Dr0930, a member of the amidohydrolase superfamily in Deinococcus radiodurans, was cloned, expressed and purified to homogeneity. The enzyme crystallized in the space group P3121 and the structure was determined to a resolution of 2.1 Å. The protein folds as a (β/α)7β-barrel and a binuclear metal center is found at the C-terminal end of the β-barrel. The purified protein contains a mixture of zinc and iron and is intensely purple at high concentrations. The purple color was determined to be due to a charge transfer complex between iron in the β-metal position and Tyr-97. Mutation of Tyr-97 to phenylalanine or complexation of the metal center with manganese abolished the absorbance in the visible region of the spectrum. Computational docking was used to predict potential substrates for this previously unannotated protein. The enzyme was found to catalyze the hydrolysis of δ- and γ-lactones with an alkyl substitution at the carbon adjacent to the ring oxygen. The best substrate was δ-nonanoic lactone with a kcat/Km of 1.6 × 106 M−1 s−1. Dr0930 was also found to catalyze the very slow hydrolysis of paraoxon with values of kcat and kcat/Km of 0.07 min−1 and 0.8 M−1 s−1, respectively. The amino acid sequence identity to the phosphotriesterase (PTE) from Pseudomonas diminuta is ~30%. The eight substrate specificity loops were transplanted from PTE to Dr0930 but no phosphotriesterase activity could be detected in the chimeric PTE-Dr0930 hybrid. Mutation of Phe-26 and Cys-72 in Dr0930 to residues found in the active site of PTE enhanced the kinetic constants for the hydrolysis of paraoxon. The F26G/C72I mutant catalyzed the hydrolysis of paraoxon with a kcat of 1.14 min−1, an increase of 16-fold over the wild type enzyme. These results support previous proposals that phosphotriesterase activity evolved from an ancestral parent enzyme possessing lactonase activity.

Rapid evolution of enzymes provides unique molecular insights into the remarkable adaptability of proteins and helps to elucidate the relationship between amino acid sequence, structure and function. We interrogated the evolution of the phosphotriesterase from Pseudomonas diminuta (PdPTE), which hydrolyzes synthetic organophosphates with remarkable catalytic efficiency. PTE is thought to be an evolutionarily “young” enzyme and it has been postulated that it has evolved from members of the phosphotriesterase-like lactonase (PLL) family that show promiscuous organophosphate degrading activity. Starting from a weakly promiscuous PLL scaffold (Dr0930 from Deinococcus radiodurans), we designed an extremely efficient organophosphate hydrolase (OPH) with broad substrate specificity using rational and random mutagenesis in combination with in vitro activity screening. The OPH activity for seven organophosphate substrates was simultaneously enhanced by up to five orders of magnitude, achieving absolute values of catalytic efficiencies up to 106 M−1 s−1. Structural and computational analyses identified the molecular basis for the enhanced OPH activity of the engineered PLL variants and demonstrated that OPH catalysis in PdPTE and the engineered PLL differ significantly in the mode of substrate binding.