A dynamic loop module enables phosphotriesterase function in cysteine-dependent hydrolases

Massive DNA sequencing datasets are providing an unprecedented thesaurus of protein sequences. Still, when sequence homology to known enzymes is the only guide, their functional assignment and delineation of their catalytic strategies and mechanisms are lagging. The incremental nature of homology exploration is overcome by ultrahigh-throughput functional genomics, where ‘jumps’ into unknown sequence space provide annotations without precedent. As a case in point, recent work has identified novel metal-free phosphotriesterases with a cysteine-containing triad in the active site capable of rate accelerations of up to 10 ¹³ in k _cat / K _M . Here we expand this exploration and observe sequence-structure-function relationships of a range of homologous proteins from the dienelactone hydrolase (DLH) family, revealing 10 new phosphotriesterases. Four new crystal structures provide clues to mechanism, suggesting – based on phylogenetic and structural analysis – that phosphotriesterase activity is mediated by ‘lid’ loops surrounding the active site with activity correlated over 4 orders of magnitude to loop flexibility. These insights allow protein engineering by loop grafting across homologues, resulting in increased phosphotriesterase activity in a human enzyme. This exploration provides an annotation of starting points as well as an engineering strategy for the development of new reagents for bioremediation or treatment of organophosphate poisoning.