Cloning and characterization of a norbelladine 4'-O-methyltransferase involved in the biosynthesis of the Alzheimer's drug galanthamine in Narcissus sp. aff. pseudonarcissus

Abstract

Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer's disease. This compound is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and summer snowflake (Leucojum aestivum). Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4'-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4'-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway.

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.

Figure 4. Phylogenetic analysis of NpN4OMT1.

A maximum-likelihood phylogenetic tree of characterized methyltransferases listed in Table S2 –, –, , –. Alignment constructed using MUSCLE.