Evolution of New Catalytic Mechanisms for Xenobiotic Hydrolysis

Abstract

Several different evolutionary processes allow enzymes to gain new catalytic activity, or enhance existing properties. Xenobiotic-degrading enzymes are often used to study the molecular basis for the evolution of new catalytic activity, as the substrates are often synthetic and comparatively new to the biosphere, the directionality of the evolutionary process is generally known and the evolved protein can often be traced back to a specific ancestral enzyme. However, the molecular basis behind the evolution of novel catalytic activities in different xenobiotic degrading enzymes is not well understood. In this work several different xenobiotic hydrolysing metalloenzymes were investigated to better understand new enzymatic reactions can evolve: the bacterial phosphotriesterases, triazine chlorohydrolases, and linuron/molinate hydrolases. These three enzyme families were studied using a combination of protein crystallography, enzyme kinetics, metal-ion dependence analysis, thermostability and solubility quantification, bioinformatics analyses, and critical analysis relevant literature. The conclusions that were drawn from these investigations cover fundamental concepts relating to enzymatic catalysis, including (1) the role of metal-ion cofactors in catalysis and the evolution of new function, (2) the desolvation of charged catalytic residues, the energetic cost of this desolvation and how this contributes to catalysis, and (3) the epistatic restrictions imposed on the evolution of new enzyme activity.

Various smaller milestones were accomplished as a result of these investigations. The molecular structures of hydrolases for the herbicides molinate and linuron (molinate hydrolase and phenylurea hydrolase B) have been solved for the first time and, in combination with extensive kinetic and metal analysis, a catalytic mechanism has been proposed. This information will be useful for optimization of both enzymes for the bioremediation of herbicide-contaminated sites. Detailed information outlining the structural and catalytic basis of triazine hydrolase activity and solubility was also obtained. Work towards understanding the structural basis underlying how triazine hydrolase can accumulate in a soluble, active, form and catalyse the hydrolysis of triazine based herbicides will aid the future protein engineering of more stable or active variants. The activity of phosphotriesterase was improved 5000-fold with the most widely used pesticide in the US, malathion. This phosphotriesterase variant has great potential for bioremediation applications and also provides a useful case study into the role that epistasis plays in the engineering of improved enzyme function. Finally, future research ideas are proposed, using results described in this work. Show more