Structure-Function Studies of the Methyltransferase Enzymes involved in Benzylisoquinoline Alkaloid Metabolism
Date
2022-01-09
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Abstract
Plants represent a vast natural repository of unique and often potent compounds of pharmaceutical and industrial importance. Opium poppy (Papaver somniferum) is one of the first cultivated and most well studied species, particularly for its ability to produce an assortment of benzylisoquinoline alkaloids (BIAs) including the analgesic morphine and antitussive noscapine. A substantial body of functional and genomic studies have elucidated the dominant pathway and associated subset of enzymes responsible for BIA biosynthesis within opium poppy. The ability of many BIA enzymes to accept multiple substrates remains of particular interest, primarily for their potential to produce novel or rare BIAs as scaffolds for drug design. However, broad substrate promiscuity and lack of concerted mechanism of catalysis for homologous BIA enzymes prevents structure-function predictions by primary sequence alone. Tertiary structure determination of the BIA enzymes will help identify common motifs of substrate recognition and functionally important structure-activity relationships. Enzymatic methylation is one of the most frequent steps to the derivatization of BIAs in planta. Several methyltransferases have been identified which target the central nitrogen (NMTs) and peripheral oxygen atoms (OMTs) common amongst all BIAs. This thesis presents the crystal structure (at a dmin ranging from 1.6-2.5 Å) of Glaucium flavum tetrahydroprotoberberine N-methyltransferase (TNMT) in complex with a variety of preferred and alternative substrates. Structural determinants of substrate binding and catalysis are compared to the previously published structures of coclaurine and pavine NMT. The ability of the TNMT to only bind (S)-cis tetrahydroprotoberberine substrates is linked to the stereospecificity of the enzyme. Additional enzyme-substrate complexes highlight the role of specific residues in catalytic mechanism utilized by the BIA NMTs. Unlike the NMTs, the BIA OMTs possess broad evolutionary origins and distinct substrate binding poses. Homodimeric OMT structures highlight common motifs of BIA O-methylation, including a central dimerization domain which partially defines the substrate pocket. This thesis presents the first characterized heterodimeric BIA O-methyltransferase and resultant novel enzyme activity. Further characterization of the structural determinants required for heterodimerization and the effect of heterodimerization on differential substrate binding are presented. Homology modelling, activity assays, differential scanning fluorimetry, and crystallization trials provide a pathway for future structural studies by X-ray diffraction.
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Keywords
benzylisoquinoline alkaloid, biosynthesis, methyltransferase, protein structure, x-ray crystallography, enzyme mechanism, Opium poppy, biocatalysis
Citation
Lang, D. (2022). Structure-Function Studies of the Methyltransferase Enzymes involved in Benzylisoquinoline Alkaloid Metabolism (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.