The long term goal of this research is to thoroughly define the precise chemical mechanisms of each enzyme in the meta-fission pathway. Studies of these enzymes have considerable intellectual as well as practical implications. These studies should elucidate the role of mechanism and stereochemistry in the evolution of catabolic pathways as they adapt to specific substrates. In addition, biodegradation of aromatic compounds represent one potential solution to incumbent problem of toxic waste pollution. During this funding period we will focus our efforts on three key enzymes c,f this pathway: 4-oxalocrotonate tautornerase, 4- oxalocrotonate decarboxylase, and vinylpyruvate hydratase. These enzymes represent the essence of what appears to be a common chemical strategy used to degrade compounds by the meta-fission route. These exploratory studies will serve as a foundation for subsequent future detailed mechanistic studies and preliminary studies of the 2-HOD hydrolase branch.
Our specific aims, listed in order of priority, during this funding period will be: (1) Unequivocal identification of the proposed intermediates of the 4-oxalocrotonate branch of the meta-fission pathway. (2) Characterization of the non-enzymatic chemistry of the intermediates of the 4-oxalocrotonate branch. (3) Determination of the stereospecificity of 4-oxalocrotonate tautomerase. (4) Determination of the role of vinylpyruvate hydratase: is it a hydratase or hydrolase? (5) Determination of the stereochemical course of the 4- oxalocrotonate decarboxylase. These basic studies should serve as a foundation for genetic manipulation of microorganisms to meet specific detoxification needs. Furthermore, the enzymes of the meta-fission pathway appear to be the model for bacterial construction of novel degradative pathways. These studies will set the stage for study of the more complex variations of the meta-fission pathway found in other organisms. Finally, these studies will increase knowledge about some basic and intriguing enzymological processes.
