Fig. 2: In vitro coupled biochemical assay that examines the coumarin synthase activity of AtCOSY. | Nature Communications

Fig. 2: In vitro coupled biochemical assay that examines the coumarin synthase activity of AtCOSY.

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

Fig. 2

a Reaction schematic of the conversion of 6-hydroxyferulic acid (6OHFA) to scopoletin using 4-coumarate-CoA ligase 1 from A. thaliana (At4CL1) and coumarin synthase (AtCOSY). b Combined LC-MS extracted ion chromatograms (XICs) of 165.05556 m/z and 209.04549 m/z; 1, and 191.03443 m/z; 2. As scopoletin production is spontaneous, assay carried out in absence of AtCOSY serves as the baseline control for the coumarin synthase activity measurement. 4CL1 + AtCOSY sample shows complete conversion of 6OHFA into scopoletin. c Total ion count of 6OHFA and scopoletin from LC-MS for the enzymatic assays in triplicates. Data are presented as mean values +/− standard error of the mean. Relevant quantitative data underlying this figure is provided as a Source data file. d Reconstructed pose of trans−6OHFCoA in the active site of the CoA-bound AtCOSY structure. Phe40, Tyr42, His161, Cys 164, Trp371, and backbone atoms of Gly166 are highlighted as they make close contact with the substrate. e Relative coumarin synthase activity of wild-type and mutant AtCOSY. Log2 fold-change between the LC-HRAM-MS peak areas of coumarin ([M-H]− = 191.03443 m/z) in 4CL1 + AtCOSY (WT or mutant) samples and that of 4CL1-only samples are shown. We performed the mutant assays for the following mutants (H161A, H161Q, W371H, W371A, W371V, W371M, Y42F, F40T, C164A, F40T/Y42S, Y42F/H161A, G166A, G166L, Y373A, Y373F, L374A, and “Loop-swap”). All assays were performed in triplicates and the error bars represent standard error of the mean (except for Y373A, which was performed in duplicate due to experimental error). Relevant quantitative data underlying this figure is provided as a Source data file. f Structural alignment of CoA-bound (blue) and scopoletin-bound (green) AtCOSY structures highlighting the dynamic loop containing Tyr373 and Leu374. Arrows indicate change in orientation of Tyr373 and Leu374 in CoA-bound structure compared to scopoletin-bound structure. Source data are provided as a Source Data file.

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