The addition of a methyl moiety to a small chemical is a common transformation in the biosynthesis of natural products across all three domains of life. These methylation reactions are most often catalysed by S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTs). MTs are categorized based on the electron-rich, methyl accepting atom, usually O, N, C, or S. SAM-dependent natural product MTs (NPMTs) are responsible for the modification of a wide array of structurally distinct substrates, including signalling and host defense compounds, pigments, prosthetic groups, cofactors, cell membrane and cell wall components, and xenobiotics. Most notably, methylation modulates the bioavailability, bioactivity, and reactivity of acceptor molecules, and thus exerts a central role on the functional output of many metabolic pathways. Our current understanding of the structural enzymology of NPMTs groups these phylogenetically diverse enzymes into two MT-superfamily fold classes (class I and class III). Structural biology has also shed light on the catalytic mechanisms and molecular bases for substrate specificity for over fifty NPMTs. These biophysical-based approaches have contributed to our understanding of NPMT evolution, demonstrating how a widespread protein fold evolved to accommodate chemically diverse methyl acceptors and to catalyse disparate mechanisms suited to the physiochemical properties of the target substrates. This evolutionary diversity suggests that NPMTs may serve as starting points for generating new biocatalysts.

Summary(S)-Norcoclaurine synthase (NCS) (EC 4.2.1.78) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids such as morphine, sanguinarine, and berberine, in plants. A molecular clone encoding NCS was isolated from a meadow rue (Thalictrum flavum ssp. glaucum) cell suspension culture cDNA library. Heterologous expression of the NCS cDNA, truncated to remove a putative signal peptide, produced a recombinant protein with NCS activity. Recombinant NCS showed sigmoidal saturation kinetics for dopamine (Hill coefficient = 1.98), hyperbolic saturation kinetics for 4-HPAA (K m of 700 lM), and pH and temperature optima of 7.0 and 40°C, respectively, all similar to the purified, plant-derived enzyme. NCS exhibits 28-38% identity, and putative structural homology, with the Bet v 1 allergen and pathogenesis-related (PR)10 protein families. NCS also displays 35% identity with the enzyme (HYP1) responsible for hypericin biosynthesis in St John's wort (Hypericum perforatum). The novel catalytic functions of NCS and HYP1 define a new class of plant secondary metabolic enzymes within the Bet v 1 and PR10 protein families. Weaker homology was also detected between NCS and proteins identified in the latex of Papaver somniferum (opium poppy), and in Arabidopsis thaliana. A family of three to five NCS genes is abundantly expressed in the rhizome, followed by petioles and roots of T. flavum. NCS transcripts were localized to the immature endodermis and pericycle in roots, and the protoderm of leaf primordia in rhizomes; thus, the sites of NCS gene expression and berberine accumulation are temporally and spatially separated in roots and rhizomes respectively.

Norcoclaurine synthase catalyzes an asymmetric Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde to give (S)-norcoclaurine. This is the first committed step in the biosynthesis of the benzylisoquinoline alkaloids that include morphine and codeine. In this work, the gene encoding for the Thalictrum flavum norcoclaurine synthase is highly overexpressed in Escherichia coli and the resulting His-tagged recombinant enzyme is purified for the first time. A continuous assay based on circular dichroism spectroscopy is developed and used to monitor the kinetics of the enzymatic reaction. Dopamine analogues bearing a methoxy or hydrogen substituent in place of the C-1 phenolic group were readily accepted by the enzyme whereas those bearing the same substituents at C-2 were not. This supports a mechanism involving a two-step cyclization of the putative iminium ion intermediate that does not proceed via a spirocyclic intermediate. The reaction of [3,5,6-2H]dopamine was found to be slowed by a kinetic isotope effect of 1.7 +/- 0.1 on the value of kcat/KM. This is interpreted as showing that the deprotonation step causing rearomatization is partially rate determining in the overall reaction.