Báo cáo Y học: Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids pot

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. In vitro deglucosylation of strictosidine by R. serpentina SG proceeds by the same mechanism as has been shown for the C. roseus enzyme 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. serpentina and 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 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 ¨ 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 cDNA sequence 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) Eur. J. Biochem. 269, 2204–2213 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02878.x 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 5 min, followed by 35 cycles of 94 °Cfor1min,60°Cfor 1.5 min, 72 °Cfor2min,thenheldat72°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¢-and5¢ 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 Marathon TM cDNA Amplification Kit and Advant- ageÒ cDNA polymerase from Clontech (Heidelberg, Germany) according to the manufacturer’s manual. The full-length cDNA was amplified by PCR using AdvantageÒ cDNA polymerase from Clontech under following condi- tions: 94 °C for 1 min, followed by 35 cycles of 94 °Cfor 0.5 min, 60 °Cfor1.5min,72°C for 3 min, then held at 72 °C for 5 min. The primer pairs NcoI(5¢-GGTG GTCCAT GGACAATACTCAAGC-3¢,forward)–PstI (5¢-CTGCA GTTAGGTTTTTTGCCTCTTGACTAAC- 3¢,reverse)andNdeI(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 pGEMÒ-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 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- fuged (4500 g, 10 min), the cells taken up in 1 mL sterile H 2 O and crashed with ultrasonic. The supernatant after centrif- ugation for 30 min at 35 000 g wasusedtotestSGactivity. The pure heterologously expressed SG was obtained using IMPACT TM -CN system (New England Biolabs) according to the manufacturer’s manual. The full-length SG cDNA was ligated in the NdeIandSapI sites of the pTYB1 vector and transformed into E. coli strain ER2566. Transformants were selected on Luria–Bertani medium supplemented with 50 mgÆL )1 ampicillin. 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 28 °C. When D 600 ¼ 0.5, IPTG (final concentration 0.5 m M )wasadded to induce expression. Cells were harvested after 13 h of cultivation at 28 °C by centrifugation for 15 min at 5000 g and taken up in 50 mL of cell break buffer (20 m M Tris/HCl, pH 8.0; 1 m M 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 intein tag. SG was eluted with column buffer (fraction size 0.5 mL). Fractions 3–22 with protein concentration higher than 15 lg ÆmL )1 were combined and dialyzed against 2 · 1 L of Tris/EDTA buffer (20 m M Tris/HCl, pH 8.0; 1m M EDTA) 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. A typical assay contained appropriate enzyme activities between 1 and 8 pkat and 20 nmol of strictosidine in 5 lL MeOH in total volume of 50 lL0.1 M citrate/phosphate buffer (pH 5.0) and was incubated for 15 or 30 min at 30 °C. 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 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 505 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 lgof 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 (K m and V max ) 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 0.1 M citrate/phosphate buffer (pH 5.0), 15 min incubation at 30 °C. The substrate con- centrations tested were: 10 l M )500 l M of strictosidine, 50 l M )250 l M 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 ajmaline, 1 m M serpentine and 1 m M CuSO 4 was studied by incubation of 12.5 nmol of strictosidine with 26 ng of SG in 0.1 M citrate/phosphate buffer (pH 5.0) for 30 min at 30 °C. Size-exclusion chromatography was conducted with Superdex 75 HR 10/30 column (Pharmacia) (CV 30 mL). The proteins were eluted with 20 m M Tris/HCl buffer, pH 8.0, containing 2 m M Na 2 EDTA, 10% glycerol and 10 m M 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 F 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 3 CN and HCHO [30]. Synthesis and identification of deglucosylation products Strictosidine (1 mg) dissolved in 100 lLMeOHwas incubated in H 2 O(totalvol.1mL)with450lg crude enzyme preparation from transgenic E. coli for 1 h at 30 °C. 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 1 H-NMR as well as by HR-FAB-MS: m/z 351.1720 ([M + H] + ,calc.forC 21 H 23 O 3 N 2 , 351.1709), 503.2037 ([M + NBA] + ,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 3 CN for 15 min at 30 °C. 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 KBH 4 were carried out analogously in 1 M KP i buffer (pH 7.5–8.0). Deglucosylation of dolichantoside Dolichantoside (30 mg, 55 lmol) was incubated with 39 lg SG in 30 mL 0.1 M citrate/phosphate buffer (pH 5.0) overnight (16–18 h) at 30 °C. 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 R f 0.64 showing blue fluorescence at 366 nm after spraying with CAS was eluted yielding 1 mg of (9)(2.6lmol, 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.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 (600 MHz, pyridine-d 5 ): d 1.50 (1H, m, H-14b), 1.57 (3H, d, J ¼ 6.5, H 3 -18), 1.73 (1H, m, H-14a), 2.39 (3H, s, Nb-CH 3 ), 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, J ¼ 11.5, H-5a), 3.67 (3H, s, CO 2 CH 3 ), 3.82 (1H, m, H-15), 3.87 (1H, dd, 13.7, 6.4, H-3), 4.36 (1H, dq, J ¼ 9.3, 6.5, H-19), 6.48 (1H, s, H-21), 7.26 (1H, dd, J ¼ 7.7, 7.7, H-10), 7.30 (1H, dd, J ¼ 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, J ¼ 7.7, H-12). 13 C NMR (determined from HSQC and HMBC spectra, 600 MHz, pyridine-d 5 ): 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-CH 3 ), 46.0 (d, C-20), 50.4 (q, CO 2 CH 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 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- tina SG deduced amino-acid sequence revealed no regions predicted to form transmembrane helices. In contrast to the 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 Ó FEBS 2002 Cloning of strictosidine glucosidase from Rauvolfia (Eur. J. Biochem. 269) 2207 bound 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 50 °C. The temperature optimum for SG from C. roseus cell suspensions was reported to be 30 °C[18], although the enriched Catharanthus enzyme was highly stable up to 50 °C [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 50 °C [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 m M Cu 2+ 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 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 Enrichment factor 120 60.2 ND 250 K m 0.2 m M (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% 1m M Serpentine 50% 25.2% M r 230 kDa (I) >1500 kDa 63.043 kDa (calculated); 60.881 kDa (calculated); >450 kDa (II) >660 kDa >450 kDa pI (calculated) ND ND 5.73 6.01 Fig. 3. Alignment of deduced amino acid sequences of three glucosidases involved in indole alkaloid biosynthesis. SG_Rs: SG from R.serpentina,SG_Cr:SGfromC. 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. Ó FEBS 2002 Cloning of strictosidine glucosidase from Rauvolfia (Eur. J. Biochem. 269) 2209 is 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-glucoside, 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- 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 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 (NaBH 3 CN and KBH 4 )whichare expected to reduce aldehyde groups in (5) and thus prevent it from further conversion. When a twofold excess of NaBH 3 CN 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 NaBH 3 CN 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 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 methyl group at d 1.57 correlated in the 1 H- 1 HCOSY 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;lane2,molecular mass marker; lane 3, eluted active strictosidine glucosidase. 2210 I. Gerasimenko et al. (Eur. J. Biochem. 269) Ó FEBS 2002 spectrum 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. Ó FEBS 2002 Cloning of strictosidine glucosidase from Rauvolfia (Eur. J. Biochem. 269) 2211 The 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 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 in the same end product cathenamine and involves the same intermediate 4,21-dehydrocorynantheine aldehyde. The formation of the 3-isocorreantine A after 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. roseus cell 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 cDNA sequence 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. ACKNOWLEDGEMENTS 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. 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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. Ó FEBS 2002 Cloning of strictosidine glucosidase from Rauvolfia (Eur. J. Biochem. 269) 2213 . Heterologous expression of a Rauvol a cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids Irina Gerasimenko,. (5¢-GGAGGGTGGCAGCATGTCGTTCCTTGG GG-3¢,forward),GSP 5a( 5¢-GTGGCTTCTTGAGTCAT AGAATCGTGGATGAC-3¢, reverse) and GSP5b (5¢-GT GCATACAACGAAGGCAATCGAGGTCC-3¢, reverse) using Marathon TM cDNA Amplification Kit and Advant- ageÒ cDNA polymerase from Clontech