Structure–function studies of tetrahydroprotoberberine N-methyltransferase reveal the molecular basis of stereoselective substrate recognition

Abstract

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.

Molecular Origins of Functional Diversity in Benzylisoquinoline Alkaloid Methyltransferases

“…In the available crystal structures, up to 13 direct or water-mediated hydrogen bonds appear to position the cosubstrate ( Figure 4 ). The implicated residues are almost perfectly conserved in all cloned BIA NMTs, with the exception of one non-conservative substitution (Gln to His) in PsRNMT ( Supplementary Figure 6 ) (Torres et al, 2016; Bennett et al, 2018; Lang et al, 2019). Comparison of the apoenzyme and binary complexes (e.g., TfPavNMT versus TfPavNMT + SAH) revealed only a minor hinge movement of domains relative to that seen in the OMTs.…”

“…An initial investigation was reported for T. flavum pavine NMT (PDB 5KOK) and, recently, for C. japonica coclaurine NMT (PDB 6GKV) (Torres et al, 2016; Bennett et al, 2018). A third report concerning the tetrahydroprotoberberine NMT from G. flavum was accepted for publication during the preparation of this manuscript (PDB 6P3O) (Lang et al, 2019). Taken together, these three studies cover much of the functional range reported to date for BIA NMTs and reveal many of the molecular determinants of function.…”

“…(A) Key features of BIA NMTs are labeled according to their relative location along the T. flavum pavine N -methyltransferase (TfPavNMT) polypeptide with residues denoted by ovals and larger features by rectangles. (B) Crystal structures of TfPavNMT with bound SAH and ( R,S )-tetrahydropapaverine are reported by Torres et al (2016) (PDB 5KOK), (E) C. japonica coclaurine NMT with bound SAH and N -methylheliamine is reported by Bennett et al (2018) (PDB 6GKV), and (H) G. flavum tetrahydroprotoberberine NMT with bound SAH and ( S )- cis - N -methylstylopine is reported by Lang et al (2019) (PDB 6P3O). Domains correspond to those shown in (A) and Supplementary Figure 6 .…”

“…The Rossmann fold SAM-binding domains of various BIA NMTs are structurally quite similar, yielding RMSD values of ∼0.4–0.5 Å when aligned to each other (Torres et al, 2016; Lang et al, 2019). Binding of the cosubstrate occurs via sequence motifs largely conserved with other plant SAM-dependent MTs (Kozbial and Mushegian, 2005; Gana et al, 2013).…”

“…In all BIA NMTs, the binding pocket is lined with many hydrophobic residues and apparently gated by a loop, which becomes ordered and partially helical upon substrate binding. While a limited number of hydrogen bonds are recognizable in the GfTNMT crystal structure, as a general rule, it appears that substrate recognition and binding depends primarily on steric effects, Van der Waals interactions, and aromatic interactions ( Figure 4 ) (Torres et al, 2016; Lang et al, 2019).…”