Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids Irina Gerasimenko, Yuri Sheludko, Xueyan Ma
Trang 1Heterologous 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
cDNAsequences of SG from Catharanthus roseus and
raucaffricine glucosidase (RG) from Rauvolfia serpentina,
primers for RT-PCR were designed and the cDNAencoding
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 is predicted to lack an
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
In vitrodeglucosylation of strictosidine by R serpentina SG proceeds by the same mechanism as has been shown for the
C roseusenzyme preparation The reaction gives rise to the end product cathenamine and involves 4,21-dehydrocory-nantheine aldehyde as an intermediate The enzymatic hydrolysis of dolichantoside (Nb-methylstrictosidine) leads
to several products One of them was identified as a new compound, 3-isocorreantine A From the data it can be concluded that the divergence of the biosynthetic pathways leading to different classes of indole alkaloids formed in
R serpentinaand C roseus cell suspension cultures occurs at
a later stage than strictosidine deglucosylation
Keywords: strictosidine b-D-glucosidase; heterologous expression; Rauvolfia serpentina; ajmaline; indole alkaloid biosynthesis
Elucidation of the biosynthesis of natural plant products
has been a matter of investigation for over half a century
Although there have been major efforts in the field, only
very few biosynthetic pathways have been delineated in
detail at the enzymatic level Knowing the enzymes involved
is, however, a prerequisite for understanding the
biosyn-thetic mechanisms The next steps are to search for the
participating genes and to clarify the regulation of product
synthesis, with the aim of influencing the biosynthesis on a
rational basis The best known pathways comprise those of
the flavonoid biosynthesis [1,2], the isoquinoline alkaloid
formation [3,4] and the biosynthesis of indole alkaloids [5,6]
The key intermediate in the biosynthesis of all
mono-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 strictstrict-osidine 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¨ckigt, unpublished results) and the raucaffricine glucosidase (RG) [15,16] It is one of our future aims to heterologously express the entire biosynthetic pathway
Correspondence to J Sto¨ckigt, Lehrstuhl fu¨r Pharmazeutische
Biologie, Institut fu¨r Pharmazie, Johannes Gutenberg-Universita¨t
Mainz, Staudinger Weg 5, 55099 Mainz, Germany.
Fax: + 49 6131 3923752, Tel.: + 49 6131 3925751,
E-mail: stoeckig@mail.uni-mainz.de
Abbreviations: CAS, ceric ammonium sulfate reagent; IPTG, isopropyl
thio-b- D -galactoside; NBA, 3-nitrobenzylalcohol; RG, raucaffricine
glucosidase; SG, strictosidine glucosidase; SS, strictosidine synthase;
PNAE, polyneuridine aldehyde esterase.
Enzyme: strictosidine b- D -glucosidase (EC 3.2.1.105).
Note: the cDNAsequence of SG from Rauvolfia serpentina was
submitted to the GenBank under accession number AJ302044.
(Received 18 November 2001, revised 6 March 2002,
accepted 12 March 2002)
Trang 2leading from strictosidine to ajmaline In the present article,
we report on cloning and heterologous expression in
Escherichia coli of the cDNAfrom 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
M A T E R I A L S A N D M E T H O D S
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 RNAfrom 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 (T15-NNN) and RLM
reverse transcriptase (Promega, Mannheim, Germany) were
used for first strand cDNAsynthesis PCR was carried out
in Genius thermocycler (Techne, Burkhardtsdorf,
Germany) with Taq DNApolymerase from Gibco
(Karls-ruhe, Germany) under the following conditions: 94C for
5 min, followed by 35 cycles of 94C for 1 min, 60 C for
1.5 min, 72C for 2 min, then held at 72 C for 5 min The
1311-bp fragment was amplified with primers F5 (5¢-CAAT
TTGTACAAGGAAGATATC-3¢, forward) and R2 (5¢-TT
AGTATTTTTGCTTCTTGAC-3¢, reverse) The 3¢- and 5¢
RACE PCR was carried out with gene specific primers
GSP3 (5¢-GGAGGGTGGCAGCATGTCGTTCCTTGG
GG-3¢, forward), GSP5a (5¢-GTGGCTTCTTGAGTCAT
AGAATCGTGGATGAC-3¢, reverse) and GSP5b (5¢-GT
GCATACAACGAAGGCAATCGAGGTCC-3¢, reverse)
using MarathonTMcDNA Amplification Kit and
Advant-age cDNApolymerase from Clontech (Heidelberg,
Germany) according to the manufacturer’s manual The
full-length cDNAwas amplified by PCR using Advantage
cDNApolymerase from Clontech under following
condi-tions: 94C for 1 min, followed by 35 cycles of 94 C for
0.5 min, 60C for 1.5 min, 72 C for 3 min, then held at
72C for 5 min The primer pairs NcoI (5¢-GGTG
GTCCAT GGACAATACTCAAGC-3¢, forward) – PstI
(5¢-CTGCA
GTTAGGTTTTTTGCCTCTTGACTAAC-3¢, reverse) and NdeI (5¢-CACATATGGACAATACTCA
AGCTGA GCC-3¢, forward) – SapI (5¢-TGCTCTTCC
GCAGGTTTTTTGCCTCTTGAC-3¢, reverse) were used
to introduce respective restriction sites at the ends of the ORF After ligation into pGEM-T Easy Vector (Prome-ga), both strands of the obtained fragment were sequenced
by primer walking using the dideoxy chain termination method [22]
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 byPSORTserver [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 cDNAwas inserted in the NcoI and PstI 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)1ampicillin at 37C Acontrol 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-fuged (4500 g, 10 min), the cells taken up in 1 mL sterile H2O and crashed with ultrasonic The supernatant after centrif-ugation for 30 min at 35 000 g was used to test SG activity The pure heterologously expressed SG was obtained using IMPACTTM-CN system (New England Biolabs) according
to the manufacturer’s manual The full-length SG cDNA was ligated in the NdeI and SapI sites of the pTYB1 vector and transformed into E coli strain ER2566 Transformants were selected on Luria–Bertani medium supplemented with
50 mgÆL)1ampicillin For purification of SG 50 mL of fresh grown bacterial culture were inoculated into 2.5 L of the above nutrition medium and incubated at 28C When
D600¼ 0.5, IPTG (final concentration 0.5 mM) was added
to induce expression Cells were harvested after 13 h of cultivation at 28C by centrifugation for 15 min at 5000 g and taken up in 50 mL of cell break buffer (20 mMTris/HCl,
pH 8.0; 1 mM EDTA; 0.5M 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 mMTris/HCl, pH 8.0; 1 mMEDTA; 0.5MNaCl) 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 mMTris/HCl, pH 8.0; 1 mM EDTA; 0.5M NaCl; 50 mM dithiothreitol) The flow was stopped and the column kept for 23 h at 4C for cleavage of
Fig 1 The key role of strictosidine in the
biosynthesis of different classes of
monoterpe-noid indole alkaloids.
Trang 3intein tag SG was eluted with column buffer (fraction size
0.5 mL) Fractions 3–22 with protein concentration higher
than 15 lgÆmL)1 were combined and dialyzed against
2· 1 L of Tris/EDTAbuffer (20 mM Tris/HCl, pH 8.0;
1 mMEDTA) yielding a solution with protein concentration
of 13 lgÆmL)1 and specific SG activity of 350 pkatÆlg)1
protein The purity of SG was analyzed on Coomassie and
silver stained SDS/PAGE
Protein determination and enzyme assays
Protein concentrations were measured by the method of
Bradford [27] using bovine serum albumin (Merck,
Darms-tadt, Germany) as standard Strictosidine glucosidase
activity was calculated on the basis of strictosidine decrease
measured by HPLC Atypical assay contained appropriate
enzyme activities between 1 and 8 pkat and 20 nmol of
strictosidine in 5 lL MeOH in total volume of 50 lL 0.1M
citrate/phosphate buffer (pH 5.0) and was incubated for 15
or 30 min at 30C The reaction was terminated by
addition of 100 lL MeOH After centrifugation (11 000 g,
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 mM NaH2PO4 (pH 2.5), gradient 15 : 85fi 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)1flow 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, 0.1Mcitrate/phosphate buffer, pH 5.0) containing
putative substrates (400 nmol in 20 lL MeOH) and 0.13 lg
strictosidine glucosidase (45.5 pkat with strictosidine) was
incubated at 30C overnight (16 h) The reaction was
terminated with 200 lL MeOH, and 100 lL of the resulting
mixture were added to 1 mL of the Glucose Trinder
Reagent (Sigma, Deisenhofen, Germany) The D505 was
measured after 20 min Control incubations were carried
out without the enzyme To check the stability of SG during
the over night reaction, the sample containing 0.13 lg of
enzyme without substrate was incubated in the same
conditions, 20 nmol of strictosidine were added after 16 h,
the reaction mixture incubated for further 1 h and SG
activity analyzed by HPLC
Properties of the enzyme
Enzyme kinetic parameters (Kmand Vmax) were determined
in presence of 13 ng (with strictosidine), 26 ng (with
5a-carboxystrictosidine) or 65 ng (with 19,20-dihydro- and
Nb-methylstrictosidine) of SG in 0.1Mcitrate/phosphate buffer
(pH 5.0), 15 min incubation at 30C The substrate
con-centrations tested were: 10 lM)500 lM of strictosidine,
50 lM)250 lMof 5a-carboxystrictosidine, 100 lM)250 lM
of 19,20-dihydrostrictosidine, and 25 lM)250 lM of
Nb-methylstrictosidine The pH optimum was determined
by incubation of 20 nmol of strictosidine with 26 ng of SG
for 30 min at 30C in different buffers: 0.1M citrate/
phosphate (pH 3.8–7.0), 0.1MKPi(pH 5.8–8.0), and 0.1M
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 0.1Mcitrate/phosphate buffer (pH 5.0) for
30 min at different temperatures (13–65C) Inhibition by 0.25 mMcathenamine, 0.25 mMajmaline, 1 mMserpentine and 1 mMCuSO4was studied by incubation of 12.5 nmol
of strictosidine with 26 ng of SG in 0.1Mcitrate/phosphate buffer (pH 5.0) for 30 min at 30C
Size-exclusion chromatography was conducted with Superdex 75 HR 10/30 column (Pharmacia) (CV 30 mL) The proteins were eluted with 20 mM Tris/HCl buffer,
pH 8.0, containing 2 mM Na2EDTA, 10% glycerol and
10 mM 2-mercaptoethanol at a flow rate of 24 mLÆh)1 collecting 0.1 mL fractions for SG activity test
General experimental procedures For thin layer chromatography (TLC), 0.2-mm or 0.5-mm silica gel 60 F254 plates, 20· 20 cm (Merck, Darmstadt, Germany) were used with the solvent systems SS1/petro-leum ether/acetone/diethylamine (7 : 2 : 1) or SS2/CHCl3/ MeOH (8 : 2) Substances were detected by measuring the
A254and 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.1H-NMR spectra were measured using AMX 400 and DRX 600 instruments (Bruker, Karlsruhe, Germany) with CDCl3 and
pyridine-d5 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 NaBH3CN and HCHO [30]
Synthesis and identification of deglucosylation products
Strictosidine (1 mg) dissolved in 100 lL MeOH was incubated in H2O (total vol 1 mL) with 450 lg crude enzyme preparation from transgenic E coli for 1 h at
30C For control assays the enzyme preparation was heated in a boiling water bath for 20 min After centrifu-gation (11 000 g, 5 min) the pellet (formed after incubation with the active enzyme only) was freeze-dried The main component was identified as cathenamine (8) by EI-MS and
1H-NMR as well as by HR-FAB-MS: m/z 351.1720 ([M + H]+, calc for C21H23O3N2, 351.1709), 503.2037 ([M + NBA]+, calc for C28H29O6N3, 503.2056)
Identification of intermediate under reducing conditions
(a) Strictosidine (225 nmol) was incubated in 0.1Mcitrate/ phosphate buffer (pH 5.0) (total volume 1.5 mL) with
132 lg crude transgenic E coli protein in presence of
450 nmol NaBH3CN for 15 min at 30C The reaction mixture was extracted with ethyl acetate The organic phase was evaporated and the residue analyzed by 2D-TLC with
Trang 4solvent 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 0.1M
citrate/phosphate buffer (pH 5.0) (total vol 1.5 mL) with
132 lg crude transgenic E coli protein in presence of
900 lmol NaBH3CN for 15 min at 30C The reaction
mixture was extracted with ethyl acetate After evaporation
of the organic phase the remaining residue was analyzed by
2D-TLC with solvent system SS1 Two products located at
Rf 0.34 and 0.44 were identified as sitsirikine (10) and
isositsirikine (11) by their EI-MS data Experiments with
4000-fold excess of KBH4were carried out analogously in
1MKPibuffer (pH 7.5–8.0)
Deglucosylation of dolichantoside
Dolichantoside (30 mg, 55 lmol) was incubated with 39 lg
SG in 30 mL 0.1M citrate/phosphate buffer (pH 5.0)
overnight (16–18 h) at 30C The reaction mixture was
extracted with an equal volume of EtOAc, pH of the water
phase adjusted to 8.0 with 25% ammonia and extraction
with equal volume of EtOAc repeated The organic phases
were evaporated and chromatographed using solvent
sys-tem SS2 The product at Rf0.64 showing blue fluorescence
at 366 nm after spraying with CAS was eluted yielding 1 mg
of (9) (2.6 lmol, 4.7%) 3-isocorreantine A(9): EI-MS m/z
(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 for C22H26O4N2, 382.1893), 367.1681
(M+-CH3, calc for C21H23O4N2, 367.1658) 1H NMR
(600 MHz, pyridine-d5): d 1.50 (1H, m, H-14b), 1.57 (3H, d,
J¼ 6.5, H3-18), 1.73 (1H, m, H-14a), 2.39 (3H, s, Nb-CH3),
2.56 (1H, m, H-20), 2.60 (1H, m, H-5b), 2.68 (1H, m, H-6b),
3.02 (1H, dd, 14.2, 2.7, H-6a), 3.52 (1H, d, J¼ 11.5, H-5a),
3.67 (3H, s, CO2CH3), 3.82 (1H, m, H-15), 3.87 (1H, dd,
13.7, 6.4, H-3), 4.36 (1H, dq, J¼ 9.3, 6.5, H-19), 6.48 (1H,
s, H-21), 7.26 (1H, dd, J¼ 7.7, 7.7, H-10), 7.30 (1H, dd,
J¼ 7.7, 7.7, H-11), 7.61 (1H, d, J ¼ 7.7, H-9), 7.82 (1H, s,
H-17), 7.91 (1H, d, J¼ 7.7, H-12).13C NMR (determined
from HSQC and HMBC spectra, 600 MHz, pyridine-d5): d
18.3 (q, C-18), 24.9 (t, C-14), 29.1(d, C-15), 32.4 (t, C-6),
41.8 (q, Nb-CH3), 46.0 (d, C-20), 50.4 (q, CO2CH3), 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, CO2CH3)
Import-ant NOE correlations: H-3–H-14a; H-15–H-19;
H-21–H-12, H3-18, H-19, H-20
R E S U L T S A N D D I S C U S S I O N
Cloning of cDNA encoding strictosidine glucosidase
Primers for PCR were designed on the basis of comparison
of cDNAsequences 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-PCR
experi-ments yielded a 1311-bp long DNAfragment with a high
homology of 79.9% to C roseus SG After successful
amplification of cDNAends containing start and stop
codons, the full-length cDNAwas 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
of 60.881 kDa and an isoelectric point of 6.01 differing from the C roseus SG (Table 1) The deduced amino-acid sequence shows 70% homology to SG from C roseus followed by RG from R serpentina (56%) and other plant b-glucosidases The presence of a family 1 glycosyl hydrol-ase N-terminal signature (position 47–61) allows assignment
of the SG from R serpentina to this enzyme family [31] It is noteworthy that in the second signature of glycosyl hydrolases family 1 (position 412–419), which contains the putative nucleophile catalytic glutamic acid [32,33], in position 417 asparagine is changed to serine This position
is also modified in SG from C roseus, where glutamic acid
is followed by cysteine (Fig 3) Region-directed mutagen-esis of b-glucosidase from Agrobacterium faecalis indicated that this asparagine residue does not play a critical role in catalysis [33] In the SGs this residue is not conserved, supporting the above mentioned results In contrast, the next glycine proved to be essential for enzyme activity probably due to its small size necessary for the right conformation of the active site [33] This residue is indeed conserved in both SGs and RG The second catalytic glutamic acid acting as proton donor is suggested to be located upstream of the nucleophile in the highly conserved motif NEP (position 206–208) [34,35] The sequence DxxRxxY near the C-terminus (position 435–441) is also conserved in family 1 of glycosyl hydrolases, although it was shown that only aspartic acid plays an important, but not critical, role in catalysis [33] Analysis of the R serpen-tinaSG deduced amino-acid sequence revealed no regions predicted to form transmembrane helices In contrast to the
SG from C roseus, the R 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 usingPSORTsoftware [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 roseuswas 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 cDNAcloned from R serpentina indeed encoded the SG, it was expressed in E coli Crude extracts of the bacteria transformed with pSE280 vector containing SG cDNAshowed high strictosidine glucosidase activity (2.4 pkatÆlg)1total 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 cDNAindeed encodes SG from R serpentina Properties of heterologously expressed SG
To achieve simple and efficient purification of the enzyme,
SG was expressed in fusion with the intein tag [37] and
Trang 5bound on a chitin column After self-cleavage of the intein
sequence in presence of thiol, native SG without any
additional amino acids was eluted from the column The
enzyme became enriched 250-fold and showed a single band
on silver stained SDS/PAGE (Fig 4) This solution
con-taining pure SG was used for further determination of
enzyme properties
Optimum catalytic activity was expressed at a
tempera-ture of 50C The temperature optimum for SG from
C roseus cell suspensions was reported to be 30C [18],
although the enriched Catharanthus enzyme was highly
stable up to 50C [19] Whether the high temperature
optimum of R serpentina SG may be attributed specifically
to Rauvolfia cells is uncertain But another enzyme isolated from the same cell suspension culture, arbutin synthase, also displayed an optimum catalytic activity at 50C [38,39] SG showed a pH optimum at 5.2 with activity of 50% of the maximum at pH 4.2 and slowly decreasing up to pH 8.0 These results are in contrast with those reported for SG from C roseus by different authors (Table 1) They resem-ble, however, values known for nonspecific plant b-D -glucosidases [40] Similar to the SG from C roseus, the
R serpentina enzyme was inhibited by 1 mM Cu2+ and
1 mM serpentine, although at a significant lower degree (Table 1), indicating a close relationship of both enzymes The K value for strictosidine was 0.12 mM, 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.
Trang 6ponds well to the data of two SG enzymes characterized
from C roseus cell cultures [18], although the Km 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 mM
concentration
Size-exclusion chromatography on Superdex-75 column
revealed that the purified heterologously expressed SG from
R serpentinahas a molecular mass > 450 kDa, as it has
been demonstrated earlier for the Catharanthus enzyme
(Table 1)
Substrate specificity For the first time the pure SG was incubated with a great variety of b-D-glucosides, 34 in total, most of them being natural products of different classes Five of these com-pounds were converted at a rate of 0.8–90% compared to strictosidine (Table 2) Except of ipecoside which derives from enzymatic condensation of secologanin and dopamine [41] and is deglucosylated at a low rate of 0.8%, all other accepted substrates possess the basic skeleton of strictosi-dine The a(S) configuration at C3 is essential for SG from
R serpentina as well as for glucosidase from C roseus [18,19] Whereas vincoside, the 3b(R) epimer of strictosidine,
Table 1 Comparison of properties of strictosidine glucosidases from R serpentina and C roseus ND, not determined.
SG from C roseus cell suspension cultures [18]
SG from C roseus cell suspension cultures [19]
SG from C roseus expressed in yeast [20]
SG from R serpentina expressed in E coli
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)
M r 230 kDa (I) >1500 kDa 63.043 kDa (calculated); 60.881 kDa (calculated);
>450 kDa (II) >660 kDa >450 kDa
Fig 3 Alignment of deduced amino acid
sequences of three glucosidases involved in
indole alkaloid biosynthesis SG_Rs: SG from
R serpentina, SG_Cr: SG from C roseus,
RG_Rs: RG from R serpentina Identical
amino acids are shaded Motifs conserved in
members of glycosyl hydrolases family 1 are
highlighted black, the putative catalytic
glu-tamate residues are marked A, proton donor.
B, nucleophile.
Trang 7is not accepted, the 5a-carboxystrictosidine with 3a(S)
configuration has a relative conversion rate of 90%
Changing the structure of strictosidine by acetylation of
the b nitrogen leads to more significant decrease of
conversion than methylation of the b nitrogen or
hydro-genation of the isolated 18,19-double bond (Table 2) Indole
alkaloids possessing a sarpagine or ajmaline ring system
were not accepted (Table 2), as well as 21 nonindole
glucosides tested (secologanin, loganin,
p-nitrophenylglu-coside, arbutin, vanillin-glup-nitrophenylglu-coside,
vanillylalcohol-phenyl-glucoside, picein, salicin, amygdalin, avetiin,
6-bromo-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-tinaenzyme 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 1H 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 catalysed by SG from C roseus [43]
In order to intercept putative precursors of cathenamine (8) formed immediately after hydrolysis of strictosidine (1) (Fig 5), the enzymatic reaction was carried out in presence
of reducing agents (NaBH3CN and KBH4) which are expected to reduce aldehyde groups in (5) and thus prevent it from further conversion When a twofold excess
of NaBH3CN was added, only tetrahydroalstonine (12) was detected This result supports the identification of (8)
as the end product of the cell-free strictosidine degluco-sylation, as the reduction of (8) leads to tetrahydroalsto-nine When the concentration of NaBH3CN was increased
to a 2000-fold excess, the two products sitsirikine (10) and isositsirikine (11) were identified, which demonstrates that 4,21-dehydrocorynantheine aldehyde (7) is involved in the indole alkaloid biosynthesis in R serpentina as well as it has been shown earlier for C roseus [42]
Further experiments to identify other intermediates applying hydroxylamine and thiols were unsuccessful, as well as attempts to impede the bond rotation necessary for the ring D closure by conducting the enzymatic reaction at low temperature in presence of reducing agents (data not 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 C22H26O4N2 But the 1H-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) has reacted 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 methyl group at d 1.57 correlated in the 1H-1H COSY spectrum to H19 at d 4.36 suggested the closure of the ring
E, which is confirmed by the shift of H-17 (d 7.82) H-21 appears as a singlet at d 6.48 correlated on NOESY
Fig 4 Silver stained SDS/PAGE of heterologously expressed SG.
Lane 1, crude protein extract from transgenic E coli; lane 2, molecular
mass marker; lane 3, eluted active strictosidine glucosidase.
Trang 8spectrum to one of the aromatic protons (H-12 at d 7.91)
indicating that C-21 bears an hydroxyl function and is
adjacent to Na The structure elucidation of the new
alkaloid was completed by HSQC, HMBC and NOESY measurements, which enabled the determination of the chemical shifts of carbons and the relative stereochemistry
Fig 5 Enzymatic conversion of strictosidine
and its Nb-methyl derivative by heterologously
expressed strictosidine glucosidase from
R serpentina cell suspension cultures.
Table 2 Substrate specificity of pure
heterolo-gously expressed SG form R serpentina ND,
not determined a Determined by HPLC.
Trang 9The novel compound is the 3-isomer of correantine A,
which has been isolated from Psychotria correae [44]
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 Aidentified in this
study was, however, not detected in these experiments,
although when treated with an unspecific b-glucosidase,
3-isodolichantoside gave correantine Aand 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 Km
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
C O N C L U S I O N S
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
in the same end product cathenamine and involves the same
intermediate 4,21-dehydrocorynantheine aldehyde The
formation of the 3-isocorreantine Aafter hydrolysis of
dolichantoside is an indication that the deglucosylation
product is released from the enzyme before the ring D is
closed From these data, it can be concluded that the
divergence of the biosynthetic pathways leading to different
classes of indole alkaloids formed in R serpentina and
C roseuscell suspension cultures occurs at a later stage than
strictosidine deglucosylation, i.e after formation of
4,21-dehydrogeissoschizine, which has been shown to be
con-verted into ajmalicine type alkaloids or geissoschizine by the
enzyme preparations from C roseus [46] The knowledge of
the cDNAsequence and the possibility of obtaining high
amounts of pure active SG may help to identify and
characterize further enzyme(s) of ajmaline biosynthesis
converting the reactive intermediates formed after
strictosi-dine hydrolysis
A C K N O W L E D G E M E N T S
Financial support from Deutsche Forschungsgemeinschaft (Bonn, Bad
Godesberg, Germany) and by the Fonds der Chemischen Industrie
(Frankfurt/Main, Germany) is highly appreciated X M is very
grateful to BASF Company (Ludwigshafen, Germany) for providing a
scholarship We also thank Mr H Kolshorn (Institute of Organic
Chemistry, Mainz, Germany) for NMR measurements and helpful
discussion, Dr J Gross (Institute of Organic Chemistry, Heidelberg,
Germany) for FD-, HR-FAB- and HR-EI-MS spectra, and to
Dr D Strand (Mainz, Germany) for linguistic advice.
R E F E R E N C E S
1 Winkel-Shirley, B (2001) Flavonoid biosynthesis Acolorful model for genetics, biochemistry, cell biology, and biotechnology Plant Physiol 126, 485–493.
2 Forkmann, G & Martens, S (2001) Metabolic engineering and applications of flavonoids Curr Opin Biotechnol 12, 155–160.
3 Zenk, M.H (1995) Chasing the enzymes of alkaloid biosynthesis.
In Organic Reactivity: Physical and Biological Aspects (Golding, B.T., Griffin, R.J & Maskill, H., eds), pp 89–109 The Royal Society of Chemistry, Newcastle upon Tune, UK.
4 Grothe, T., Lenz, R & Kutchan, T.M (2001) Molecular char-acterization of the salutaridinol 7-O-acetyltransferase involved in morphine biosynthesis in opium poppy Papaver somniferum.
J Biol Chem 276, 30717–30723.
5 Sto¨ckigt, J (1995) Biosynthesis in Rauwolfia serpentina – modern aspects of an old medicinal plant In The Alkaloids Chemistry and Pharmacology (Cordell, G.A., ed.), pp 115–172 Academic Press, New York.
6 Sto¨ckigt, J (1998) Alkaloid metabolism in plant cell culture In Natural Product Analysis (Schreier, P., Herderich, M., Humpf, H.M & Schwab, W., eds), pp 313–325 Vieweg Braunschweig, Wiesbaden.
7 Sto¨ckigt, J & Zenk, M.H (1977) Strictosidine (isovincoside): the key intermediate in the biosynthesis of monoterpenoid indole alkaloids J Chem Soc Chem Commun 646–648.
8 Sto¨ckigt, J & Zenk, M.H (1977) Isovincoside (strictosidine), the key intermediate in the enzymatic formation of indole alkaloids FEBS Lett 79, 233–237.
9 Rueffer, M., Nagakura, N & Zenk, M.H (1978) Strictosidine, the common precursor for monoterpenoid indole alkaloids with 3a and 3b configuration Tetrahedron Lett 1593–1596.
10 Nagakura, N., Rueffer, M & Zenk, M.H (1979) The Biosynthesis
of monoterpenoid indole alkaloids from strictosidine J Chem Soc Perkin Trans I, 2308–2312.
11 Hampp, N & Zenk, M.H (1988) Homogeneous strictosidine synthase from cell suspension cultures of Rauvolfia serpentina Phytochemistry 27, 3811–3815.
12 Kutchan, T.M., Hampp, N., Lottspeich, F., Beyreuther, K & Zenk, M.H (1988) The cDNAclone for strictosidine synthase from Rauvolfia serpentina DNASequence determination and expression in Escherichia coli FEBS Lett 237, 40–44.
13 Kutchan, T.M (1989) Expression of enzymatically active cloned strictosidine synthase from the higher plant Rauvolfia serpentina in Escherichia coli FEBS Lett 257, 127–130.
14 Dogru, E., Warzecha, H., Seibel, F., Haebel, S., Lottspeich, F & Sto¨ckigt, J (2000) The gene encoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesis in plants is
an ortholog of the a/b hydrolase super family Eur J Biochem.
267, 1397–1406.
15 Warzecha, H., Obitz, P & Sto¨ckigt, J (1999) Purification, partial amino acid sequence and structure of the product of raucaffricine-O-b- D -glucosidase from plant cell cultures of Rauvolfia serpentina Phytochemistry 50, 1099–1109.
16 Warzecha, H., Gerasimenko, I., Kutchan, T.M & Stockigt, J (2000) Molecular cloning and functional bacterial expression of a plant glucosidase specifically involved in alkaloid biosynthesis Phytochemistry 54, 657–666.
17 Gerasimenko, I., Sheludko, Y., Ma, X & Sto¨ckigt, J (2001) Properties of heterologously expressed strictosidine glucosidase from Rauvolfia serpentina cell suspension culture In Traits, Tracks and Traces, 49 th Annual Congress of the Society for Medicinal Plant Research, Abstracts 2.5, Germany.
18 Hemscheidt, T & Zenk, M.H (1980) Glucosidases involved in indole alkaloid biosynthesis of Catharanthus cell cultures FEBS Lett 110, 187–191.
19 Luijendijk, T.J.C., Stevens, L.H & Verpoorte, R (1998) Purifi-cation and characterisation of strictosidine b- -glucosidase from
Trang 10Catharanthus roseus cell suspension cultures Plant Physiol
Bio-chem 36, 419–425.
20 Geerlings, A., Iban˜ez, M.M.L., Memeling, J., van der Heiden, R &
Verpoorte, R (2000) Molecular cloning and analysis of
strictosi-dine b- D -glucosidase, an enzyme in terpenoid indole alkaloid
bio-synthesis in Catharanthus roseus J Biol Chem 275, 3051–3056.
21 Linsmaier, E.M & Skoog, F (1965) Organic growth factor
requirements of tobacco tissue cultures Physiol Plant 18, 100–
127.
22 Sanger, F., Nicklen, S & Coulson, A.R (1977) DNA sequencing
with chain-terminating inhibitors Proc Natl Acad Sci USA 74,
5463–5467.
23 Hofmann, K., Bucher, P., Falquet, L & Bairoch, A (1999) The
PROSITE database, its status in 1999 Nucleic Acid Res 27, 215–
219.
24 Tusnady, G.E & Simon, I (1998) Principles governing amino acid
composition of integral membrane proteins: applications to
topology prediction J Mol Biol 283, 489–506.
25 Sonnhammer, E.L.L., von Heijne, G & Krogh, A (1998) A
hidden Markov model for predicting transmembrane helices in
protein sequences In Proceedings of the of Sixth International
Conference on Intelligent Systems for Molecular Biology (Glasgow,
J., Littlejohn, T., Major, F., Lathrop, R., Sankoff, D & Sensen,
C., eds), pp 175–182 Menlo Park, CA: AAAI Press.
26 Nakai, K & Kanehisa, M (1992) Aknowledge base for predicting
protein localization sites in eukaryotic cells Genomics 14, 897–911.
27 Bradford, M.M (1976) Arapid and sensitive method for the
quantification of microgram quantities of protein utilizing the
principle of protein-dye binding Anal Biochem 72, 248–254.
28 Pfitzner, U & Zenk, M.H (1982) Immobilisation of strictosidine
synthase from Catharanthus cell cultures and preparative synthesis
of strictosidine Planta Med 46, 10–14.
29 Sheludko, Y., Gerasimenko, I & Platonova, O (2000) Divergence
of the indole alkaloid pattern in two somatic hybrid plant cell
subcultures of Rauvolfia serpentina · Rhazya stricta Planta Med.
66, 656–659.
30 Achenbach, H & Benirschke, M (1997) Constituents of tropical
medicinal plants 81 Confirmation of the absolute configuration
of dolichantoside and isodolichantoside by synthesis from
(–)-secologanin Phytochemistry 44, 1387–1390.
31 Henrissat, B & Davies, G (1997) Structural and sequence-based
classification of glycoside hydrolases Curr Opin Struct Biol 7,
637–644.
32 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
mechanism of a retaining glycosidase J Am Chem Soc 112,
5887–5889.
33 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.
34 Baird, S.D., Hefford, M.A., Johnson, D.A., Sung, W.L., Yaguchi,
M & Seligi, V.L (1990) The Glu residue in the conserved Asn-Glu-Pro sequence in two highly divergent endo-beta-1,4-gluca-nases is essential for enzymatic activity Biochem Biophys Res Commun 169, 1035–1039.
35 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) II Identification of Glu-198
as an active site carboxylate group with acid catalytic function Arch Biochem Biophys 315, 323–330.
36 Stevens, L.H., Blom, T.J.M & Verpoorte, R (1993) Subcellular localization of tryptophan decarboxylase, strictosidine synthase and strictosidine glucosidase in suspension cultured cells of Catharanthus roseus and Tabernamontana divaricata Plant Cell Report 12, 573–576.
37 Chong, S., Mersha, F.B., Comb, D.G., Scott, M.E., Landry, D., Vence, L.M., Perler, F.B., Benner, J., Kuchera, R.B., Hirvonen, C.A., Pelletier, J.J., Paulus, H & Xu, M.Q (1997) Single column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element Gene 192, 277– 281.
38 Arend, J., Warzecha, H & Sto¨ckigt, J (2000) Hydroquinone: O-glucosyltransferase from cultivated Rauvolfia cells: enrichment and partial amino acid sequences Phytochemistry 53, 187–193.
39 A rend, J., Warzecha, H., Hefner, T & Sto¨ckigt, J (2001) Utilizing genetically engineered Escherichia coli to produce plant specific glucosides Biotechn Bioeng 76, 126–131.
40 Dey, P.M & Del Campillo, E (1984) Biochemistry of the multiple forms of glucosidases in plants In Advances in Enzymology and Related Areas of Molecular Biology, 56 (Meister, A., ed.), pp 141–
249 Willy-Interscience, New York.
41 De-Eknamkul, W., Suttipanta, N & Kutchan, T.M (2000) Pur-ification and characterization of deacetylipecoside synthase from Alangium lamarckii Thw Phytochemistry 55, 177–181.
42 Sto¨ckigt, J., Rueffer, M., Zenk, M.H & Hoyer, G.A (1978) Indirect identification of 4,21-dehydrocorynantheine aldehyde as
an intermediate in the biosynthesis of ajmalicine and related alkaloids Planta Med 33, 188–192.
43 Sto¨ckigt, J (1979) Enzymatic formation of intermediates in the biosynthesis of ajmalicine: strictosidine and cathenamine Phyto-chemistry 18, 965–971.
44 Achenbach, H., Lottes, M., Waibel, R., Karikas, G.A., Correa, M.D & Gupta, M.P (1995) Alkaloids and other compounds from Psychotria correae Phytochemistry 38, 1537–1545.
45 Brandt, V., Tits, M., Penelle, J., Frederich, M & Angenot, L (2001) Main glucosidase conversion products of the gluco-alkaloids dolichantoside and palicoside Phytochemistry 57, 653–659.
46 Rueffer, M., Kan-Fan, C., Husson, H.P., Sto¨ckigt, J & Zenk, M.H (1979) 4,21-Dehydrogeissoschizine, an intermediate in het-eroyohimbine alkaloid biosynthesis J Chem Soc Chem Com 1016–1018.