PHP-Family Diesterase\nfrom <i>Novosphingobium</i> with Broad Specificity and High\nCatalytic Efficiency against Organophosphate\nFlame-Retardant Derived Diesters

Organophosphate flame retardants have been widely used\nin plastic\nproducts since the early 2000s. Unfortunately, these compounds leach\nout of the plastics over time and are carcinogenic, developmental\ntoxins, and endocrine disruptors. Due to the high usage levels and\nstable nature of the compounds, widespread contamination of the environment\nhas now been observed. Despite their recent introduction into the\nenvironment, bacteria from the <i>Sphingomonadaceae</i> family\nhave evolved a three-step hydrolytic pathway to utilize these compounds.\nThe second step in this pathway in <i>Sphingobium</i> sp.\nTCM1 is catalyzed by <i>Sb</i>-PDE, which is a member of\nthe polymerase and histidinol phosphatase (PHP) family of phosphatases.\nThis enzyme is only the second case of a PHP-family enzyme capable\nof hydrolyzing phosphodiesters. Bioinformatics analysis has now been\nused to identify a second PHP diesterase from <i>Novosphingobium</i> sp. EMRT-2 (<i>No</i>-PDE). Kinetic characterization of <i>Sb</i>-PDE and <i>No</i>-PDE with authentic organophosphate\nflame-retardant diesters demonstrates that these enzymes are true\ndiesterases with more than 1000-fold selectivity for the diesterase\nactivity seen in some cases. Synthesis of a wide array of authentic\nflame-retardant diesters has allowed the substrate specificity of\nthese enzymes to be determined, and mutagenic analysis of the active\nsite residues has identified key residues that give rise to the high\nlevels of diesterase activity. Despite high sequence identity, <i>No</i>-PDE is found to have a broader substrate specificity\nagainst flame-retardant derived diesters, and <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> values greater than 10<sup>4</sup> M<sup>–1</sup> s<sup>–1</sup> are seen with\nthe best substrates.