Fig. 2: Functional characterization of AtCXE15 and AtCXE20.

a, b Melting temperature curves of AtCXE15 (a, red) and AtCXE20 (b, green) in the presence or absence of rac-GR24 (brown) were determined through DSF analysis. The graph represents the average of three technical replicates. c Limited trypsin digestions of AtCXE15 and AtCXE20 are shown in the presence or absence of rac-GR24. All proteins were resolved via SDS-PAGE and visualized by Coomassie stain. d YLG hydrolysis assay of AtCXE15 (red), AtCXE15^E271A (blue), and AtCXE15^S169A (green). e Comparative YLG hydrolysis assay of CXE15 (red) and CXE20 (brown). Colored lines represent non-linear regression curve fit based on the averaged raw data points plotted using GraphPad Prism 10.0. f Initial kinetic rate constants (k_cat (s⁻¹), K_m (μΜ), k_cat/K_m (s⁻¹ μΜ⁻¹) of CXE15 and CXE20. g, h Electrostatic surface representation of AtCXE15 and AtCXE20 (left). Zoom in view of CXE15 (g) and CXE20 (h) catalytic pockets (middle), and sequence conservation of selected residues (right) within the corresponding catalytic pocket. The electrostatic potential is calculated by PyMOL and APBS with the non-linear Poisson–Boltzmann equation contoured at ±5 kT/e. Negative and positively charged surface areas are colored in red and blue, respectively. Naming scheme, At A. thaliana, Bn Brassica napus, Cr Caspella rubella, Cs Camelina sativa, Tc Theobroma cacao, Gh Gossypium hirsutum, Ma Mercurialis annula, Ah Arachis hypohaea, Cp Carcia papaya, Rc Ricinus communis, Cb Capsicum baccatum, Mc Momordica charantia, Tw Tripterygium wilfordii, Pa Populus alba, Dz Durio zibethinus. All experiments in (a–f) were repeated three times. Source data are provided as a Source Data file.