Facchini, PeterFarrow, Scott Cameron2015-11-192015-11-192015http://hdl.handle.net/11023/2648Benzylisoquinoline alkaloids (BIAs) are a large and structurally diverse group of plant specialized metabolites with several possessing pharmacological properties including the analgesic morphine, the cough suppressant codeine, and the vasodilator papaverine. In opium poppy, the antepenultimate and final steps in morphine biosynthesis are catalyzed by oxidoreductases belonging to the 2-oxoglutarate/Fe(II)-dependent dioxygenase gene family, namely thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM). When assayed with a wider range of BIAs, CODM, T6ODM, and the functionally unassigned paralogs DIOX2 and DIOX7, renamed protopine O-dealkylase (PODA) and papaverine 7-O-demethylase (P7ODM), respectively, showed novel and efficient O-dealkylation activities, including regioand substrate-specific O-demethylation and O,O-demethylenation. Preferred substrates for O,Odemethylenation by CODM and PODA were protopine alkaloids that serve as intermediates in the biosynthesis of benzo[c]phenanthridine and rhoeadine derivatives. Virus-induced gene silencing (VIGS) 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 revealed their indirect involvement in the formation of the antimicrobial benzo[c]phenanthridine sanguinarine and certain rhoeadine alkaloids in opium poppy. Furthermore, the efficient substrate-and regio-specific 7-O-demethylation of papaverine by P7ODM yielding pacodine suggests an unexpected biosynthetic route to pacodine. In addition to these findings, my thesis investigated oxidoreductases from the cytochromes P450 (CYP) and aldo-keto reductase (AKR) gene families. Using the functionally characterized codeinone reductase (COR) AKR translated nucleotide sequence as a query, we identified a COR paralogue from an opium poppy transcriptome database that was fused and in frame with a CYP. The resulting protein fusion catalyzed the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The fusion protein, renamed reticuline epimerase (REPI), was detected in opium poppy and in Papaver bracteatum, which accumulates the morphinan alkaloid thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. Suppression of REPI transcripts using VIGS in opium poppy reduced levels of (R)reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.BiochemistryOpium poppyBenzylisoquinoline alkaloidsOxidoreductasesdoctoral thesis10.11575/PRISM/26041