This renewal application seeks continued support for our investigations of the chemistry and enzymology of the enzymes of the beta-ketoadipate pathway. During the current funding period we have made considerable progress on nearly all of our initially stated goals. In addition, our mechanistic and stereochemical findings have revealed some unusual interrelationships among a number of enzymes in this pathway and have suggested that some of these enzymes may have evolved to accommodate preferred mechanistic pathways and not on the basis of structural features of common substrate classes. During the next funding period, we have put together a multidisciplinary program which includes a microbiologist (L.N. Ornston) and two crystallographers (A. Goldman and D.A. Ollis). We intend to interface our chemical and enzymological studies with the genetic and structural studies of these research groups. We are also moving our work aggressively into the area of the metabolism of halogenated aromatic compounds by a variety of enzymes in the beta-ketoadipate pathway. Specifically, we will be investigating the following: I. Chemical synthesis of disubstituted muconate derivatives. II. Chemical lactonization of substituted muconates. III. Studies on 3-carboxymuconate cyloisomerase (CMLE) from P. putida. IV. Studies on muconolactone delta-isomerase and 4-carboxymuconolactone decarboxylase. V. Studies on muconate cyloisomerases from P. sp. B13 and T. cutaneum. In light of the increasing importance of these metabolic pathways to environmental chemistry, we believe that the new chemistry and enzymology uncovered will be of great value to microbiologists, chemists and biochemists concerned with the detoxification of polyhalogenated aromatics.
