| Organophosphate compounds belong to a lethal class of chemicals that have been employed as agricultural pesticides and chemical warfare agents. As they are a major threat to human health, there is an urgent need for enzymes that will efficiently degrade and detoxify particular organophosphate compounds. A naturally occurring enzyme, organophosphorus hydrolase (OPH), has been shown to hydrolyze paraoxon, an organophosphorus pesticide, at a nearly diffusion-limited rate. OPH displays weak activity against other pesticides, such as methyl parathion and coumaphos, as well as nerve agents, such as soman, sarin, and Russian VX. Thus, OPH is an attractive target for site-directed mutagenesis aimed at achieving sufficient levels of activity for bioremediation and detoxification.; In this dissertation, a universal gene engineering system, terminator overhang PCR cloning (TOPC), is used to construct a customized OPH expression plasmid and generate a library of OPH variants. With the addition of automated protein expression and assay, TOPC represents a high-throughput, iterative process of design, production, and testing. Knowledge gained during each iteration is used to modify design parameters for the next iteration. In the initial iteration of the OPH project, described here, 86 single amino acid substitutions were engineered into the OPH gene. The resulting variants were tested for their ability to cleave the preferred OPH substrate, paraoxon, and two inefficiently cleaved substrates, methyl parathion and coumaphos. A single iteration of the process was sufficient to produce three variants that cleave methyl parathion with markedly increased efficiency. In contrast, a variant that efficiently cleaves coumaphos was not identified. One variant, H254R, cleaved methyl parathion with an initial velocity 12-fold greater than the natural enzyme. Kinetic analysis of this variant revealed that substrate specificity (Kcat/K m) was increased 33-fold, mainly attributable to a 24-fold increase in Kcat. One round of protein engineering was sufficient to increase substrate specificity against methyl parathion to within 13% of the substrate specificity demonstrated by the wild type enzyme against paraoxon. Additional rounds of large-scale protein mutagenesis using the TOPC strategy should allow for further productive rational engineering of OPH. |
