Abstract
Benzylisoquinoline alkaloids are a diverse class of plant specialized metabolites that includes the analgesic morphine, the antimicrobials sanguinarine and berberine, and the vasodilator papaverine. The two-electron oxidation of dihydrosanguinarine catalyzed by dihydrobenzophenanthridine oxidase (DBOX) is the final step in sanguinarine biosynthesis. The formation of the fully conjugated ring system in sanguinarine is similar to the four-electron oxidations of (S)-canadine to berberine and (S)-tetrahydropapaverine to papaverine. We report the isolation and functional characterization of an opium poppy (Papaver somniferum) cDNA encoding DBOX, a flavoprotein oxidase with homology to (S)-tetrahydroprotoberberine oxidase and the berberine bridge enzyme. A query of translated opium poppy stem transcriptome databases using berberine bridge enzyme yielded several candidate genes, including an (S)-tetrahydroprotoberberine oxidase-like sequence selected for heterologous expression in Pichia pastoris. The recombinant enzyme preferentially catalyzed the oxidation of dihydrosanguinarine to sanguinarine but also converted (RS)-tetrahydropapaverine to papaverine and several protoberberine alkaloids to oxidized forms, including (RS)-canadine to berberine. The K(m) values of 201 and 146 μm for dihydrosanguinarine and the protoberberine alkaloid (S)-scoulerine, respectively, suggested high concentrations of these substrates in the plant. Virus-induced gene silencing to reduce DBOX transcript levels resulted in a corresponding reduction in sanguinarine, dihydrosanguinarine, and papaverine accumulation in opium poppy roots in support of DBOX as a multifunctional oxidative enzyme in BIA metabolism.
Highlights
Oxidized forms of benzylisoquinoline alkaloids occur in plants
Isolation of FAD-dependent oxidoreductase (FADOX) Gene Candidates—Opium poppy genes encoding FADOXs were identified from stem and elicitortreated cell culture transcriptome databases queried using opium poppy Berberine bridge enzyme (BBE). 16 non-redundant contigs were identified, including eight full-length or nearly full-length sequences
Except for FADOX2 and FADOX6, which contain stop codons and a deletion in the Open reading frames (ORFs), respectively, amino acid sequences of other FADOX candidates were aligned to opium poppy BBE1
Summary
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. We report the isolation and functional characterization of an opium poppy (Papaver somniferum) cDNA encoding DBOX, a flavoprotein oxidase with homology to (S)-tetrahydroprotoberberine oxidase and the berberine bridge enzyme. Berberine bridge enzyme (BBE) is a well characterized flavoprotein oxidase that catalyzes the stereo-specific conversion of the central intermediate (S)-reticuline to (S)-scoulerine [4] (Fig. 1). Corresponding genes for neither DBOX nor DHBO have been isolated Both sanguinarine and berberine are quaternary ammonium cations, which occur at other points in BIA metabolism, including the oxidation of (S)-reticuline to 1,2-dehydroreticuline (Fig. 1). Our work describes the isolation and functional characterization of an opium poppy cDNA encoding DBOX, a flavoprotein oxidase catalyzing the terminal oxidations in sanguinarine and papaverine biosynthesis
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