Structural and Functional Studies of Pavine N-Methyltransferase from Thalictrum flavum Reveal Novel Insights into Substrate Recognition and Catalytic Mechanism

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 ). Additional structures were determined for the native protein (d(min) = 2.0 ) as well as binary complexes with SAM (d(min) = 2.3 ) or the reaction product S-adenosylhomocysteine (d(min) = 1.6 ). 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 ) 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.