Structure of Papaver somniferum O-Methyltransferase 1 Reveals Initiation of Noscapine Biosynthesis with Implications for Plant Natural Product Methylation

Abstract

The opium poppy, Papaver somniferum, has been a source of medicinal alkaloids since the earliest civilizations; ca. 3400 B.C. The benzylisoquinoline alkaloid noscapine is produced commercially in P. somniferum for use as a cough suppressant and it also has potential as an anticancer compound. The first committed step in the recently elucidated noscapine biosynthetic pathway involves the conversion of scoulerine to tetrahydrocolumbamine by 9-O-methylation, catalysed by O-methyltransferase 1 (PSMT1). We demonstrate, through protein structures (obtained through rational crystal engineering at resolutions from 1.5 to 1.2Å for the engineered variants) across the reaction coordinate, how domain closure allows specific methyl transfer to generate the product. SAM-dependent methyl transfer is central to myriad natural products in plants, analysis of amino-acid sequence, now taking the threedimensional structure of PSMT1 and low identity homologs into account, begins to shed light on the structural features that govern substrate specificity in these key, ubiquitous, plant enzymes. We propose how thus allowing prediction across the wide genomic resource.

Molecular Origins of Functional Diversity in Benzylisoquinoline Alkaloid Methyltransferases

“…Comparison of apo-enzyme and enzyme–substrate complexes further reveals that the residue equivalent to Thr170 in Tf6OMT (Ser211 in PsSOMT1) interacts with the co-substrate upon binding and contributes to a substantial conformational change (16° hinge movement) of the enzyme likely to be crucial for catalysis (Robin et al, 2016). Intriguingly, binding of SAH was sufficient to induce the closing movement in Tf6OMT, whereas the equivalent movement was only observed after binding of both SAH and the BIA substrate in PsSOMT1 (Cabry et al, 2019). Despite this minor difference, the available evidence suggests that SAM binding occurs in a very similar manner for all BIA OMTs.…”

“…To date, structures have been reported for T. flavum norcoclaurine 6OMT (Tf6OMT; PDB 5ICE) and P. somniferum scoulerine O -methyltransferase 1 (PsSOMT1; PDB 6I6K) (Robin et al, 2016; Cabry et al, 2019). While these structures have allowed for the generation of compelling hypotheses concerning substrate binding and catalysis, relatively little experimental work (e.g., site-directed mutagenesis) is presently available in support of their validity.…”

“…(A) Key features of BIA OMTs are labeled according to their relative location along the T. flavum norcoclaurine-6- O -methyltransferase (Tf6OMT) polypeptide with residues denoted by ovals and larger features by rectangles. Crystal structures of (B) Tf6OMT with bound SAH and ( S )-norlaudanosoline by Robin et al (2016) (PDB 5ICE), and (E) P. somniferum scoulerine OMT1 (PsSOMT1) with bound SAH and ( S )-scoulerine reported by Cabry et al (2019) (PDB 6I6K). Domains correspond to those shown in (A) and Supplementary Figure 3 .…”

“…Binding of the alkaloid substrate occurs at a location proximal to the SAM binding site and via residues overlapping the aforementioned conserved SAM-binding motifs ( Figure 3 ; Supplementary Figure 3 ). Notably, the relative orientation of the bound BIA molecule is flipped between Tf6OMT and PsSOMT1, such that isoquinoline and benzyl moieties or their derivatives in protoberberines ( Supplementary Figure 4 ; Supplementary Figure 5 ) generally make reciprocal interactions in either enzyme (Robin et al, 2016; Cabry et al, 2019). A histidine residue (His256 in Tf6OMT), in which hydrogen bonds with the target hydroxyl group, is perfectly conserved across BIA OMTs.…”

Over 100 Million Years of Enzyme Evolution Underpinning the Production of Morphine in the Papaveraceae Family of Flowering Plants

“…In addition to PSMT1, PSMT2 and PSMT3 are also involved in noscapine biosynthesis. They are all members of the same O -methyltransferase family, with PSMT2 and PSMT3 being more closely related to each other than to PSMT1 ( Cabry et al., 2019 ). PSMT2 and PSMT3 share the same exon/intron structure ( Winzer et al., 2012 ).…”