Publications

• Purification, crystallization and X-ray diffraction analysis of pavine N-methyltransferase from Thalictrum flavum.

  Jain A., Ziegler J., Liscombe D.K., Facchini P.J., Tucker P.A., Panjikar S.

  A cDNA from the plant Thalictrum flavum encoding pavine N-methyltransferase, an enzyme belonging to a novel class of S-adenosylmethionine-dependent N-methyltransferases specific for benzylisoquinoline alkaloids, has been heterologously expressed in Escherichia coli. The enzyme was purified using a ... >> More

  A cDNA from the plant Thalictrum flavum encoding pavine N-methyltransferase, an enzyme belonging to a novel class of S-adenosylmethionine-dependent N-methyltransferases specific for benzylisoquinoline alkaloids, has been heterologously expressed in Escherichia coli. The enzyme was purified using affinity and gel-filtration chromatography and was crystallized in space group P2(1). The structure was solved at 2.0 A resolution using a xenon derivative and the single isomorphous replacement with anomalous scattering method. << Less

  Acta Crystallogr Sect F Struct Biol Cryst Commun 64:1066-1069(2008) [PubMed] [EuropePMC]

• Structural and functional studies of pavine N-methyltransferase from Thalictrum flavum reveal novel insights into substrate recognition and catalytic mechanism.

  Torres M.A., Hoffarth E., Eugenio L., Savtchouk J., Chen X., Morris J.S., Facchini P.J., Ng K.K.

  Benzylisoquinoline alkaloids (BIAs) are produced in a wide variety of plants and include many common analgesic, antitussive, and anticancer compounds. Several members of a distinct family of S-adenosylmethionine (SAM)-dependent N-methyltransferases (NMTs) play critical roles in BIA biosynthesis, b ... >> More

  Benzylisoquinoline alkaloids (BIAs) are produced in a wide variety of plants and include many common analgesic, antitussive, and anticancer compounds. Several members of a distinct family of S-adenosylmethionine (SAM)-dependent N-methyltransferases (NMTs) play critical roles in BIA biosynthesis, but the molecular basis of substrate recognition and catalysis is not known for NMTs involved in BIA metabolism. To address this issue, the crystal structure of pavine NMT from Thalictrum flavum was solved using selenomethionine-substituted protein (d_min = 2.8 Å). Additional structures were determined for the native protein (d_min = 2.0 Å) as well as binary complexes with SAM (d_min = 2.3 Å) or the reaction product S-adenosylhomocysteine (d_min = 1.6 Å). The structure of a complex with S-adenosylhomocysteine and two molecules of tetrahydropapaverine (THP; one as the S conformer and a second in the R configuration) (d_min = 1.8 Å) revealed key features of substrate recognition. Pavine NMT converted racemic THP to laudanosine, but the enzyme showed a preference for (±)-pavine and (S)-reticuline as substrates. These structures suggest the involvement of highly conserved residues at the active site. Mutagenesis of three residues near the methyl group of SAM and the nitrogen atom of the alkaloid acceptor decreased enzyme activity without disrupting the structure of the protein. The binding site for THP provides a framework for understanding substrate specificity among numerous NMTs involved in the biosynthesis of BIAs and other specialized metabolites. This information will facilitate metabolic engineering efforts aimed at producing medicinally important compounds in heterologous systems, such as yeast. << Less

  J. Biol. Chem. 291:23403-23415(2016) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Targeted metabolite and transcript profiling for elucidating enzyme function: isolation of novel N-methyltransferases from three benzylisoquinoline alkaloid-producing species.

  Natural Products Genomics Resource (NAPGEN), Liscombe D.K., Ziegler J., Schmidt J., Ammer C., Facchini P.J.

  An integrated approach using targeted metabolite profiles and modest EST libraries each containing approximately 3500 unigenes was developed in order to discover and functionally characterize novel genes involved in plant-specialized metabolism. EST databases have been established for benzylisoqui ... >> More

  An integrated approach using targeted metabolite profiles and modest EST libraries each containing approximately 3500 unigenes was developed in order to discover and functionally characterize novel genes involved in plant-specialized metabolism. EST databases have been established for benzylisoquinoline alkaloid-producing cell cultures of Eschscholzia californica, Papaver bracteatum and Thalictrum flavum, and are a rich repository of alkaloid biosynthetic genes. ESI-FTICR-MS and ESI-MS/MS analyses facilitated unambiguous identification and relative quantification of the alkaloids in each system. Manual integration of known and candidate biosynthetic genes in each EST library with benzylisoquinoline alkaloid biosynthetic networks assembled from empirical metabolite profiles allowed identification and functional characterization of four N-methyltransferases (NMTs). One cDNA from T. flavum encoded pavine N-methyltransferase (TfPavNMT), which showed a unique preference for (+/-)-pavine and represents the first isolated enzyme involved in the pavine alkaloid branch pathway. Correlation of the occurrence of specific alkaloids, the complement of ESTs encoding known benzylisoquinoline alkaloid biosynthetic genes and the differential substrate range of characterized NMTs demonstrated the feasibility of bilaterally predicting enzyme function and species-dependent specialized metabolite profiles. << Less

  Plant J. 60:729-743(2009) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.