, A., Bandaranayake, H., 1998. Insertional polymorphism in
introns 4 and 10 of the maize beta-glucosidase gene Glu1.
Genome 41, 597±604.
Geerlings, A., Martinez-Lozano Ibanez, M., Memelink, J., van der
Heijden, R., Verpoorte, R., 2000. Molecular cloning and analysis
of strictosidine b-D-glucosidase, an enzyme in terpenoid indole al-
kaloid biosynthesis in Catharanthus roseus. J. Biol. Chem. 275,
3051±3059.
Gerasimenko, I., Stockigt, J., in preparation.
Hemscheidt, T., Zenk, M.H., 1980. Glucosidases involved in indole
alkaloid biosynthesis of Catharanthus cell cultures. FEBS Lett.
110, 187±191.
Henrissat, B., 1991. A classi®cation of glycosyl hydrolases based on
amino acid sequence similarities. Biochem. J. 280, 309±316.
Henrissat, B., Davies, G., 1997. Structural and sequence-based classi-
®cation of glycoside hydrolases. Curr. Opin. Struct. Biol. 7, 637±
644.
Keresztessy, Z., Kiss, L., Hughes, M.A., 1994. Investigation of the
active site of the cyanogenic b-D-glucosidase (linamarase) from
Manihot esculenta Crantz (cassava). I. Evidence for an essential

666
H. Warzecha et al. / Phytochemistry 54 (2000) 657±666
carboxylate and a reactive histidine residue in a single catalytic
center. Arch. Biochem. Biophys. 314, 142±152.
Schubel, H., Stockigt, J., Feicht, R., Simon, H., 1986. Partial puri®-
cation and characterization of rauca€ricine b-D-glucosidase from
plant cell-suspension cultures of Rauwol®a serpentina Benth.
Helv. Chim. Acta 69, 538±547.
Kutchan, T.M., 1998. Molecular genetics of plant alkaloid biosyn-
thesis. In: Cordell, G.A. (Ed.), The Alkaloids Ð Chemistry and
Biology, 50. Academic Press, San Diego, pp. 257±316.
Linsmaier, E.M., Skoog, F., 1965. Organic growth factor require-
ments of tobacco tissue cultures. Physiol. Plant 18, 100±
127.
Shen, Z., Eisenreich, W., Kutchan, T.M., 1998. Bacterial biotrans-
formation of 3a(S)-strictosidine to the monoterpenoid indole al-
kaloid vallesiachotamine. Phytochemistry 48, 293±296.
Stockigt, J., 1979. Non-involvement of 5a-carboxystrictosidine and -
vincoside in the biosynthesis of sarpagine- and ajmaline-type al-
kaloids. Tetrahedron Lett. 28, 2615±2618.
Luijendijk, T.J.C., Stevens, L.H., Verpoorte, R., 1998. Puri®cation
and characterisation of strictosidine b-D-glucosidase from
Catharanthus roseus cell suspension cultures. Plant Physiol.
Biochem. 36, 419±425.
Stockigt, J., 1995. Biosynthesis in Rauwol®a serpentina, modern
aspects of an old medicinal plant. In: Cordell, G.A. (Ed.), The
Alkaloids, 47. Academic Press, San Diego, pp. 115±172.
Stockigt, J., Zenk, M.H., 1977. Strictosidine (Isovincoside): the key
intermediate in the biosynthesis of monoterpenoid indole alka-
loids. J. Chem. Soc. Chem. Commun., 646±648.
Poulton, J.E., 1990. Cyanogenesis in plants. Plant Physiol. 94, 401±
405.
Rask, L., Andreasson, E., Ekbom, B., Eriksson, S., Pontoppidan, B.,
Meijer, J., 2000. Myrosinase: gene family evolution and herbivore
defense in Brassicaceae. Plant Mol. Biol. 42, 93±113.
Rue€er, M., Nagakura, N., Zenk, M.H., 1978. Strictosidine, the
common precursor for monoterpenoid indole alkaloids with 3a
and 3b con®guration. Tetrahedron Lett. 18, 1593±1596.
Ruyter, C.M., Schubel, H., Stockigt, J., 1988. Novel glucoalkaloids
from Rauwol®a cell cultures Ð acetylrauglucine and related glu-
cosides. Z. Naturforsch. 43c, 479±484.
Trimbur, D.E., Warren, R.A.J., Withers, S.G., 1992. Region-directed
mutagenesis of residues surrounding the active site nucleophile in
b-glucosidase from Agrobacterium faecalis. J. Biol. Chem. 267,
10248±10251.
Warzecha, H., Obitz, P., Stockigt, J., 1999. Puri®cation, partial
amino acid sequence and structure of the product of rauca€ri-
cine-O-b-D-glucosidase from plant cell cultures of Rauwol®a ser-
pentina. Phytochemistry 50, 1099±1109.
Ruyter, C.M., Stockigt, J., 1991. Enzymatic formation of rauca€ri-
cine, the major indole alkaloid in Rauwol®a serpentina cell-sus-
pension cultures. Helv. Chim. Acta 74, 1707±1712.
Withers, S.G., Warren, R.A.J., Street, I.P., Rupitz, K., Kempton,
J.B., Aebersold, R., 1990. Unequivocal demonstration of the
involvement of a glutamate residue as a nucleophile in the mech-
anism of a ``retaining'' glycosidase. J. Am. Chem. Soc. 112, 5887±
5889.
Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. In: Molecular
Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York.