Noscapine biosynthesis in opium poppy involves three characterized O-methyltransferases (OMTs) and a fourth responsible for the 4'-methoxyl on the phthalide isoquinoline scaffold. The first three enzymes are homodimers, whereas the latter is a heterodimer encoded by two linked genes (OMT2 and OMT3). Neither OMT2 nor OMT3 form stable homodimers, but yield a substrate-specific heterodimer when their genes are co-expressed in Escherichia coli. The only substrate, 4'-O-desmethyl-3-O-acetylpapaveroxine, is a seco-berbine pathway intermediate that undergoes ester hydrolysis subsequent to 4'-O-methylation leading to the formation of narcotine hemiacetal. In the absence of 4'-O-methylation, a parallel pathway yields narcotoline hemiacetal. Dehydrogenation produces noscapine and narcotoline from the corresponding hemiacetals. Phthalide isoquinoline intermediates with a 4'-hydroxyl (i.e. narcotoline and narcotoline hemiacetal), or the corresponding 1-hydroxyl on protoberberine intermediates, were not accepted. Norcoclaurine 6OMT, which shares 81% amino acid sequence identity with OMT3, also formed a functionally similar heterodimer with OMT2. Suppression of OMT2 transcript levels in opium poppy increased narcotoline accumulation, whereas reduced OMT3 transcript abundance caused no detectable change in the alkaloid phenotype. Opium poppy chemotype Marianne accumulates high levels of narcotoline and showed no detectable OMT2:OMT3 activity. Compared with the active subunit from the Bea's Choice chemotype, Marianne OMT2 exhibited a single S122Y mutation in the dimerization domain that precluded heterodimer formation based on homology models. Both subunits contributed to the formation of the substrate-binding domain, although site-directed mutagenesis revealed OMT2 as the active subunit. The occurrence of physiologically relevant OMT heterodimers increases the catalytic diversity of enzymes derived from a smaller number of gene products.

Edited by Joseph M. Jez Benzylisoquinoline alkaloids (BIAs) are a structurally diverse class of plant-specialized metabolites that have been particularly well-studied in the order Ranunculales. The N-methyltransferases (NMTs) in BIA biosynthesis can be divided into three groups according to substrate specificity and amino acid sequence. Here, we report the first crystal structures of enzyme complexes from the tetrahydroprotoberberine NMT (TNMT) subclass, specifically for GfTNMT from the yellow horned poppy (Glaucium flavum). GfTNMT was co-crystallized with the cofactor S-adenosyl-L-methionine (d min ‫؍‬ 1.6 Å), the product S-adenosyl-L-homocysteine (d min ‫؍‬ 1.8 Å), or in complex with S-adenosyl-L-homocysteine and (S)-cis-N-methylstylopine (d min ‫؍‬ 1.8 Å). These structures reveal for the first time how a mostly hydrophobic L-shaped substrate recognition pocket selects for the (S)-cis configuration of the two central six-membered rings in protoberberine BIA compounds. Mutagenesis studies confirm and functionally define the roles of several highly-conserved residues within and near the GfTNMT-active site. The substrate specificity of TNMT enzymes appears to arise from the arrangement of subgroup-specific stereospecific recognition elements relative to catalytic elements that are more widely-conserved among all BIA NMTs. The binding mode of protoberberine compounds to GfTNMT appears to be similar to coclaurine NMT, with the isoquinoline rings buried deepest in the binding pocket. This binding mode differs from that of pavine NMT, in which the benzyl ring is bound more deeply than the isoquinoline rings. The insights into substrate recognition and catalysis provided here form a sound basis for the rational engineering of NMT enzymes for chemoenzymatic synthesis and metabolic engineering.

A focused library of virtual heterobifunctional ligands was generated in silico and a set of ligands with recombined fragments was synthesized and evaluated for binding to Clostridium difficile toxins. The position of the trisaccharide fragment was used as a reference for filtering docked poses during virtual screening to match the trisaccharide ligand in a crystal structure. The peptoid, a diversity fragment probing the protein surface area adjacent to a known binding site, was generated by a multi-component Ugi reaction. Our approach combines modular fragment-based design with in silico screening of synthetically feasible compounds and lays the groundwork for future efforts in development of composite bifunctional ligands for large clostridial toxins.

Plants such as the opium poppy have been extensively studied for their production of benzylisoquinoline alkaloids (BIAs), a class of specialized metabolites with many useful and potent pharmacological properties. Naturally occurring BIAs such as morphine and noscapine have been widely used since ancient times, but recent advances in synthetic biology and protein engineering provide exciting new opportunities for creating novel compounds with novel or improved pharmacological properties. N-methyltransferases (NMTs) play key roles in several different branches of BIA-metabolism. Hundreds of BIA NMT gene sequences from a wide variety of plants can be separated into three general groups based on function and sequence identity. Recent work from our group led to the determination of the first molecular structure of an NMT involved with BIA biosynthesis (pavine-NMT from Thalictrum flavum, M.A. Torres et al, J. Biol. Chem. 291:23403, 2016). More recently, we have determined the structures of enzymes from the two other groups of NMT's involved with BIA biosynthesis. Using pavine-NMT from T. flavum as a search model, we used molecular replacement to solve the structures of tetrahydroprotoberberine-NMT (52% sequence identity, dmin = 1.8 Å) and coclaurine-NMT (63% sequence identity, dmin = 2.2 Å) from Glaucium flavum. These structures reveal a high level of structural conservation in the overall protein fold, arrangement of catalytic residues at the active site and substrate-binding site for S-adenosylmethionine. A subset of residues within the binding site for the methyl-acceptor alkaloid substrate appear to define the unique substrate recognition specificities of each group of NMT's. To further explore these structure-function relationships, we have undertaken mutagenesis studies in combination with differential scanning fluorimetry, enzyme activity measurements and structure determination of enzymes with alkaloid substrates or substrate-analogs. Our presentation will discuss some of our recent progress in defining structure-function relationships in NMT enzymes and initial steps towards engineering altered substrate preferences for synthetic biology applications.

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