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.

Identification of a Noroxomaritidine Reductase with Amaryllidaceae Alkaloid Biosynthesis Related Activities

“…pseudonarcissus, G. elwesii, and Galanthus sp. (17,18). This transcript was also co-expressed with N4OMT in the previously described Trinitybased assembly for Galanthus sp.…”

“…aff. pseudonarcissus with 106,450 sequences and a workflow for the identification of Amaryllidaceae alkaloid biosynthetic pathway genes from the 9,505 contigs that co-express with the accumulation of galanthamine was described (17). This resource was used to identify candidate oxidoreductases in the pathway.…”

“…aff. pseudonarcissus SDR gene that co-expresses with the previously discovered N4OMT (17). Biochemical analysis of the recombinant protein reveals that this N. sp.…”

“…Recent studies have identified enzymes catalyzing steps in the core Amaryllidaceae alkaloid biosynthesis pathway, including norbelladine 4Ј-O-methyltransferase (N4OMT) and the cytochrome P450 CYP96T1 (44). N4OMT catalyzes the 4Ј-O-methylation of norbelladine to 4Ј-O-methylnorbelladine (17). CYP96T1 is a para-paraЈ phenol-phenol coupling cytochrome P450 that generates an enantiomeric mixture of noroxomaritidine (18), which can be further modified into hemanthamine-or crinine-type alkaloids depending on the stereochemistry of noroxomaritidine (19 -21).…”

“…aff. pseudonarcissus, Galanthus sp., and Galanthus elwesii transcriptomes successfully identified N4OMT and CYP96T1 in the core Amaryllidaceae alkaloid biosynthetic pathway (17,18). For several missing steps in the hemanthamine, crinine, maritinamine, and elwesine pathways, we hypothesized that members of either aldo-keto reductase or SDR enzyme families may catalyze norbelladine synthesis and/or reduction of the noroxomaritidine enantiomers.…”