Extended Data Fig. 7: Biochemical assays of the recombinant OSD4 enzyme.
From: Complete biosynthesis of salicylic acid from phenylalanine in plants
a, Biochemical reaction catalyzed by the benzyl salicylate carboxylesterase OSD4. b, Protein expression and purification of the recombinant protein MBP-OSD4 in E. coli BL21 (DE3). Lane 1, the molecular mass ladder; Lanes 2 and 3, the crude extracts from E. coli with MBP-OSD4 without or with IPTG treatment; the insoluble fractions (Lane 4) and soluble fractions (lane 5) from sonicated E. coli with MBP-OSD4 treated with IPTG; lanes 6, the purified MBP-OSD4 protein. The arrow points to the MBP-OSD4 protein. Proteins were visualized by Coomassie Blue R-250 staining. c, UV spectra of the OSD4 enzymatic product and SA standard. d and e, The LC total ion chromatogram and MS1 pattern (d), and specific ion chromatograms and MS/MS patterns (e) at multiple collision energies. The obtained molecular ion [M-H]− at m/z 137.0247 was an indicative peak for SA formation. The structure and proposed fragments of OSD4 enzymatic products were shown in (e). f and g, Effects of pH value (f) and temperature (g) on enzyme activities of MBP-OSD4. The optimal temperature is around 20–50 °C and the optimal pH value is 8.0. h, Kinetics of MBP-OSD4 protein on BS. The enzymatic activity obeys the Michaelis-Menten equation. i, The enzyme substrate specificities assays of recombinant MBP-OSD4 toward different substrates including benzyl salicylate, phenethyl salicylate, phenyl salicylate, methyl salicylate, ethyl salicylate, phenylethyl benzoate, benzyl benzoate, methyl benzoate, ethyl benzoate. Data are means ± s.d.; n = 3 (f, g, h, and i) independent samples. BS, benzyl salicylate; BAlc, benzyl alcohol; SA, salicylic acid.
