The ORCA-ome as a key to understanding alkaloid biosynthesis in Catharanthus roseus
Hasnain, G.
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Hasnain, G. (2010, June 29). The ORCA-ome as a key to understanding alkaloid biosynthesis in Catharanthus roseus. Retrieved from
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Chapter 4
Cloning and functional analysis of cathenamine reductase from Catharanthus roseus
Ghulam Hasnain, Teus J. C. Luijendijk, Robert Verpoorte, and Johan Memelink
Institute of Biology, Leiden University, P.O. Box 9505, 2300 RA Leiden, The Netherlands
Abstract
Catharanthus roseus is a medicinal plant which synthesizes a class of secondary metabolites known as terpenoid indole alkaloids (TIA). The monomeric alkaloids ajmalicine and serpentine are used in the treatment of cardiac and circulatory diseases, and the dimeric alkaloids vinblastine and vincristine are potent anti-tumour drugs. Starting from the primary metabolites tryptophan and geraniol the biosynthesis of bisindole alkaloids in C. roseus is thought to involve at least 35 intermediates and a similar number of enzymes. Fourteen enzyme-encoding genes have been isolated to date. Two transcription factors called ORCA2 and ORCA3 which regulate the MeJA-responsive expression of at least half of the isolated genes have been described. A genome-wide screen using the cDNA- AFLP technique for ORCA target genes described in Chapter 2 has resulted in the identification of several dozens of new genes, many of which are predicted to encode enzymes. One of the transcript tags, CR-75, was upregulated by overexpression of either ORCA2 or ORCA3. Metabolite analysis described in Chapter 3 showed that overexpression of either ORCA2 or ORCA3 resulted in increased ajmalicine levels, indicating that the gene encoding Cathenamine Reductase (CR) must be regulated by both ORCAs. Since the CR-75 tag gave a TBLASTX hit to aldo/keto oxidoreductase enzymes we investigated the possibility that CR-75 corresponds to cathenamine reductase. Recombinant CR-75 protein produced in E. coli was able convert cathenamine to ajmalicine using NADPH as a cofactor, thereby identifying CR-75 as cathenamine reductase.
Introduction
Catharanthus roseus or Madagascar periwinkle, belonging to the family of Apocynaceae, produces a class of secondary metabolites termed terpenoid indole alkaloids (TIAs). Possible functions of these compounds in the plant include antimicrobial or antifeeding activity, UV protection or nitrogen storage and transport. Their synthesis has mainly been studied out of pharmaceutical interest. The monomeric alkaloids ajmalicine and serpentine are used in the treatment of cardiac and circulatory diseases, and the dimeric alkaloids vinblastine and vincristine are potent anti-tumour drugs. Although a lot is known about the pharmacological effects of many TIAs, little is known about how plants synthesize them. This is mainly due to the complex structure of the compounds, and to the regulatory complexity.
The central intermediate in TIA biosynthesis is strictosidine formed by coupling of the iridoid glycoside secologanin which is derived from geraniol, and tryptamine, produced from tryptophan (Fig. 1). Following synthesis by the enzyme strictosidine synthase (STR) strictosidine is converted by the enzyme strictosidine β-D-glucosidase (SGD) giving rise to cathenamine, 4,21-dehydrogeissoschizine and epi-cathenamine (Fig. 1) (Heinstein et al., 1979; Stevens, 1994).
Cathenamine equilibrates to 4,21-dehydrogeissoschizine, although the equilibrium is favored towards cathenamine (Heinstein et al., 1979; Kan-Fan and Husson, 1979). The formation of a small amount of 4,21-dehydrogeissoschizine after strictosidine conversion has important implications because 4,21-dehydrogeissoschizine is a precursor for many indole alkaloids via unknown mechanisms.
Cathenamine exists in an equilibrium of four stereoisomers, cathenamine, 19-epi-cathenamine, 3-isocathenamine, and 3-iso-19-epicathenamine. The equilibrium that exists in the test tube
between the different forms of cathenamine, between cathenamine and its carbinolamine form, and between cathenamine and 4,21-dehydrogeissoschizine has been called ‘La Ronde’ (Lounasmaa and Hanhinen, 1998). How these equilibriums behave in vivo is not known. It is likely that the chemical equilibriums are shifted by enzymes that catalyze the conversion of one of the intermediates from
“La Ronde”.
Obvious, however, is that after strictosidine conversion by SGD many closely related products are being formed via spontaneous chemical reactions. Probably from this point the biosynthesis diverges towards the different kind of indole alkaloids found in nature.
Cathenamine and epi-cathenamine can be reduced by the enzyme cathenamine reductase (CR) to form ajmalicine and epi-ajmalicine, respectively. For C. roseus two types of reductase have been described in literature that act separately in the biosynthesis of ajmalicine isomers. Cathenamine reductase (CR) was described by Stoeckigt et. al. (1983), and tetrahydroalstonine synthase (THAS) by (Hemscheidt and Zenk, 1985). Both enzymes specifically use NADPH as a cofactor, and both were partially purified in a similar fashion. The substrates for these enzymes are claimed to be different, epi-cathenamine for THAS, and cathenamine for CR. Product formation was shown to be stereospecific. THAS only catalyses the formation of tetrahydroalstonine, while CR yields both ajmalicine and epi-ajmalicine. Specificity is demonstrated by the discovery of cell cultures that only produced tetrahydroalstonine but no other heteroyohimbine alkaloids and that did not contain any CR activity (Hemscheidt and Zenk, 1985).
Figure 1. Biosynthesis of ajmalicine and tetrahydroalstonine in C. roseus.Enzymes for which corresponding genes have been isolated are shown in a black box and enzymes which have been studied biochemically with unidentified genes are shown in a gray box. Direction of reaction is shown by arrows, double arrows indicate equilibrium. All conversion steps lacking an enzyme abbreviation are thought to occur spontaneously. STR: strictosidine synthase, SGD: strictosidine β-D- glucosidase, CR: cathenamine reductase, THAS: tetrahydroalstonine synthase. Diagram modified from (Stevens, 1994;
Geerlings, 1999; Sundberg and Smith, 2002; Loyola-Vargas et al., 2007)
Starting from the amino acid tryptophan and the monoterpenoid geraniol, the biosynthesis of bisindole alkaloids in C. roseus is thought to involve at least 35 intermediates and a similar number of enzymes (van der Heijden et al., 2004). Thirteen enzyme-encoding genes have been isolated. Two transcription factors called ORCA2 and ORCA3 which regulate the MeJA-responsive expression of at least half of the isolated genes (Chapter 2) have been isolated. The genome- wide screen using the cDNA-AFLP technique for ORCA target genes described in Chapter 2 has resulted in the identification of several dozens of new genes, many of which are predicted to encode enzymes. One of the transcript tags, CR-75, was upregulated by overexpression of either ORCA2 or ORCA3 (Chapter 2). Metabolite analysis showed that overexpression of either ORCA2 or ORCA3 resulted in increased ajmalicine levels (Chapter 3), indicating that the gene encoding cathenamine reductase must be regulated by both ORCAs. Since the CR-75 tag gave a TBLASTX hit to aldo/keto oxidoreductase enzymes we investigated the possibility that CR-75 corresponds to cathenamine reductase. We isolated the full-length gene. The recombinant protein isolated from E. coli was able convert cathenamine to ajmalicine using NADPH as a cofactor.
Materials and Methods
Cell cultures, treatments, RNA extraction and Northern blotting
C. roseus MP183L wild type and transgenic cell lines were maintained as described in Chapter 2.
Treatments were performed 4 d after transfer. MeJA (Bedoukian) was diluted in DMSO and added to a final concentration of 10 µM. Yeast extract (Difco) was dissolved in water, autoclaved, passed through an ultra-filter with a molecular weight cut-off of 3 kDa (Millipore) to remove chitin, and was added at a final concentration of 400 µg/ml to cells. For the induction of transgenes 10 µM estradiol (dissolved in DMSO) was added. Induced cells were harvested at 24 h after induction.
Control cultures were treated with DMSO at a final concentration of 0.1% (v/v). Harvested samples were frozen in liquid nitrogen and stored at -80 ºC. RNA extraction and Northern blot hybridization was performed as described in Chapter 2.
Isolation of the CR-75 cDNA and plasmid construction
Transcript tag CR-75 was 210 bp long (Chapter 2). A BLASTN search of a C. roseus EST database gave 100% identity with an EST sequence with accession number EG562635. Based on the EST sequence a forward primer (5`TCA CTT CAG ATT CTT GGG GTT G-3`) and a reverse primer (5`TAC GTT GTG GGA TTT CAT GAA TTT G -3`) were designed to isolate 5` sequences and 3`sequences by PCR with a gene-specific primer and a vector primer using a pACTII cDNA library of YE-treated MP183L cells (Menke et al., 1999). The CR-75 open reading frame (ORF) was PCR amplified with the primers 5` ATT GGT ACC AAT GGA AAA GCA AGT TGA GAT CCC TG-3`
and 5` GCT CTC GAG CAC AAG TCT CCA TCC CAA AGC TC-3` using the pACTII library as a template and cloned in the pGEM-T Easy vector (Promega). The ORF was excised from pGEM-T Easy with KpnI/XhoI and cloned in the protein expression vector pASK-IBA45 plus (IBA, Göttingen, Germany) digested with KpnI/XhoI. Thus when expressed in E. coli the recombinant
protein contains a Strep tag at the N-terminus and a hexahistidine tag at the C-terminus.
Neighbor-joining phylogenetic analysis
Multiple sequence alignments were done with ClustalX2.0.11 (http://www.ddbj.nig.ac.jp) using the default settings (Thompson et al., 1997) and homology searches were done with BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi), in the National Center for Biotechnology Information database. For phylogenetic analysis the amino acid sequences of one representative from each subfamily of the AKR family (URL: http://www.med.upenn.edu/akr/) were aligned with the deduced amino acids of CR-57. The tree was constructed by use of the majority rule and strict consensus algorithm implanted in PHYLIP (Felsenstein, 1989). Terminal gaps were removed prior to running the analysis, while the internal alignment gaps were left and analysis conducted scoring gaps as characters or as missing characters. The neighbor joining method was used to create the tree.
Bootstrap analysis was conducted using 1000 bootstrap replicates (Felsenstein, 1989). TreeView (v.1.6.6) was used to display resulting trees (Page, 1996). The NADPH-binding domain and the putative active CR site were recognized as described by (Jez et al., 1997).
Recombinant protein isolation and Western blotting
Plasmid pASK-IBA45 plus carrying CR-75 was transformed to E. coli strain BL21 (DE3) pLysS.
The cells harboring the plasmid were cultured to an A600 of 0.5 in Luria-Bertani medium containing 50 µg/ml chloramphenicol and 200 µg/ml carbenicillin at 37 oC. The expression of the recombinant protein was then induced by adding 0.04 μg/ml anhydrotetracycline. After cultivation at 29 °C for 3.5 h and harvesting by centrifugation at 4500 g for 10 min at 4 °C, the bacterial pellet was resuspended in 20 ml Ni-NTA binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl pH 8.0), frozen in liquid nitrogen and stored at −80 °C. All further steps were performed at 4 °C.
Cells were lysed by thawing and DNA was sheared by 5 bursts of ultrasound (10 s, 40 W) using an ultrasonic tip. The suspension was centrifuged (18,000 g, 30 min, 4 °C) and the clear supernatant, containing the soluble fraction, was filtrated through a 0.45 µm membrane (Nalgene). The affinity- tagged CR-75 protein was purified using a 1 ml bed volume of Ni-NTA agarose (Qiagen). The bound proteins were eluted within the first 2 ml of elution buffer (1M imidazole, 0.5 M NaCl, 20 mM Tris-HCl pH8) collected in 0.25 ml fractions. The first six fractions were pooled and further purified using 1 ml bed volume of StrepTactin-sepharose (IBA, Göttingen, Germany). The bound proteins were eluted with elution buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA pH 8.0, 2.5 mM desthiobiotin). The eluate was collected in 0.25 ml fractions and the first six fractions were pooled and desalted using a PD-10 column (Amersham Pharmacia Biotech), equilibrated in 50 mM potassium phosphate (pH 6.6). Protein concentrations were measured using Bio-Rad protein assay reagent. Protein samples were separated by 10% (w/v) SDS-PAGE and transferred to Protran nitrocellulose (Whatman). Western blots were probed with anti-penta-His-HRP conjugated antibody (Qiagen) according to the manufacturer’s instructions. Antibody binding was detected by incubation in 250 µM sodium luminol (Sigma), 0.1 M Tris-HCl pH 8.8, 3 mM H2O2, 67 µM p-coumaric acid (Sigma) and exposure to X-ray film.
Substrate preparation enzyme assay
Strictosidine was prepared enzymatically using STR immobilized on a CNBr-activated Sepharose column (Pfitzner and Zenk, 1982). STR was purified from C. roseus suspension cultured cells as follows: 800-1200 g of freshly harvested cells were extracted by the described procedure (see enzyme assay), using 1:1 (v/w) 0.15 M sodium phosphate buffer (pH 7.0), with 2 mM EDTA and 3 mM DTT. PVPP was added to 5% (w/w). After clarification of the extract by centrifugation (30 min 9000 g, 4oC), a 30-50-% ammonium sulphate pellet was prepared, which was taken up in a small volume of 50 mM sodium phosphate buffer (pH 7.5). This was used for hydrophobic interaction chromatography over Phenyl-Sepharose CL-4B.Active fractions were pooled, and concentrated using an Amicon unit with Diaflo YM-10 filter, to ca. 20 ml. The concentrate was subjected to size exclusion chromatography using a 1 m column (2.6 id) of Sephacryl S-200 HR with 50 mM sodium phosphate (pH 7.5) as buffer. Purified STR was coupled to CNBr-activated Sepharose, following the instructions given by the manufacturer and the STR column was then used for coupling of tryptamine and secologanin ( Aldrich, Milwaukee, WI, USA). After the substrate solution was passed through the column the eluate was frozen at – 80 oC and subsequently lyophilized.
Enzyme assay
The glucose moiety of strictosidine was removed by incubating it with crude protein extract. For crude protein extraction wild type MP183L suspension cells were harvested by separation over a glass filter, washed with demineralised water, and frozen in liquid nitrogen. Frozen cells were grinded with pestle and mortar cooled with liquid nitrogen. Polyvinylpolypyrrolidone (PVPP, 5%
w/w) was added and stirred through the powdered material. Extraction buffer consisted of 0.1 M sodium phosphate (pH 6.3), with 3 mM EDTA and 6 mM DTT, and was added in a 1:1 ratio (v/w) and mixed to homogenous slurry. To accelerate thawing a water bath at 60 °C was used, with frequent stirring to prevent local thawing. The slurry was passed through a 0.45 µm membrane (Nalgene) at 4 oC. If not used directly, protein preparations were frozen in liquid nitrogen and stored at – 80 oC.
Cathenamine was prepared by incubating 25 µl of protein extract with 0.625 mM strictosidine in 0.1 M NaH2PO4 (pH 6.3) for 60 min at 30 oC in 500 µl. The mixture was centrifuged at 1600 g and the precipitate was washed with water, freeze dried and dissolved in dimethylformamide (2 mg/ml).
Ten µl of this solution were added to 20 mM sodium phosphate buffer (pH 6.6), 500 µM NADPH, and 2 to 10 µg recombinant protein or 5 µl of crude protein extract in 100 µl. After incubation for 30 min at 30 oC the reaction was stopped by the addition of 20 µl 1 M Na2CO3 solution pH 10. Control reactions were also set up without NADPH and with boiled protein.
Separation of alkaloids by HPLC
The alkaloid separation was performed on a 4 (i.d.) X 125 mm Lichrospher 60 (4 × 125 mm, 5 µm particles) RP-select B column (Merck). Eluent (0.75 ml/min) was delivered by means of a Waters 600S gradient controller pump (Waters, Milford, MA, USA). Eluent contained methanol : water in the proportions (72 : 28 v/v), 0.35% NaH2PO4 .H2O (v/w) and 0.2 % SDS (v/w). The alkaloids were eluted within 25 min. The online detection was realized with a Waters 990 photodiode-array
(PDA) detector recording from 210 to 350 nm. Identification of alkaloids was done by comparison of retention times and UV spectra with those of our reference library created from authentic samples.
Results
Full-length CR-75 cDNA encodes a protein belonging to the AKR superfamily
The genome-wide screen using the cDNA-AFLP technique for ORCA target genes described in Chapter 2 has resulted in the identification of several dozens of new genes, many of which are predicted to encode enzymes. Metabolite analysis showed that overexpression of either ORCA2 or ORCA3 resulted in increased ajmalicine levels (Chapter 3), indicating that the gene encoding the enzyme responsible for the conversion of cathenamine to ajmalicine (Fig. 1) must be regulated by both ORCAs. The enzyme described in literature for the conversion of this step is an NADPH- dependent oxido-reductase which was named cathenamine reductase (CR) (Stoeckigt et al., 1983). We searched in our cDNA-AFLP data set of tags upregulated by both ORCA2 and ORCA3 induction (Chapter 2; Fig. 5) for putative oxido-reductases and found transcript tag CR-75. Search of the NCBI non-redundant gene database with the CR-75 sequence using the TBLASTX algorithm gave a best hit to an aldo/keto reductase (AKR) gene from Sesbania rostrata (accession number CAA11226). AKRs are monomeric proteins, about 320 amino acid residues in size, that bind the cofactor NADPH (Wilson et al., 1992; Hoog et al., 1994; Wilson et al., 1995). We thought that this gene might encode
cathenamine reductase, and decided to isolate the full-length gene to investigate its function. PCR amplification of a cDNA library with CR-75 specific primers resulted in a cDNA sequence of 1141 bp, containing an ORF encoding a protein of 323 amino acids. The deduced amino acid sequence showed 40 -58 % identity with other AKRs from plant origin. The highest identity of 58% was observed with a putative chalcone reductase (AKR4B1) from S. rostrata, whereas 48% identity was found with codeinone reductase (AKR4B2-3) from Papaver somniferum, 43% with D-galacturonate reductase (AKR4B4) from Fragaria ananassa, 47-48% with deoxymugineic acid synthases from Zea mays, Oryza sativa, Triticum aestivum and Hordeum vulgare (AKR4B5-8) (Fig. 2). CR-75 protein also shares similarities with mammalian AKRs, and contains most of the active-site residues conserved in the AKR superfamily enzymes: the catalytic tetrad (Asp53, Tyr58, Lys88, and His121) and the residues for NADPH binding (Asp53, Ser167, Asn168, Gln189, Ser216, Leu218, Lys265, Ser266, Arg271, and Asn275) (Fig. 2). On the basis of sequence homology, CR-75 protein is a member of the AKR4 group and CR-75 protein was assigned the number AKR4B9 (Fig. 3).
The CR-75 transcript accumulates in response to ORCA overexpression and MeJA It was evident from cDNA-AFLP analysis that the CR-75 transcript tag is regulated by both ORCA2 and ORCA3 (Fig. 4a). The expression of CR-75 was verified by northern blot hybridization in ORCA2 and ORCA3 cell lines and in wild type cell lines. The CR-75 transcript was upregulated in ORCA cell lines when treated with estradiol compared to DMSO-treated samples. The expression of CR-75 was not affected in GFP cell lines treated with estradiol. When wild type cell lines were
Figure 2. Sequence alignment of C. roseus aldo-keto reductase with other members of the 4B subfamily of the AKR superfamily. 4B1, Sesbania rostrata chalcone reductase (CAA11226); 4B2, Papaver somniferum codeinone reductase (AAF13739); 4B3, Papaver somniferum codeinone reductase (AAF13736); 4B4, Fragaria ananassa D-galacturonate reductase (AAB97005); 4B5, Zea mays deoxymugineic acid synthase1 (BAF03164); 4B6 Oryza sativa deoxymugineic acid synthase1 (BAF03161 ); 4B7, Hordeum vulgare deoxymugineic acid synthase1 (BAF03162 ); 4B8, Triticum aestivum deoxymugineic acid synthase1 (BAF03163 ); CR-75 (= 4B9), Catharanthus roseus cathenamine reductase. The predicted catalytic residues (Asp53, Tyr58, Lys88, and His121) are marked with , and the residues for the NADPH binding with . For the predicted secondary structure α-helices (rectangles), β-strands (arrows), and loops (lines) are diagrammed.
treated with the plant hormone MeJA the expression was upregulated compared to the DMSO- treated cells or to non-treated cells (Fig. 4b). Yeast elicitor also induced the expression of CR-75.
The regulation of CR-75 mRNA accumulation by ORCA2 and ORCA3 and its responsiveness to MeJA and YE clearly are consistent with a possible role in the TIA biosynthesis pathway. Besides CR, another NADPH-dependent enzyme in the TIA pathway for which the corresponding gene have not been cloned is the related tetrahydroalstonine synthase (THAS) (Fig. 1).
Recombinant CR-75 protein has cathenamine reductase activity
The CR-75 protein was expressed in E. coli strain BL21 with a C-terminal His-tag and an N-terminal Strep-tag and purified by consecutive Ni-NTA and Strep-Tactin affinity chromatography steps with a yield of around 1 mg of recombinant protein from 1 g of E. coli cell pellet. Western blot analysis revealed the presence of a major band around 40 kDa, which was the expected size of the CR-75 protein.
The substrate cathenamine is a highly unstable compound which is not commercially available. Therefore we used an alternative strategy. Strictosidine was synthesized enzymatically from commercial tryptamine and secologanin, using STR purified from C. roseus cells suspension culture and immobilized on a CNBr-activated Sepharose column (Pfitzner and Zenk, 1982). The purified strictosidine was then incubated with crude protein extract from C. roseus cells. During
Figure 3. Unrooted phylogenetic tree of the aldo-keto reductase superfamily. Representative sequences from each subfamily of the AKR superfamily were aligned with ClustalX and the tree was displayed with TreeView The details and accession numbers of AKR proteins are at the AKR Superfamily page at the University of Pennsylvania (http://www.med.upenn.edu/
akr/). Catharanthus roseus AKR was assigned the member number AKR4B9.
hydrolysis of strictosidine by the crude extract, first a white precipitate was formed which was previously shown to mainly consist of cathenamine and its carbinolamine form (Stevens, 1994). The color of the precipitate then gradually turned bright yellow or orange. The yellow color is due to degradation products of 4,21-dehydrogeissoschizine which include 5,6-dihydroflavopereirine (Fig.
1) (Geerlings et al., 2000). The incubation mixture was extracted with chloroform and the chloroform phase was evaporated under reduced pressure. The precipitate was dissolved in dimethylformamide and was used as a source of substrate in the enzyme assay without further purification. Incubation of the mixture with recombinant protein produced ajmalicine in an NADPH+ dependent manner (Fig.
6). We did not observe the formation of tetrahydroalstonine in the enzyme assay. When we used crude protein extract as an enzyme source we also observed only ajmalicine formation. It might be that the cell line MP183L which we used for protein extraction contains only CR and lacksTHAS.
The product formed during the CR-75 assay was identified by comparison of its retention time with that of authentic ajmalicine and by its spectrum as determined using a photodiode array detector.
Ajmalicine and tetrahydroalstonine have identical UV spectra but the retention time was different as ajmalicine was eluted at 9.8 min while tetrahydroalstonine eluted at 12.4 min. Cathenamine, with identical UV spectrum as tetrahydroalstonine and ajmalicine, eluted with a retention time around 18.5 min, but no authentic standard for this compound was available for confirmation.
Figure 4. CR-75 transcript accumulates in response to ORCA overexpression, MeJA and YE. (A) Expression pattern of CR-75 transcript tag detected by cDNA-AFLP with primers BC1 and M22. Each line was treated with DMSO (D) or 10 µM estradiol (E). (B) Expression pattern of CR-75 transcript detected by Northern blot hybridization. Wild type (WT) cells were treated with MeJA (MJ), DMSO (D) or yeast elicitor (YE) for 24 hours. T0 corresponds to a sample taken at time zero. O2 and O3 indicate samples from ORCA2 and ORCA3 cell lines respectively. Samples from negative control lines are indicated with GFP. The bottom panel shows the ethidium bromide-stained gel prior to blotting.
Since cathenamine cannot be obtained in pure form, we could not determine enzyme kinetic parameters for the CR-75 recombinant protein. In order to get some indication whether the CR-75 cathenamine reductase is a specific enzyme in TIA biosynthesis or a common alcohol-dehydrogenase type enzyme from primary metabolism which can accept cathenamine as a substrate, formaldehyde, benzaldehyde, methanol, ethanol and propanol were tested as substrates. None of these compounds acted as substrate for the CR-75 protein, indicating that the enzyme is has at least some degree of
Figure 5. Analysis of recombinant CR-75 protein.The purified protein (1 µg) was separated by 10% SDS-PAGE and either stained with Coomassie Brilliant Blue (lane 1), or visualized after Western Blotting using anti-His antibodies (lane 2). Sizes of marker (M) bands are indicated in kDa.
Figure 6. CR-75 enzyme catalyzes the formation of ajmalicine. HPLC chromatograms of (A) authentic ajmalicine with a retention time of 9.968 min at 280 nm, (B) reaction products with the substrate mixture and CR-75 protein in the presence of NADPH, (C) reaction products with the substrate mixture and CR-75 protein without NADPH and (D) reaction products with the substrate mixture and crude protein extract from C. roseus cells in the presence of NADPH. The insets show UV spectra of the peaks with retention times around 10 min
specificity. We also used tetrahydroalstonine, ajmalicine, catharanthine, and tabersonine (15 µM) as substrates and did not observe any enzyme activity as measured by NADPH oxidation under these conditions.
Discussion
The cDNA-AFLP analysis of ORCA2 and ORCA3 over-expressing cell lines resulted in sets of transcript tags which were either upregulated by both ORCAs or specifically by one of the ORCAs.
The tag CR-75 was upregulated by both ORCAs (Chapter 2). Metabolite analysis showed that overexpression of either ORCA2 or ORCA3 resulted in increased ajmalicine levels (Chapter 3), indicating that the gene encoding CR must be regulated by both ORCAs. Since the CR-75 tag gave a TBLASTX hit to aldo/keto oxidoreductase enzymes we investigated the possibility that CR-75 corresponds to CR.
The full-length cDNA was isolated. Northern blot analysis confirmed that CR-75 transcript accumulated in response to overexpression of either ORCA2 or ORCA3 and in response to MeJA or yeast elicitor. All of the known TIA pathway genes are responsive to MeJA (Chapter 2), and therefore the expression pattern of the CR-75 transcript was consistent with a role of the encoded enzyme in TIA biosynthesis.
The CR-75 protein belongs to the aldo-keto reductase enzyme family. Aldo-keto reductases are found in all organisms and metabolize a diverse range of substrates, including aliphatic and aromatic aldehydes, monosaccharides, steroids, prostaglandins, polycyclic aromatic hydrocarbons and isoflavonoids (Jez et al., 1997). The AKR superfamily contains more than 120 enzymes, which are divided in 15 families (AKR1-AKR15) (Yokochi et al., 2004), nine of which contains multiple subfamilies (Hyndman et al., 2003). The proteins possess the α/β barrel motif, which provides a common scaffold for NAD(P)(H)-dependent catalytic activity, with the substrate specificity determined by a variety of loops on the C-terminal side of the barrel (Jez et al., 1997). The NADPH binding domain is highly conserved in all AKRs, even among proteins with less than 30% amino acid identity (Jez et al., 1997). The majority of known AKRs are monomeric proteins of about 320 amino acids in length, but multimeric forms also exist (Hyndman et al., 2003).
Phylogenetic analysis revealed that the CR-75 is a member of the AKR4B subfamily, and it was assigned the unique number AKR4B9 being the ninth identified subfamily member.
Interestingly the AKR4B subfamily also includes codeinone reductase from opium poppy which is a key enzyme in the biosynthesis of the alkaloid morphine (Unterlinner et al., 1999). Based on the correlation between the effects of ORCA overexpression on ajmalicine biosynthesis and on accumulation of the CR-75 transcript, we hypothesized that CR-75 corresponds to CR. An alternative NADPH-dependent reaction that may be catalyzed by CR-75 protein is the reduction of epicathenamine to tetrahydroalstonine (Loyola-Vargas et al., 2007).
To test our hypothesis that CR-75 corresponds to CR we needed cathenamine substrate.
Cathenamine is an unstable molecule which exists in equilibrium with 4,21-dehydrogeissoschizine after deglucosylation of strictosidine (Geerlings et al., 2000). To obtain cathenamine we coupled
tryptamine and secologanin enzymatically using STR . The formed strictosidine then was incubated with crude protein extract from C. roseus cell culture, which contains high SGD enzyme activity.
Products of this reaction were used as a source of substrate for the CR enzyme assay. We observed the formation of ajmalicine in an NADPH-dependent manner. Incubation of the substrate mixture with crude protein extract also resulted in the formation of ajmalicine. We did not observe the formation of tetrahydroalstonine in either case. This may be because the substrate epi-cathenamine is not present in our crude mixture. We could not determine kinetic parameters of the reaction due to of the fact that pure cathenamine cannot be obtained. We checked several other compounds from primary metabolism and secondary metabolism as substrates for CR-75 protein and found that none of them were metabolized as shown by lack of oxidation of NADPH.
With the identification of CR, all enzymes are now available for the production of ajmalicine from secologanin and tryptophan in heterologous organisms. Around 5000 kg of ajmalicine is purified annually from C. roseus leaves with a market price of about $2000 per kg. It has been previously shown that yeast cells fed with tryptamine and a crude extract of Symphoricarpus albus berries, which is a rich and cheap source of secologanin, are able to produce cathenamine when engineered to express STR and SGD (Geerlings et al., 2001). It would be interesting to see whether yeast cells engineered to express TDC, STR, SGD and CR are able to produce ajmalicine when grown on extracts from S. albus berries. Depending on the production level such a heterologous production system could form an economically viable alternative to ajmalicine extraction from C.
roseus leaves.
Acknowledgments
We are thankful to Professor Trevor Penning (University of Pennsylvania School of Medicine) for assignment of C. roseus CR to AKR subfamily 4B9.
We also thank Barbora Pomahačová for help with HPLC analysis, Ward de Winter is acknowledged for expert help with cell suspension cultures. G.H. was partially supported by Institute of Biotechnology and Genetic Engineering NWFP Agricultural University Peshawar, Pakistan and by a van der Leeuw grant from the Netherlands Organization for Scientific Research (NWO) awarded to J.M.
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