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Title: Characterization of Oxidative Enzymes Involved in the Biosynthesis of Benzylisoquinoline Alkaloids in Opium Poppy (Papaver somniferum)
Author: Beaudoin, Guillaume Arthur Welch
Advisor: Facchini, Peter James
Keywords: Biology--Molecular;Plant Physiology;Biochemistry
Issue Date: 16-Mar-2015
Abstract: Benzylisoquinoline alkaloids (BIAs) are a large group of nitrogen-containing specialized metabolites. Opium poppy (Papaver somniferum) is an important pharmaceutical plant and has been cultivated for thousands of years for its analgesic constituents: the morphinan BIAs codeine and morphine. In addition, opium poppy produces other BIAs with biological activities, such as the vasodilator papaverine, the potential anti-cancer drug noscapine and the antimicrobial agent sanguinarine. The objective of this work was to identify and characterize oxidative enzymes involved in BIA biosynthesis, with a specific focus on cytochromes P450 (P450s). The biosynthesis of sanguinarine from (S)-reticuline requires four P450s. Our first objective was to identify these unidentified P450s in opium poppy. Our approach was based on the coordinated induction of all sanguinarine biosynthetic enzyme transcripts and proteins in elicited opium poppy cell suspension cultures. Using data from a previous microarray study, we identified 12 uncharacterized inducible P450s. Recombinant expression in Saccharomyces cerevisiae revealed that 4 of these enzymes (CYP719A20, CYP719A25, CYP82N3 and CYP82N4) were involved in sanguinarine biosynthesis. Unexpectedly, in planta silencing using virus-induced gene silencing showed that plants suppressed in these P450s transcripts have significantly increased levels of sanguinarine and dihydrosanguinarine. Many changes in the accumulation of sanguinarine biosynthetic intermediates were also detectable. In CYP82N3- and CYP82N4-silenced plants, we detected a significant accumulation of some alkaloids in roots and a significant reduction of some of these in latex. Previous silencing of the penultimate step in morphine biosynthesis, codeinone reductase (COR), showed a dramatic decrease in morphinan alkaloids, as well as a significant increase in reticuline, the central BIA branch point intermediate. A search for similar enzymes that may be co-silenced revealed a P450 reductase fusion. Further in vitro and in planta characterization showed that this enzyme is responsible for the final unidentified and first committed steps in morphine biosynthesis, the epimerization of (S)-reticuline. Papaver rhoeas, which does not accumulate morphinan alkaloids, possesses this enzyme as two polypeptides. The significance of this difference remains to be studied. We were also able to identify a paralog of COR which can catalyze the isomerization of many morphinans, potentially opening new routes to manufacture novel opiates.
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