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.
Highlights
Amaryllidaceae alkaloids are a group of alkaloids with many documented biological activities
Papaverine hydrochloride, S-adenosyl methionine (AdoMet), cobalt chloride, zinc chloride and manganese chloride were obtained from Fisher Scientific
The Illumina sequencing of daffodil leaf, bulb and inflorescence tissues resulted in 65 million paired-end reads that were used to make the daffodil transcriptome assembly
Summary
Amaryllidaceae alkaloids are a group of alkaloids with many documented biological activities. This makes them valuable potential medicines several examples are the anti-cancer compounds hemanthamine and lycorine and the anti-viral compound pancratistatin [1,2,3]. Galanthamine is an alkaloid discovered in 1953 that is produced by members of the Amaryllidaceae family [4] It reduces the symptoms of Alzheimer’s disease through acetylcholine esterase inhibition and nicotinic receptor binding. No committed biosynthetic genes have been identified [7,8]. Limited enzyme kinetic characterization has been done on plant protein extracts enriched for the norbelladine 49-O-methyltransferase (N4OMT) of Nerine bowdenii, but the underlying gene was never identified [9]
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