Download MendeleyStructure-function studies of tetrahydroprotoberberineN-methyltransferase reveal the molecular basis of stereoselective substrate recognition.
Lang, D.E., Morris, J.S., Rowley, M., Torres, M.A., Maksimovich, V.A., Facchini, P.J., Ng, K.K.S.(2019) J Biol Chem 294: 14482-14498
- PubMed: 31395658
- DOI: https://doi.org/10.1074/jbc.RA119.009214
- Primary Citation of Related Structures:
6P3M, 6P3N, 6P3O - PubMed Abstract:
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 Gf TNMT from the yellow horned poppy ( Glaucium flavum ). Gf TNMT 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 Gf TNMT-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 Gf TNMT 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.
Organizational Affiliation:
Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
