Enzyme Catalysis of Toluene Degradation and Unusual DNA Photoproduct Repair

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Enzynnes utilize organic radicals to catalyze a variety of important nnetabolic reactions. The overall goal of this research is to delineate the mechanistic details of radical generation and control by these enzymes. This research in the ROO phase will focus on a DNA repair enzyme named spore photoproduct lyase (SPL). SPL utilizes S-adenosylmethionine (SAM) coupled by a unique [4Fe-4S] cluster to generate the reactive organic radicals to repair the unique T-T crosslink 5-thyminyl-5, 6-dihydrothymine (commonly called spore photoproduct, SP) formed upon UV irradiation. SPL exists in the spores of bacteria such as B. subtilis and B. anthracis. It adopts a "direct reverse" strategy to repair SP, meaning that the UV damage is quickly reversed with neither removal nor replacement of the damaged thymine bases. It thus represents a unique DNA repair pathway in Nature. In addition, the efficient DNA repair catalyzed by SPL makes UV irradiation no longer lethal to the spore-forming bacteria. To understand the SPL mediated DNA repair reaction, chemical, kinetic, spectroscopic, and mutagenic methods will be employed. The objectives include: investigating the SPL activity using substrates with a wide range of DNA secondary structures, probing the reaction mechanism by SP analogues (mechanism-based enzyme inhibitors), and examining the kinetic isotope effects and reaction reversibility. In addition, the redox potential of the [4Fe-4S] cluster will be determined and the influence of SAIV! and key amino acids to the redox potential will be investigated. Understanding the enzyme mechanism will help us identify potential SPL inhibitors. As SPL is the key enzyme to repair the UV damage in endospore-forming bacteria, inhibiting its activity in vivo will prevent the bacteria from fixing these damages at the germination phase. In combination with the SPL inhibitor, UV irradiation will regain its power as a cheap and convenient tool for sterilization purpose.