A dynamic loop module enables phosphotriesterase function in cysteine-dependent hydrolases
A large disparity has emerged between the availability of massive metagenomic DNA sequence data and the actual functional annotation of enzyme catalysts, their mechanistic classification and insight into catalytic stragies. Exploration by sequence homology is intrinsically conservative, but ultrahigh-throughput functional metagenomics offers the chance to jump into unknown sequence space to provide novel catalysts without precedent. Having identified a novel metal-free phosphotriesterase with an active site cysteine-containing triad, here, we launch an exploration of sequence-structure-function relationships of a range of related proteins from the dienelactone hydrolase (DLH) family, revealing 10 new phosphotriesterases. Four crystal structures provide clues to mechanism, suggesting - based on phylogenetic and structural analysis - that phosphotriesterase activity is mediated by loops surrounding the active site with activity changes over 4 orders of magnitude correlated to loop flexibility. These insights allow protein engineering by loop grafting across family members, resulting in 8-fold increased phosphotriesterase activity in a human enzyme. We thus demonstrate that identification of a single pioneer enzyme can provide starting points for activity engineering strategy yielding new reagents for bioremediation or treatment of organophosphate poisoning.