Figure 4

(a) Comparison of ligand (α-methyl trans-cinnamate) conformations taken from the 4ZA7 crystal structure (violet) with the lowest energy docking pose (green), and with the reported transition state geometry²⁴ (orange) of 1,3-dipolar cycloaddition. I. – top view - the alignment of the double bond with respect to the prFMN cofactor in the docking conformation is in good agreement with the transition state geometry obtained by QM/MM study; II. – front view – substrate orientation related to prFMN and key residues R175, E280 and E285. (b) The energetically favoured, inactive orientation of 1v (purple) (similar positioning obtained also in case of 1w) - with E285 and prFMN distant from carboxyl group and the acrylic double bond of the substrate – compared to the active orientation of 1a (green). (c) Surface representation (blue) of the FDC1 binding pocket with substrate 1t, that displays the role of residues Q192 and I330 in narrowing the binding site in the proximity of the substrate’s aryl group; (d) binding of 1w in the active site of mutant I330A mutant (green), with the double bond in favourable position related to the C1′ and C4a of prFMN, compared to its inactive positioning in the wt-FDC1 (deep purple); steric hindrance between residue I330 (purple, paled) and the aryl group of the properly positioned 1w (green) occurs and is removed through mutation I330A.