Extended Data Fig. 2: CsPT4-T characterization. | Nature

Extended Data Fig. 2: CsPT4-T characterization.

From: Complete biosynthesis of cannabinoids and their unnatural analogues in yeast

Extended Data Fig. 2

a, CsPT4-T localized to the purified microsomal fraction. All LC–MS chromatograms were selected for the theoretical m/z values of the respective compounds of interest (Extended Data Table 3). Chromatography gradient for cannabinoid analogues was used. Signals were compared to genuine standards. We incubated boiled microsomal fraction (mic ΔT), soluble fraction (sol) and microsomal fraction (mic) of yCAN14 in the presence of 500 µM olivetolic acid and 500 µM GPP for 1 h at 30 °C and observed GOT activity only in the microsomal fraction. b, CsPT4-T olivetolic acid kinetics. Using nonlinear regression to fit the Michaelis–Menten kinetic model for varied olivetolic acid (0.25 µM–0.56 mM) and constant GPP (1.67 mM) concentrations revealed a Km(olivetolic acid) = 6.73 ± 0.26 µM (mean ± s.d.) (n = 4 technically independent samples; measurements were plotted individually). c, CsPT4-T GPP kinetics. Eadie–Hofstee linearization of Michaelis–Menten model showed non-Michaelis–Menten type behaviour for CsPT4-T for varied GPP (0.25 µM–1.67 mM) and constant olivetolic acid (1.67 mM) concentrations. Measurements do not fall on a line as would be expected (R2, coefficient of determination; n = 3 technically independent samples; measurements were plotted individually). d, Phylogenetic tree of Cannabis and related prenyltransferases. The numbers indicate the bootstrap value (%) from 1,000 replications. Grey dashed lines depict branch offsets to accommodate labels. The scale bar shows the amino acid substitution ratio. Prenyltransferases cluster by biosynthetic pathway. CsPT4 catalyses CBGA production. The function of the other Cannabis prenyltransferases remains unknown.

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