Structure-function studies of tetrahydroprotoberberine N -methyltransferase reveal the molecular basis of stereoselective substrate recognition
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 GfTNMT from the yellow horned poppy (Glaucium flavum). GfTNMT was co-crystallized with the cofactor S-adenosyl-l-methionine (dmin = 1.6 Å), the product S-adenosyl-l-homocysteine (dmin = 1.8 Å), or in complex with S-adenosyl-l-homocysteine and (S)-cis-N-methylstylopine (dmin = 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 catalyt...Continue Reading
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A single residue determines substrate preference in benzylisoquinoline alkaloid N-methyltransferases
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