Structural and Functional Diversity Among Alkaloid N-Methyltransferases

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dc.contributor.advisorFacchini, Peter J.
dc.contributor.authorMorris, Jeremy Stephane
dc.contributor.committeememberHarrison, Joe
dc.contributor.committeememberJez, Joseph
dc.contributor.committeememberLing, Chang-Chun
dc.contributor.committeememberNg, Kenneth K.-S.
dc.date2019-11
dc.date.accessioned2019-07-05T22:19:54Z
dc.date.available2019-07-05T22:19:54Z
dc.date.issued2019-07-02
dc.description.abstractAlkaloids 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).en_US
dc.identifier.citationMorris, J.S. (2019). Structural and Functional Diversity Among Alkaloid N-Methyltransferases (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.en_US
dc.identifier.doihttp://dx.doi.org/10.11575/PRISM/36713
dc.identifier.urihttp://hdl.handle.net/1880/110593
dc.language.isoengen_US
dc.publisher.facultyScienceen_US
dc.publisher.institutionUniversity of Calgaryen
dc.rightsUniversity 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.en_US
dc.subjectBiochemistryen_US
dc.subjectBenzylisoquinolineen_US
dc.subjectAlkaloiden_US
dc.subjectPlanten_US
dc.subjectSpecializeden_US
dc.subjectMetabolismen_US
dc.subjectBiosynthesisen_US
dc.subjectSyntheticen_US
dc.subjectPhenylalkylamineen_US
dc.subjectPhenethylamineen_US
dc.subjectEphedrineen_US
dc.subjectEphedraen_US
dc.subjectOpiumen_US
dc.subjectPoppyen_US
dc.subjectMethyltransferaseen_US
dc.subjectMagnoflorineen_US
dc.subjectReticulineen_US
dc.subjectMutagenesisen_US
dc.subjectStructuralen_US
dc.subject.classificationBotanyen_US
dc.subject.classificationPlant Physiologyen_US
dc.subject.classificationBiochemistryen_US
dc.titleStructural and Functional Diversity Among Alkaloid N-Methyltransferasesen_US
dc.typedoctoral thesisen_US
thesis.degree.disciplineBiological Sciencesen_US
thesis.degree.grantorUniversity of Calgaryen_US
thesis.degree.nameDoctor of Philosophy (PhD)en_US
ucalgary.item.requestcopytrueen_US
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