Figure 4

pXd-INV-D80A mutant complexes with phenolic inhibitors. A detail of the active site showing key residues for binding represented as sticks (the catalytic Asp221, Glu334 and the mutated D80A coloured in prune) and relevant water molecules as red spheres. The 2Fo-Fc electron density at the bound molecules has been contoured at RMSD of 0.9–1 ơ. Crystals were soaked into β-D-fructose and then into: (A) p-nitrophenol (PNP), (B) catechol (CAT), (C) epigallocatechin gallate (EGCG). A trapped ethylene glycol (EG) molecule was found in the catechol-soaked crystals. Main polar interactions of each inhibitor with the residues at the active site are represented as dashed lines, those corresponding to fructose (white sticks) being common and omitted for clarity. (D) Structural superimposition of the inhibitors positions from the catechol (green) vs. the EGCG (purple) ‒soaked crystals, showing the common fructose at subsite ‒1 in white. The catechol is located at the same position that the diphenolic moiety of benzopyrane from EGCG. (E) Structural comparison of the bound catechol and p-nitrophenol positions, compared to the pXd-INV complexed with sucrose³⁰, PDB code 5FIX. p-Nitrophenol is bound at a position close to glucose from sucrose, therefore competing with this donor substrate and blocking hydrolysis. (F) Structural comparison of the catechol and p-nitrophenol positions, compared to the two positions of the HT acceptor found in the reported pXd-INV-fructose complex⁵⁶, PDB code 5NSL. Images created with software Pymol 1.7 (http://www.pymol.org/).