By overlaying the structures of wtPTE, AE, and neoPTE with the structure of A. radiobacter PTE in complex with the paraoxon analogue diethyl 4-methoxyphenyl phosphate (see Figure 2), regions important for paraoxon binding could be identified and the effect of mutations derived. The loss of phosphotriesterase in AE and its restoration in neoPTE is achieved mainly by changes in shape complementarity between enzyme and substrate, changes in hydrophobicity, and π-π stacking (A–C). It is likely that the movement of the β-metal also influences catalysis, although the exact effects of the metal displacement on catalysis are as yet unclear (D). (A) Interaction between paraoxon and residues 306 and 308. Substitution of the bulky Phe306 by Ile improves 2NH binding in AE, but results in a loss of interaction with paraoxon. In neoPTE, rather than reversion of f306I, s308 is mutated to the more hydrophobic Cys, improving interaction with the para-nitrophenyl group (Figure 2 pink region, Figure 7A). (B) Steric hindrance between Phe271 and paraoxon. Substitution of Leu271 to the larger Phe improves 2NH binding in AE, but causes steric hindrance with paraoxon. In neoPTE, in addition to reversion to the ancestral Leu, repositioning of the loop through a combination of remote mutations results in a ‘downward’ movement of Leu271, further enlarging the pocket (see also Figure 2 orange region, Figure 7A). (C) Shift in position of Leu106/Trp131/Leu132 (Figure 2 purple region, Figure 7B). While wt- and neoPTE feature edge-to-face π-π stacking between Trp131 and the para-nitrophenyl ring, in AE the shift in position brings Trp131 closer to the partially positive edges of the ring, resulting in electrostatic repulsion. Moreover, the shift in Leu106 brings it closer to the ethoxy group of the substrate in AE. (D) Shift in position of the β-metal. The inter-metal distance is reduced from 3.8 Å in wtPTE to 3.3 Å in AE through a movement of His201 and the β-metal. In neoPTE, the original spacing seen in wtPTE is restored (Figure 2 light blue region, Figure 7C), perhaps because the decreased distance in AE destabilized the transition state for paraoxon hydrolysis.