Fig. 4: Crystal structure of AtCXE15^GR24 complex and structural investigation of SL binding and catalysis.

a Crystal structure of CXE15^GR24 (pale green) with distinct NTH (light pink) shown in different angles. b Overall structure of CXE15^GR24 bound to D-ring (sticks, light orange) of GR24 (left). Close-up view of D-ring interaction within the catalytic cavity. The F_o–F_c map of the D-ring is shown at 1σ cut-off (middle-right). The γ-oxygen of the catalytic S169 of CXE15 is covalently linked to the carboxylate group of the D-ring. c Comparative structural analysis of CXE15^apo (pale cyan) and CXE15^GR24 (light green) complex (left). Close-up view of catalytic pocket showing the differences in the orientation of amino-acid side chains when bound to D-ring of GR24 (right). d Ligplot interaction diagram of CXE15^GR24 bound to D-ring (light orange). The covalently linked residues are shown as straight lines, the hydrogen bonds are represented as dashed lines and the arc represents the hydrophobic interaction. e Electrostatic representation of CXE15 bound to D-ring (left). Zoom in view into the catalytic pocket of CXE15^GR24 bound D-ring (sticks). Dashed lines indicate the catalytic cavity. 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. f Proposed schematic representation of the mechanism of action of CXE15 towards SLs in plants. S169 is deprotonated by histidine (H302) which is stabilized in its charged form by the presence of Glutamic acid (E271). This deprotonated S169 then undergoes a nucleophilic attack on the carbonyl of the substrate (GR24). The resulting tetrahedral transition state is stabilized by an oxyanion hole (G85–G86). A water molecule subsequently performs a nucleophilic attack, protonating the leaving group (ABC-ring of GR24) and forming a covalent enzyme-substrate intermediate. In the second phase of the reaction, another water molecule attacks the covalent intermediate, resulting in the formation of the product and the restoration of the catalytic triad to its native state. Source data are provided as a Source Data file.