Browsing by Author "Morris, Jeremy Stephane"

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    Structural and Functional Diversity Among Alkaloid N-Methyltransferases
    (2019-07-02) Morris, Jeremy Stephane; Facchini, Peter J.; Harrison, Joe; Jez, Joseph; Ling, Chang-Chun; Ng, Kenneth K.-S.
    Alkaloids are a group of nitrogenous specialized metabolites occurring in approximately one fifth of all plant species and which includes over 20 000 individual molecules with diverse structures and potent biological activities. In particular, plants containing benzylisoquinoline alkaloids (BIAs; eg. morphine, noscapine, sanguinarine) and phenylalkylamine protoalkaloids (PAAs; eg. pseudoephedrine) have been integral elements of human medicinal practice for thousands of years. To produce these molecules, plants employ a number of enzyme-catalyzed reactions including methylation of the nitrogen atom which is, by definition, present in all alkaloids. With respect to BIA biosynthesis in opium poppy (Papaver somniferum) and related species, three homologous yet functionally distinct subtypes of N-methyltransferase (NMT) enzymes are implicated (Coclaurine, Tetrahydroprotoberberine, and Pavine NMTs). This thesis describes the isolation and functional characterization of Reticuline NMT (RNMT), which defines a BIA NMT subtype preferentially accepting tertiary 1-BIA and aporphine substrates. Gene silencing reveals that RNMT catalyzes the ultimate or penultimate step in the biosynthesis of taxonomically widespread alkaloid (S)-magnoflorine. Despite the classification of BIA NMTs into the aforementioned subtypes, homologs show substantial functional variation with important physiological and biotechnological consequences. This thesis describes seven additional NMTs which contribute to the unique BIA content reported in meadow rue (Thalictrum flavum) and yellow horned poppy (Glaucium flavum). In contrast to work on BIAs, PAA biosynthesis has received much less attention and no dedicated enzymes have been reported. This thesis describes the identification and functional characterization of a novel member of the BIA NMT-like enzyme family (Phenylkalkylamine NMT; PaNMT) which is implicated in (pseudo)ephedrine biosynthesis in Ephedra sinica. To highlight the biotechnological potential of PaNMT, a heterologous pathway yielding (pseudo)ephedrine was reconstituted in E. coli. To better understand the structural features underlying functional diversity in BIA NMTs, structure-guided mutagenesis of three representatives was carried out and the resulting insights into catalysis and substrate recognition are reported herein. Notably, structural variants tightly correlated with either Coclaurine or Reticuline NMT activities were identified, and reciprocal mutagenesis experiments showed that a single residue is responsible for the functional dichotomy (preference for secondary or tertiary amine substrates).