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1.
Plant J ; 77(2): 173-84, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24708518

ABSTRACT

The final step in the biosynthesis of the phthalideisoquinoline alkaloid noscapine involves a purported dehydrogenation of the narcotinehemiacetal keto moiety. A short-chain dehydrogenase/reductase (SDR), designated noscapine synthase (NOS), that catalyzes dehydrogenation of narcotinehemiacetal to noscapine was identified in opium poppy and functionally characterized. The NOS gene was isolated using an integrated transcript and metabolite profiling strategy and subsequently expressed in Escherichia coli. Noscapine synthase is highly divergent from other characterized members of the NADPH-dependent SDR superfamily involved in benzylisoquinoline alkaloid metabolism, and it exhibits exclusive substrate specificity for narcotinehemiacetal. Kinetic analyses showed that NOS exhibits higher catalytic efficiency with NAD+ as the cofactor compared with NADP+. Suppression of NOS transcript levels in opium poppy plants subjected to virus-induced gene silencing resulted in a corresponding reduction in the accumulation of noscapine and an increase in narcotinehemiacetal levels in the latex. Noscapine and NOS transcripts were detected in all opium poppy organs, but both were most abundant in stems. Unlike other putative biosynthetic genes clustered in the opium poppy genome, and their corresponding proteins, NOS transcripts and the cognate enzyme were abundant in latex, indicating that noscapine metabolism is completed in a distinct cell type compared with the rest of the pathway.


Subject(s)
Noscapine/metabolism , Opium/metabolism , Oxidoreductases/metabolism , Papaver/enzymology , Base Sequence , Biocatalysis , Chromatography, High Pressure Liquid , DNA Primers , Genes, Plant , Kinetics , Ligases/genetics , Ligases/metabolism , Molecular Sequence Data , Papaver/genetics , Papaver/metabolism , Tandem Mass Spectrometry
2.
J Biol Chem ; 288(40): 28997-9012, 2013 Oct 04.
Article in English | MEDLINE | ID: mdl-23928311

ABSTRACT

In opium poppy, the antepenultimate and final steps in morphine biosynthesis are catalyzed by the 2-oxoglutarate/Fe(II)-dependent dioxygenases, thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM). Further investigation into the biochemical functions of CODM and T6ODM revealed extensive and unexpected roles for such enzymes in the metabolism of protopine, benzo[c]phenanthridine, and rhoeadine alkaloids. When assayed with a wide range of benzylisoquinoline alkaloids, CODM, T6ODM, and the functionally unassigned paralog DIOX2, renamed protopine O-dealkylase, showed novel and efficient dealkylation activities, including regio- and substrate-specific O-demethylation and O,O-demethylenation. Enzymes catalyzing O,O-demethylenation, which cleave a methylenedioxy bridge leaving two hydroxyl groups, have previously not been reported in plants. Similar cleavage of methylenedioxy bridges on substituted amphetamines is catalyzed by heme-dependent cytochromes P450 in mammals. Preferred substrates for O,O-demethylenation by CODM and protopine O-dealkylase were protopine alkaloids that serve as intermediates in the biosynthesis of benzo[c]phenanthridine and rhoeadine derivatives. Virus-induced gene silencing used to suppress the abundance of CODM and/or T6ODM transcripts indicated a direct physiological role for these enzymes in the metabolism of protopine alkaloids, and they revealed their indirect involvement in the formation of the antimicrobial benzo[c]phenanthridine sanguinarine and certain rhoeadine alkaloids in opium poppy.


Subject(s)
Benzylisoquinolines/metabolism , Biocatalysis , Dioxygenases/metabolism , Opium/metabolism , Papaver/enzymology , Benzylisoquinolines/chemistry , Berberine Alkaloids/chemistry , Berberine Alkaloids/metabolism , Chromatography, Liquid , Formaldehyde/metabolism , Gene Silencing , Kinetics , Mass Spectrometry , Methylation , Phylogeny , Substrate Specificity , Viruses
3.
J Biol Chem ; 287(51): 42972-83, 2012 Dec 14.
Article in English | MEDLINE | ID: mdl-23118227

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.


Subject(s)
Benzophenanthridines/biosynthesis , Biocatalysis , Flavoproteins/metabolism , Opium/metabolism , Oxidoreductases/metabolism , Papaver/enzymology , Papaverine/biosynthesis , Benzophenanthridines/chemistry , Enzyme Assays , Gene Expression Regulation, Plant , Gene Silencing , Genes, Plant/genetics , Genetic Association Studies , Isoquinolines/chemistry , Oxidoreductases/genetics , Papaver/genetics , Papaverine/chemistry , Phylogeny , Plant Viruses/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Substrate Specificity
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