Fig. 3: In vitro coupled enzyme assay that examines the H/D exchange at C2 of the (D2)−6OHFA substrate. | Nature Communications

Fig. 3: In vitro coupled enzyme assay that examines the H/D exchange at C2 of the (D2)−6OHFA substrate.

From: Emergence of a proton exchange-based isomerization and lactonization mechanism in the plant coumarin synthase COSY

Fig. 3

a Reaction schematic for the conversion of 3, (D2)−6-Hydroxyferulic-acid into 4, (D2)-Scopoletin by light and 5, (D1)-Scopoletin by COSY. b Combined LC-MS extracted ion chromatograms (XICs) of 167.06804 m/z; 3, 193.04747 m/z; 4 and 192.04114 m/z; 5. The colored circles represent the relevant substrate or product peaks in each XIC trace corresponding to the compound number in a. c H/D exchange rate of assays conducted with no AtCOSY, wild-type AtCOSY, and various AtCOSY mutants. H/D exchange rate is defined as the percentage of 5 out of the total ion intensity for m/z values corresponding to 4 and 5. All assays were performed with 4CL1 in duplicates with data presented as mean values. Relevant quantitative data underlying H/D exchange is provided as a Source Data file. d MS1 spectra of the relevant peaks from assays shown in b corresponding to compounds highlighted in a.

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