Figure 1.
Proposed biosynthetic pathway for galanthamine.
3,4-Dihydroxybenzaldehyde derived from phenylalanine and tyramine derived from tyrosine are condensed to form norbelladine. Norbelladine is methylated by NpN4OMT to 4′-O-methylnorbelladine. 4′-O-Methylnorbelladine is oxidized to N-demethylnarwedine. N-demethylnarwedine is then reduced to N-demethylgalanthamine. In the last step, N-demethylgalanthamine is methylated to galanthamine.
Figure 2.
Identification of NpN4OMT in the daffodil transcriptome.
(A) Venn diagram of all sequences, all OMTs and all galanthamine correlating sequences according to HAYSTACK. (B) Accumulation level of galanthamine in daffodil (C) Candidate NpN4OMT expression profile in leaf, bulb and inflorescence with the relative initial read estimate and qRT-PCR ΔΔCt on the y-axis with leaf tissue set to 1.
Figure 3.
Recombinant NpN4OMT1 purification, enzyme assay and NMR structure elucidation of the 4′-O-methylnorbelladine product.
(A) SDS-PAGE gel 10% including fractions from crude extract and the desalted protein prep. This is shown for vector only, NpN4OMT1 and Pfs preparations. (B) Enzyme assays (top to bottom): Norbelladine standard; 4′-O-Methylnorbelladine standard; Assay with E. coli vector-only crude extract added; Assay without AdoMet added; Complete methyltransferase assay. (C) NMR structure elucidation: proton chemical shifts are black, carbon chemical shifts are blue, key HMBC correlations are black arrows, and key ROESY correlations are red arrows.
Table 1.
Substrate specificity of NpN4OMT1.
Figure 4.
Phylogenetic analysis of NpN4OMT1.
A maximum-likelihood phylogenetic tree of characterized methyltransferases listed in Table S2 [22]–[27], [46]–[49], [53], [63]–[70]. Alignment constructed using MUSCLE.