Characterization of norbelladine synthase and noroxomaritidine/norcraugsodine reductase reveals a novel catalytic route for the biosynthesis of Amaryllidaceae alkaloids including the Alzheimer's drug galanthamine

Abstract

Amaryllidaceae alkaloids (AAs) are a large group of plant specialized metabolites with diverse pharmacological properties. Norbelladine is the entry compound in AAs biosynthesis and is produced from the condensation of tyramine and 3,4-dihydroxybenzaldehyde (3,4-DHBA). There are two reported enzymes capable of catalyzing this reaction in-vitro, both with low yield. The first one, norbelladine synthase (NBS), was shown to condense tyramine and 3,4-DHBA, while noroxomaritidine/norcraugsodine reductase (NR), catalyzes a reduction reaction to produce norbelladine. To clarify the mechanisms involved in this controversial step, both NBS and NR homologs were identified from the transcriptome of Narcissus papyraceus and Leucojum aestivum, cloned and expressed in Escherichia coli. Enzymatic assays performed with tyramine and 3,4-DHBA with each enzyme separately or combined, suggested that NBS and NR function together for the condensation of tyramine and 3,4-DHBA into norcraugsodine and further reduction into norbelladine. Using molecular homology modeling and docking studies, we predicted models for the binding of tyramine and 3,4-DHBA to NBS, and of the intermediate norcraugsodine to NR. Moreover, we show that NBS and NR physically interact in yeast and in-planta, that both localize to the cytoplasm and nucleus and are expressed at high levels in bulbs, confirming their colocalization and co-expression thus their ability to work together in the same catalytic route. Finally, their co-expression in yeast led to the production of norbelladine. In all, our study establishes that both NBS and NR participate in the biosynthesis of norbelladine by catalyzing the first key steps associated in the biosynthesis of the Alzheimer's drug galanthamine.

Keywords: Amaryllidaceae alkaloids; Leucojum aestivum; Narcissus papyraceus; enzyme activity; norbelladine synthase; noroxomaritidine/norcraugsodine reductase.

Figure 1

Norbelladine synthase (NBS) and noroxomaritidine/norcraugsodine reductase (NR) are involved in the proposed biosynthetic pathway for galanthamine, lycorine and haemanthamine. NBS catalyzes the condensation of tyramine and 3,4-dihydroxybenzaldehyde (3,4-DHBA) to form norcraugsodine while noroxomaritidine/norcraugsodine reductase (NR) reduces the latter to form norbelladine, the common precursor to all Amaryllidaceae alkaloids produced in plants including galanthamine, cherylline, and lycorine.

Figure 2

Homology modeling of NpNBS, NR and TR. (A) Ribbon representation of NpNBS with colored secondary structures. β-sheets are displayed yellow, α-helices red, and loops blue. Conserved glycine rich P-loop of PR10-/Betv1 enzymes is shown black, as are conserved active site residues of norcoclaurine synthase Tyr68 (Tyr108 from TfNCS), Glu71 (Glu111), Lys83 (Lys123). As TfNCS, NBS are composed by seven-stranded antiparallel β-sheets, two long C- and N-terminal helices and two short ones, enclosing a cleft with polar residues at its entrance and hydrophobic residues in its core. (B) Transparent ribbon representation of NpNBS with predicted active site pocket in transparent surface. The predicted ligand site computed by the Site Finder tool of MOE software is displayed as white and red alpha sphere centers inside the pocket. NpNBS cavities is predicted to contain an active site of 27 residues, surrounded by catalytic Tyr68, Glu71, Lys83 and Ile102 (shown as black sticks), with Tyr68 at its entrance, Lys83 at the other side and the P-loop at the bottom. (C) Ribbon representation of NpNR with highlighted secondary structures. β-sheets are displayed yellow, α-helices red and loops in blue. (D) Cartoon ribbon representation of NpNR with transparent surface view of the predicted active site forming a catalytic tunnel that crosses the enzyme. The predicted ligand site is displayed as white and red alpha sphere centers inside the pocket. NpNR tunnel active site is predicted to contain 55 residues. Conserved active site residues Cys162, Tyr173, Lys177, Phe214, Glu224, Arg265 surrounding the tunnel are shown as black sticks. In general, amino acids involved in NADPH binding by noroxomaritidine/norcraugsodine reductase (NpKANR, 5FF9) are conserved and similarly oriented, i.e., Val81 (Val83 for NpKANR); Asp80 (82), Arg55 (57), Ser54 (56), Thr32 (34), catalytic residue Tyr173 (175), catalytic residue Lys177 (179), Asn108 (110); Thr208 (210), Pro203 (205), Gly204 (206) and Ala205 (207). (E) Ribbon representation of NpTR with secondary structures. β-strands are displayed yellow, α-helices red, and loops blue. (F) Ribbon representation of NpTR with transparent surface view of predicted active site tunnel crossing the enzyme. Predicted ligand site is displayed as white and red alpha sphere centers. Conserved catalytic Tyr172 and Lys176 are shown as black sticks.

Figure 3

NpNBS docked with 3,4-dihydroxybenzaldehyde (3,4-DHBA) and tyramine. (A) Cartoon representation of NpNBS with transparent surface-active site pocket docked with 3,4-DHBA (up) and tyramine (down). Conserved catalytic residues Tyr68 (Tyr108 from TfNCS), Glu71 (Glu110), Lys83 (Lys122) and Ile102 (instead of Asp141) shaping the binding site are shown as black sticks. (B) PLIP predicted conformation of interacting residues of NpNBS (grey) docked with 3,4-DHBA (turquoise) and tyramine (yellow). (C) NpNR docked with NADPH and norcraugsodine. Ligands NADPH (left) and norcraugsodine (right) are represented as thick yellow sticks. Conserved active site residues Cys162, Tyr173, Lys177, Phe214, Glu224 and Arg265) shaping the binding site are shown as thin black sticks. (D) PLIP predicted interacting residues of docked norcraugsodine (turquoise) with NpNR (grey sticks).

Figure 4

Enzymatic activity of NBS, NR and TR. Enzymes were tested separately or together for production of norbelladine, and the reaction product was monitored using HPLC-MS/MS. (A) Extracted ion chromatograms of quantifier MRM transition 260 → 138 m/z showing the product norbelladine in different enzymatic assays. The tested substrates used were 3,4-DHBA (300 μM) and tyramine (10 μM), and panels show norbelladine standard; assay without substrates; assay with MBP tag; assay without enzyme; and the complete assay performed with recombinant NpNBS, LaNBS, NpNR, LaNR and NpTR recombinant enzymes as indicated. Parent ion mass-to-charge (m/z) of 260 for norbelladine was subjected to collision-induced dissociation using multiple reaction monitoring (MRM) analysis. (B) Comparison and relative quantification of assays shown in Figure 4A in triplicate (mean ± SD, n = 3). The norbelladine product profiles in different assays performed were analyzed by HPLC-MS/MS and the obtained amount were relatively quantified using the area ratio of norbelladine produced in the assay to the papaverine internal standard. Data are means ± SE of three biological repeats. Asterisks indicate a significant difference (Student’s t test, p < 0.05) relative to NpNBS alone enzymatic assay.

Figure 5

Physical interaction of NBS and NR in planta and in Yeast. (A-D) The indicated proteins fused to NLuc or CLuc were expressed in leaves of Nicotiana benthamiana plants via Agrobacterium tumefaciens infection. (A, B) The images show LUC images of 96-well microtiter plates containing N. benthamiana leaf discs expressing the indicated constructs. (C, D) Luciferase activity was quantified as relative luciferase units (RLU) 48 hr post-infiltration. Data are means ± SE of three biological repeats. Asterisks indicate a significant difference (Student’s t test, p < 0.05) relative to empty vector. (E, F) Yeast expressing the indicated proteins fused to the GAL4 DNA-binding domain (Bait) or to the GAL4 DNA activation domain (Prey) were grown on synthetically defined (SD) medium lacking Leu and Trp (SD-LW), SD-LW lacking histidine and adenine (SD-LWHA), or SD-LW supplemented with Aureobasidin A (SD-LW+AbA). Empty vectors (EV) were used as negative controls. * = Asterisks indicate a significant difference (Student's t test, p < 0.05).

Figure 6

In vivo biosynthesis of norbelladine in yeast. HPLC-MS/MS analysis of norbelladine produced in yeast cultures expressing NBS and NR both singly and in combination. (A) Extracted ion chromatograms of quantifier MRM transition 260 → 138 m/z showing the product norbelladine in different indicated yeast cultures. (B) Comparison and relative quantification of assays shown in Figure 6A in triplicate (mean ± SD, n = 3). The norbelladine product profiles in different assays performed were analyzed by HPLC-MS/MS and the obtained amount were relatively quantified using the area ratio of norbelladine produced in the assay to the papaverine internal standard. Data are means ± SE of three biological repeats. Asterisks indicate a significant difference (Student’s t test, p < 0.05) relative to the yeast culture expressing NpNBS alone. * = Asterisks indicate a significant difference (Student's t test, p < 0.05).

Figure 7

NBS and NR colocalize to the cell cytoplasm and nucleus. (A) The indicated fusion proteins were co-expressed with the cyan fluorescent protein (CFP) in Nicotiana benthamiana leaves via Agrobacterium tumefaciens. After 48 hr, fluorescence was monitored in epidermal cells by confocal microscopy. Bright field, chlorophyll, yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and merged fluorescence images are shown. Scale bars in images represent 50 μM. (B) Confocal micrographs of transiently expressed NBS-YFP and NR-CFP in Nicotiana benthamiana leaves showing they colocalize to the cytoplasm and nucleus. Bright field, chlorophyll, yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and merged fluorescence images are shown. Scale bars in images represent 50 μM.

Figure 8

Relative expression of NBS, and NR in different tissues of N. papyraceus and L. aestivum using reverse transcription quantitative PCR (RT-qPCR) analysis. Different plant tissues as indicated were harvested after flowering, and NpNBS (A), LaNBS (B), NpNR (C), LaNR (D) mRNA levels were measured by RT-qPCR analysis relative to expression in leaves. NpHISTONE and LaGAPDH were used as normalizer. Data are means ± SE of three biological repeats.