Biochemistry

Abstract

CS-11-5 - Novel genes involved in alkaloid metabolism : Cloning and characterization of norbelladine synthase catalyzing the first committed step in Amaryllidaceae alkaloid biosynthesis.

Monday, July 16
2:43 PM - 2:45 PM

Amaryllidaceae alkaloids (AAs) represent a diverse class of plant specialized metabolites. Over 600 AAs have been identified so far with many interesting ones displaying potent biological activity. Pharmaceutical and commercial AAs can be obtained directly from plant sources. However, the variability and low concentration of AA accumulating in plants coupled with the limited information on their metabolic pathway are prime challenges against their use as commercial drugs. Currently, only few biosynthetic enzymes have been identified, thus numerous genes and enzymes remains to be discovered. All AAs are thought to share a common metabolic precursor, named norbelladine, formed from the condensation of 3,4-dihydroxybenzaldehyde and tyramine. Norbelladine formation represents the first committed reaction of the AA metabolic pathway. Similar condensation reactions catalyzed by Pictet-Spenglerase have been reported for benzylisoquinoline and terpene indole alkaloids. Using theses Pictet-Spenglerase nucleotide sequences, BLAST searches of Narcissus pseudonarcissus transcriptome were performed. We identified several genes including a candidate Pictet-Spenglerase, renamed norbelladine synthase (NBS). NBS sequence from N. pseudonarcissus cDNA was PCR amplified with a C-terminal His-tag, cloned into an expression vector and transformed into Escherichia coli for heterologous expression. After induction, NBS was produced and purified using the His-tag. Purified NBS was assayed for enzymatic activity with 3,4-dihydroxybenzaldehyde and tyramine substrates. LC-MS/MS analysis of the reaction product confirmed to production of norbelladine. Functional analysis of NBS was also performed and will be presented. Thus, we are the first to report on the identification and characterization of the first committed reaction leading to the formation of norbelladine, the metabolic precursor to all AAs. The identification of novel genes involved in AA biosynthesis will provide invaluable tools for the development alternative ways to produce AA using biotechnological processes such as plant engineering, plant cell culture or microbial metabolic engineering.


 

Co-Authors

Aparna Singh – Université du Québec à Trois-Rivières; Laurence Tousignant – Université du Québec à Trois-Rivières

Isabel Desgagné-Penix

Professor
Université du Québec à Trois-Rivières

Presentation(s):

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CS-11-5 - Novel genes involved in alkaloid metabolism : Cloning and characterization of norbelladine synthase catalyzing the first committed step in Amaryllidaceae alkaloid biosynthesis.



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Send Email for Novel genes involved in alkaloid metabolism : Cloning and characterization of norbelladine synthase catalyzing the first committed step in Amaryllidaceae alkaloid biosynthesis.