Characterization of a Flavoprotein Oxidase from Opium Poppy Catalyzing the Final Steps in Sanguinarine and Papaverine Biosynthesis

Abstract

Background: Oxidized forms of benzylisoquinoline alkaloids occur in plants.Results: In vitro and in vivo characterization of flavoprotein oxidases led to the isolation of a novel alkaloid biosynthetic enzyme in opium poppy. Conclusion:The final conversions in sanguinarine and papaverine biosynthesis are catalyzed by a flavoprotein oxidase. Significance: We have extended the importance of flavoprotein oxidases in benzylisoquinoline alkaloid metabolism.

Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae

“…1). In reconstitution of the pathway, we omitted dihydrobenzophenanthridine oxidase because of its low activity in S. cerevisiae 26 and because dihydrosanguinarine is easily oxidized to sanguinarine ex vivo 30 . We divided the remaining nine reactions into three 'blocks' of three sequential enzymes, as illustrated in Fig.…”

“…PsBBE was previously proposed to be functionally identical to E. californica BBE 26,37 , which is enantioselective for (S)-reticuline 33 . However, more reticuline than expected was consumed assuming an equal mixture of (R)-and (S)-reticuline from (R,S)-norlaudanosoline 22 , indicating that (R)-reticuline is also consumed in our system.…”

“…The last steps in sanguinarine biosynthesis were recently elucidated, laying the groundwork for complete synthesis of this molecule in a heterologous host [25][26][27] .…”

Natural products – modifying metabolite pathways in plants

“…Coordinated transcriptional induction and co-expression analysis, augmented by virus-induced gene silencing [64], were the driving force for the discovery of functionally unique enzymes, marking the conclusion of morphine [65] and sanguinarine [66,67] biosynthetic route elucidation. Application of the latter technique helped shed light on the controversy surrounding our understanding of papaverine formation, more than a century after the pathway was first proposed [68].…”

Δ9-Tetrahydrocannabinolic acid synthase production in Pichia pastoris enables chemical synthesis of cannabinoids

“…An additional reason for the successful production THCAS in P. pastoris KM71 KE1 might also be the pool of free FAD and the binding of the cofactor to the maturing enzyme. Three proteins have been described in literature, which share the same binding motif of FAD as THCAS, namely the glucooligosaccharide oxidase (GOOX), the berberine bridge enzyme (BBE) and the dihydrobenzophenanthridine oxidase (DBOX) (Hagel et al, 2012;Huang et al, 2008;Winkler et al, 2006). In general, methylotrophic yeasts, like P. pastoris, have a high potential to produce the cofactor FAD upon methanol induction considering the amount of eight FAD molecules bound to the oligomeric AOX1 and AOX2 (Marx et al, 2008).…”