Insights into the function, thermostability, and evolution of organophosphorus hydrolase enzymes

Organophosphorus hydrolase (OPH) enzymes are a class of enzymes capable of degrading organophosphate (OP) compounds, such as many common pesticides and chemical warfare agents. This work focuses on the mutagenesis and characterization of the OPH from Deinococcus radiodurans (Dr-OPH) in order to raise its catalytic efficiency toward OP compounds to make it a viable candidate for development into practical applications for remediation of these compounds. Both structure-guided and saturation mutagenesis techniques were used in this effort and the turnover of the OP pesticide ethyl paraoxon was raised by 104-fold, with a simultaneous 559-fold increase in catalytic efficiency. The crystal structures of 5 mutants were solved and led to the conclusions that size of the active site, efficient proton shuttling, and protein dynamics may be playing a rote in enzymatic activity.;The OPH from Geobacillus stearothermophilus ( Gs10-OPH) was also isolated and kinetically characterized, demonstrating 30-fold higher ethyl paraoxon turnover than Dr-OPH. The structure of Gs10-OPH was determined for comparison to Dr-OPH. The thermostability of Gs10-OPH was also tested and it was found to be a remarkably stable enzyme. A comparison of the structures of Gs10-OPH and Dr-OPH, as well as two other OPH enzymes from the literature, led to an understanding of the factors contributing to the thermostability of OPH enzymes. We concluded that the number of beta-branched amino acids in the active site, the number of charged residues, the number of aromatic-aromatic and ionic interactions, and the number of negatively charged residues and prolines at the N-termini of helices seem to be the most influential factors in determining the thermostability of OPH enzymes.;Dr-OPH and Gs10-OPH were also characterized for their ability to hydrolyze N-acyl-homoserine lactones, a class of compounds that has been shown to be involved in quorum-sensing and biofilm production. We found that OPH enzymes possess hydrolytic activity toward these compounds and are capable of inhibiting quorum-sensing in vivo. The dual activity of Dr-OPH and Gs10-OPH makes them both viable candidates for a number of practical applications.