Cannabis sativa (McPartland and Russo, 2001)

Rea, Kevin A, Casaretto, José A., Al-Abdul-Wahid, M. Sameer, Sukumaran, Arjun, Geddes-McAlister, Jennifer, Rothstein, Steven J. & Akhtar, Tariq A., 2019, Biosynthesis of cannflavins A and B from Cannabis sativa L, Phytochemistry 164, pp. 162-171 : 166

2.3. Phylogenetic analysis of C. sativa O-methyltransferases involved in the methylation of luteolin to chryseoriol

The observation that CsPT3 preferentially prenylates chrysoeriol, in vitro, and that prenylated luteolin is apparently absent in extracts from C. sativa implies, a priori, that methylation of luteolin to chrysoeriol must occur first in the cannflavin A and/or B pathway. We reasoned that the alleged enzyme that methylates luteolin at the 3′-hydroxyl position of the flavone B-ring to yield chrysoeriol would likely fall into the class of S - adenosyl-L- methionine (AdoMet)-dependent O -methyltransferases (OMTs), which are widely distributed throughout the plant kingdom ( Ibrahim et al., 1998; Ibrahim, 2005; Kim et al., 2010). We focused our initial searches of the TSA database for C. sativa on type 1 OMTs, which specifically methylate hydroxyl moieties of phenylpropanoid-based compounds ( Noel et al., 2003). Using a previously characterized flavonoid- O -methyltransferase from Oryza sativa (OsOMT9) that methylates the 3′-hydroxyl group on a variety of flavonoids as a query ( Kim et al., 2006), this search uncovered 40 unique nucleotide sequences corresponding to partial and/or full-length transcripts that were loosely annotated as ‘caffeic acid- O -methyltransferases’. We next compared these transcript sequences via BLASTn searches against the Cannabis whole genome contig database to confirm their corresponding full-length open reading frames. This analysis revealed twenty-four unique protein sequences ( Fig. S5 ) which were subsequently annotated as C. sativa O -methyltransferases (CsOMT1-24).

A phylogenetic analysis of the CsOMT family was then performed to establish their evolutionary relatedness to various plant OMTs that have been previously identified to act on aromatic substrates. This analysis revealed that type 1 CsOMTs are distributed into four general groups ( Fig. 4 ). It should be noted however, as Schrӧder et al. (2002) and Lam et al. (2007) previously pointed out, that assigning substrate preference based on sequence similarity alone for this class of plant enzymes is precarious. For example, the first group of type 1 plant OMTs depicted in our phylogenetic analysis includes enzymes that utilize a broad array of aromatic substrates - from simple phenolic compounds, such as chavicol, guaiacol and orcinol ( Gang et al., 2002; Scalliet et al., 2006; Akhtar et al., 2013), to more complex heterocyclic aromatics, such as homoeriodictoyl, myricetin, and resveratrol ( Schrӧder et al., 2004; Schmidin et al., 2008; Schmidt et al., 2011). We found nine CsOMT family members present within this group. The second group of type 1 OMTs appear specific to the Cannabaceae family and include seven CsOMTs along with two OMTs from Humulus lupulus that are involved in the synthesis xanthohumol ( Nagel et al., 2008). The third and fourth groups represent two closely related sister clades of type 1 plant OMTs and contain the remaining members of the CsOMT family. Strikingly, all plant OMTs that are known to methylate the 3′-hydroxyl position of various flavonoids are confined to group three and include representatives from Arabidopsis , peppermint, rice, wheat, and American golden saxifrage ( Gauthier et al., 1996; Muzac et al., 2000; Willitis et al., 2004; Kim et al., 2006; Zhou et al., 2006). We found three CsOMTs (CsOMT6, 12 and 21) that fell into this group.

Figures

Figure 5. Enzymatic characterization of CsOMT21. (A) Three flavones (apigenin, luteolin and chrysoeriol) and two flavonols (quercetin and kaempferol) that typically accumulate in C. sativa were tested as potential substrates. (B) Relative enzyme activity and substrate preference. Recombinant CsOMT21 was purified by Ni2+-affinity chromatography (inset). Proteins were resolved by SDS-PAGE and stained with Coomassie Blue. Lanes 1 and 2 contain crude E. coli extracts containing CsOMT21 and the purified protein, respectively; Molecular weights (kDa) are indicated. The selected flavones and flavonols (as numbered above) were provided to recombinant CsOMT21 in enzyme assays along with [14C]-AdoMet as a methyl donor. Data are the means ± SD from three independent experiments and are presented as relative activity compared to that observed with the preferred substrate, luteolin. (C) Evidence for the conversion of luteolin to chrysoeriol by CsOMT21. Enzyme assays with CsOMT21 together with luteolin and unlabelled AdoMet as cosubstrates were extracted and analyzed by HPLC. Note the identical retention time and CID-Q-TOF mass spectral fragmentation pattern of the enzymatic product from these assays (bottom panel) with that of an authentic chrysoeriol standard (top panel). (D) Kinetic analysis of CsOMT21. Recombinant CsOMT21 was assayed under standard assay conditions at the indicated concentrations of luteolin. Kinetic parameters were determined by non-linear regression analysis using the Michaelis-Menten kinetics model of the SigmaPlot 12.3 software.

Fig. 4. Phylogenetic analysis of type 1 O-methyltransferases from C. sativa. A neighbour-joining phylogenetic tree (1000 replicates) of type 1 O-methyltransferases from diverse plants was constructed using the MEGA 6.0 software package. Included in this analysis are twenty-four unique type 1 O-methyltransferases (CsOMTs) that were identified in the C. sativa genome. Branch lengths indicate the number of amino acid substitutions per sequence and bootstrap values (> 60%) are indicated next to each branch. The amino acid sequences and species abbreviations for all of these type 1 o-methyltransferases are listed in Supplemental Fig. S1 along with their corresponding GenBank accession numbers. Note that the CsOMTs (bolded) are uniformly distributed amongst four main groups of plant type 1 proteins.