Unlocking the Diversity of Alkaloids in Catharanthus roseus: Nuclear Localization Suggests Metabolic Channeling in Secondary Metabolism

Summary The extraordinary chemical diversity of the plant-derived monoterpene indole alkaloids, which include vinblastine, quinine, and strychnine, originates from a single biosynthetic intermediate, strictosidine aglycone. Here we report for the first time the cloning of a biosynthetic gene and characterization of the corresponding enzyme that acts at this crucial branchpoint. This enzyme, an alcohol dehydrogenase homolog, converts strictosidine aglycone to the heteroyohimbine-type alkaloid tetrahydroalstonine. We also demonstrate how this enzyme, which uses a highly reactive substrate, may interact with the upstream enzyme of the pathway.


Supplemental
, related to Figure 2. Characterization of THAS product. A. 1 H NMR Comparison of the major enzymatic product (bottom trace) with authentic standard of tetrahydroalstonine (top trace), aromatic region. B. Characterization of the major product by 13 C NMR. C. Large-scale production for NMR characterisation isolated by preparative thin layer chromatography using UV detection. Reaction by products are likely decomposition products of strictosidine aglycone. D. Real time PCR showing downregulation of THAS in silenced (VIGS) leaves. Relative gene expression level of THAS in C. roseus leaves that have been inoculated with pTRV2 Empty vector (EV) and pTRV2-THAS. The decrease of THAS gene expression due to VIGS is ≈ 85% compared to EV. E. Mass spectrometry profiles of silenced and empty vector control leaves that show statistically significant decrease in heteroyohimbine levels (P value 0.00199). The two diastereomers, ajmalicine and tetrahydroalstonine, cannot be separated under a variety of conditions on LCMS (see Supplemental Results). A peak corresponding to serpentine (m/z 349), which is derived from ajmalicine, does not significantly decrease in response to silencing of THAS. While these data indicate that THAS is involved in tetrahydroalstonine biosynthesis in vivo, we do not rule out the existence of additional C. roseus enzymes that catalyze tetrahydroalstonine formation.
Supplemental Figure S3, related to Figure 3. Sequence similarity of THAS with other enzymes. A. Pairwise protein alignment of THAS with sinapyl alcohol dehydrogenase (SAD; pdb 1YQD), the nearest functionally characterized protein homolog (64% amino acid identity); green: catalytic residues; light blue: catalytic Zn 2+ ligand sphere; dark blue: structural Zn ligand sphere; gray: NADP(H) binding; red: nuclear localization signal. B. The top hits of a tBLASTn search of the THAS amino acid sequence against the C. roseus transcriptome. The function of these transcripts has not been reported. Transcripts highlighted in gray have negligible expression levels in elicited seedlings (< 30 FPKM;THAS (locus_1974) is > 500 FPKM). The transcriptome and expression values can be downloaded from http://medicinalplantgenomics.msu.edu. The tBLASTn search was also performed at the medicinal plant genomics website.
. Figure S4, related to Figure 4. Localization of THAS. A. THAS is targeted to the nucleus via a monopartite NLS. C. roseus cells were transiently cotransformed with plasmids expressing free YFP (upper row), THAS-YFP (middle row) or YFP-THAS (lower row) and plasmid encoding the nucleocytosolic CFP marker (2 nd column). Co-localization of the fluorescence signals appears in yellow when merging the two individual (green/red) false-color images (3 rd column). Cell morphology is observed with differential interference contrast (DIC) (4 th column). Free YFP exhibits a typical nucleocytosolic localization while both THAS-YFP and YFP-THAS are primarily targeted to the nucleus with a residual cytosolic localization barely detectable. Bars 10 µm. B. Pull down of THAS with strictosidine glucosidase (SGD) as bait. His-SGD (380 µg) loaded onto His-column (V=1mL) followed by a wash with buffer (5 mL total). THAS (100 µg), preloaded with NADPH (0.5 mM), was then loaded (V=1mL). Elution was performed with 250mM imidazole washes (3 mL total). C. A negative control with an upstream biosynthetic enzyme LAMT (loganic acid methyl transferase) to demonstrate the specificity of the THAS/SGD interaction. LAMT/SGD (A) and THAS/SGD (C) interactions were analyzed by BiFC in C. roseus cells transiently transformed by plasmids encoding the indicated fusions. Cell morphology (B and D) is observed with differential interference contrast (DIC). No interaction with LAMT and SGD is observed. Bars 10 µm.

A. Cloning of THAS
The gene coding THAS was amplified from C. roseus leaf cDNA using primers designed based on the sequence from the expression reads (http://medicinalplantgenomics.msu.edu). The open reading frame of the gene was amplified using the primer pair 5'-AAGTTCTGTTTCAGGGCCCGGCAATGGCTTCAAA-3' and 5'-ATGGTCTAGAAAGCTTTAATTTGATTTCAGAGTGTTC-3' (gene specific sequence in italics), and cloned into the E. coli expression vector pOPINF (Berrow et al., 2007) using the In-fusion cloning system (Takara Clontech). The identity of the sequence was confirmed by sequencing (Source Bioscience mM glycine, 500 mM NaCl, 5% glycerol, 500 mM imidazole). Eluted protein was subjected to further purification on a Superdex Hiload 26/60 S75 gel filtration column (GE Healthcare) at a flow rate of 3.2 mL/min using Buffer C (20 mM Hepes pH 7.5, 150 mM NaCl) and collected into 8 mL fractions. After analysis by SDS-PAGE, those fractions containing no traces of other contaminating proteins were pooled and concentrated in a 10 KDa cutoff Millipore filter (Merck Millipore) and concentration was measured using a BCA assay (Thermo Fisher Scientific Inc., USA).

C. Enzyme assays
Purified THAS and purified SGD were used in all assays. Strictosidine was purified by preparative reverse phase HPLC and quantified using 1 H NMR. Strictosidine aglycone was generated in situ prior to addition of THAS by incubation of strictosidine and SGD in the appropriate solution for ten minutes, at which time strictosidine was completely converted to the aglycone. Steady state kinetic analyses were performed with 50 nM of THAS and 6 nM of SGD, 50 mM phosphate buffer (pH 7.5), 200 μM NADPH and an internal standard (50 μM caffeine). SGD has been shown to have a pH optimum between 6 and 8.5 (Luijendijk et al., 1998) and therefore should exhibit optimal activity at pH = 7.5. The kinetics were performed as follows: varying concentrations of strictosidine was placed in the wells of a 96-well plate with 50 mM phosphate buffer, followed by addition of 6 nM of SGD and the necessary volume of MilliQ water to standardize the volume. Caffeine (50 μM) was added to this mix as an internal standard. Another set of wells was prepared that contained pre-mixed solutions of 50 nM of THAS and 200 μM of NADPH. After 10 minutes of incubation with SGD, the strictosidine mix was added to the THAS + NADPH mix and mixed by pipetting several times. At 0.5 minutes, one minute and two minutes, a 10 μL aliquot of the reaction was placed in 80 μL H 2 0 + 0.1% formic acid premixed with 10 μL of methanol for a 10-fold final dilution of the sample. The 96-well plate was centrifuged at 4000 rpm for 10 min to pellet the enzyme precipitate and then analyzed by LCMS.
A similar procedure was used to determine the K m for NADPH, using 300 μM strictosidine and varying the NADPH concentration. The initial rate of the reaction (V 0 ) was calculated by fitting a linear regression through the points of product formation plotted against time. Michaelis-Menten plots were performed using SigmaPlot (Systat Software Inc.).

D. UPLC-QqQ-MS/MS analysis of the enzyme assays
Ultraperformance liquid chromatography (UPLC) was performed on a Waters Acquity UPLC system (Milford, MA) consisting of a binary pump, an online vacuum degasser, an autosampler, and a column compartment.

Kinetic studies and reaction monitoring
Chromatography was performed a BEH Shield RP18 (50 x 2.1 mm; 1.7 μm) column (Waters). The solvents used were H 2 O + 0.1% formic acid as Solvent A and 100% acetonitrile as Solvent B, with a flow rate of 0.6 mL/min. Injection volume was 2 µL.
The gradient profile was 0 min, 5% B; from 0 to 3.5 min, linear gradient to 35% B; from 3.5 min to 3.75 min, linear gradient to 100% B; wash at 100% B for 1 min; from 4.75 min to 6 min, back to 5% B for 1 min to re-equilibrate the column.

Quantification of tetrahydroalstonine: ajmalicine ratio
Separation of tetrahydroalstonine from ajmalicine could be achieved on a Luna NH2 (100 x 2.0 mm; 3 μm) column (Phenomenex). The chromatography was performed in HILIC mode using isocratic elution with 1% Solvent A and 99% Solvent C (50% acetonitrile + 50% isopropanol +0.1% formic acid) at a flow rate of 0.350 mL/min. The injection volume of both the standard solutions and the samples was 1 μL.
Mass spectrometry detection was performed on a Waters Xevo TQ-S mass spectrometer (Milford, MA, USA) equipped with an electrospray (ESI) source.
Capillary voltage was 2.5 kV in positive mode; the source was kept at 150 °C; desolvation temperature was 500 °C; cone gas flow, 50 L/h; and desolvation gas flow, 900 L/h. Unit resolution was applied to each quadrupole.
Targeted methods for each compound were developed using either commercial standards (caffeine, ajmalicine and tetrahydroalstonine were purchased from Sigma-Aldrich) or enzymatically produced compounds (strictosidine and strictosidine aglycone  The TLC plate was developed in ethyl acetate : hexanes : TEA (24 : 75 : 1), twice, and allowed to air dry between runs. Visualisation of the bands was performed using UV (254 nm). A minor product with m/z 353, which had an identical R f value to ajmalicine, was also observed, but could not be isolated in sufficient quantities for NMR.

F. NMR characterization
The major product of the reaction was excised from the TLC plate with a scalpel, and the silica was extracted with 2 mL of ethyl acetate five times.

G. Virus Induced Gene Silencing
The replicate. Titration with strictosidine aglycone (also in Buffer C) did not give significant binding heat, which indicates it cannot bind to the active site before binding of NADPH.

K. Pull down of THAS with SGD
Purified THAS was prepared for pull-down assay by cleaving the His-tag using 3C protease. THAS (200 μg) was incubated with 3C protease (1 μg) overnight at 4˚C, then 1 μg of fresh 3C protease was added and the reaction was allowed to progress for another thirty minutes at room temperature.
To purify the cleaved THAS, the reaction was passed through a 0.5 mL Ni-NTi Agarose slurry (Qiagen Ltd., Manchester, UK) pre-equilibrated with Buffer D (20 mM Hepes (pH 7.5), 150 mM NaCl). The flow through was collected and an aliquot was analyzed by SDS-page gel to verify the molecular weight. Cleaved THAS was concentrated using a Millipore filter unit with a 10 KDa cutoff and the concentration measured using a BCA assay. A glass chromatography column (0.5 cm x 10 cm) was loaded with 0.5 mL of Ni-NTi Agarose slurry (Qiagen) that was washed and equilibrated with 15 mL of Buffer D. His-tagged SGD (380 μg) was loaded onto the column and 1 mL fractions were collected. The column was then washed with 5 mL of Buffer C, followed by loading of 100 μg of THAS, premixed with 0.5 mM of NADPH. The column was washed with 5 mL of Buffer D and elution was carried out with 3 mL of buffer E (20 mM Hepes (pH 7.5), 150 mM NaCl, 250 mM imidazole).
Aliquots (20 µL Transient transformation of C. roseus cells by particle bombardment and fluorescence imaging were performed following the procedures previously described (Guirimand et al., 2009;Guirimand et al., 2010). Briefly, C. roseus plated cells were bombarded with DNA-coated gold particles (1 µm) and 1,100 psi rupture disc at a stopping-screen-to-target distance of 6 cm, using the Bio-Rad PDS1000/He system. Cells were cultivated for 16 h to 38 h before being harvested and observed. The subcellular localization was determined using an Olympus BX-51 epifluorescence microscope equipped with an Olympus DP-71 digital camera and a combination of YFP and CFP filters. The pattern of localization presented in this work is representative of circa 50 observed cells. The nuclear or nucleocytosolic localizations of the different fusion proteins were confirmed by co-transformation experiments using the nuclear-CFP marker and the nucleocytosolic CFP marker (Guirimand et al., 2010). Such plasmid co-transformations were performed using 400 ng of each plasmid or 100 ng for BiFC assays. Plasmids encoding bZIP63-YFP N and bZIP63-YFP C were used as controls (Waadt et al., 2008).