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. 2021 Feb 6;33(5):1472–1491. doi: 10.1093/plcell/koab044

Figure 4.

Figure 4

Investigation of the HCT reverse reaction in vitro. A, Scheme depicting the HCT reverse reaction that leads from caffeoyl-shikimate to caffeoyl-CoA thioester. B, HPLC–UV detection of authentic caffeoyl-CoA molecule at 350 nm and associated calibration curve exhibiting linearity over at least three orders of concentration. C, Representative HPLC–UV chromatograms showing the conversion of caffeoyl-5-O-shikimate into caffeoyl-CoA by PpHCT in vitro. Control assay was performed with boiled PpHCT enzyme. D, Absorbance spectrum from 240 to 400 nm of PpHCT assay and caffeoyl-CoA standard peaks. Wavelengths corresponding to maximum absorbance are indicated. E, PpHCT reverse activity with caffeoyl-5-O-shikimate or caffeoyl-5-O-quinate as acyl donor. F, Relative level of caffeoyl-5-O-shikimate and caffeoyl-5-O-quinate left in PpHCT assay (% of control assay). Data are the mean ± SEM of four independent enzyme assays.