9723312 Spormann This research will investigate novel enzymes catalyzing the first step in anaerobic degradation of aromatic hydrocarbons. Recent work showed that the inital step in degradation of the methyl-benzenes toluene and m-xylene is the addition of the methyl carbon to the double bond of fumarate. Benzylsuccinate synthase, which catalyzes the addition of toluene to fumarate, will be studied as the prototype for this reaction. This enzymatic addition represents a novel biochemical reaction to activate aromatic hydrocarbons. Further, the activity may also define a novel class of enzymes that catalyze the formation of carbon-carbon bonds by a heretofore unknown reaction mechanism. Benzylsuccinate synthases may be of primary importance in anaerobic mineralization pathways in a range of different bacterial species. Research in this proposals directed towards gaining a better understanding of these unusual enzymatic reactions. This project will include purification of benzylsuccinate synthase and investigation of the enzyme's reaction mechanism. In addition to characterizing a novel class of enzymes, the results of this research will have important implications for our understanding of anaerobic pathways used by petroleum hydrocarbon-mineralizing bacteria. Because of the absence of molecular oxygen under typical conditions in fuel-contaminated aquifers, anaerobic bacteria capable of degrading petroleum hydrocarbons, such as toluene and the xylene isomers, are of key importance. Investigations of the key enzymes of these pathways may lead to the development of molecular tools that may assist in monitoring file abundance and activity of relevant microbes in situ. This research will investigate novel enzymes catalyzing the first step in anaerobic degradation of aromatic hydrocarbons. These compounds are significant environmental pollutants. Until recently it was thought that degradation of such compounds required oxygen. Recent work showed that the inital step in degradation of toluene and m-xylene, novel. In addition to characterizing this novel class of enzymes, the results of this research will have important implications for our understanding of anaerobic pathways used by petroleum hydrocarbon-mineralizing bacteria. Because of the absence of molecular oxygen under typical conditions in fuel-contaminated aquifers, anaerobic bacteria capable of degrading petroleum hydrocarbons, such as toluene and the xylene isomers, are of key importance. Investigations of the key enzymes of these pathways may lead to the development of molecular tools that may assist in monitoring the abundance and activity of relevant microbes in situ, and aid in bioremediation.
