9630081 Ensign The objective of this project is to investigate the metabolic pathway(s) of acetone metabolism by microorganisms and the biochemical properties of the enzyme(s) which initiate acetone degradation. Acetone is a toxic molecule which is a product of microbial fermentation and mammalian starvation. Acetone is also a proposed intermediate metabolite in the aerobic oxidation of propane and isopropanol by some bacteria; it is an important industrial commodity synthesized in large quantities by the chemical industry. Studies of aerobic and anaerobic acetone-degrading bacteria have provided evidence for the existence of two distinct routes for acetone degradation: an initial hydroxylation reaction producing acetol (hydroxyacetone), or an initial carboxylation reaction producing acetoacetate. However, no acetone-degrading enzyme has been purified or studied in vitro, and consequently, little is known about the properties of these enzymes or the mechanisms they employ for acetone conversions. Our studies are focused primarily on Xanthobacter strain Py2, an aerobic bacterium capable of utilizing acetone and other ketones as carbon and energy sources. Preliminary studies performed in our laboratory have demonstrated that acetone metabolism in Xanthobacter Py2 proceeds by a carboxylation reaction producing acetoacetate as the product. We have recently succeeded in reconstituting this carboxylation reaction in vitro. This represents the first demonstration of in vitro acetone carboxylation in a bacterial system. The specific objectives for this project period are to (1) purify to homogeneity and biochemically characterize the acetone carboxylase of Xanthobacter Py2; (2) kinetically and mechanistically characterize acetone carboxylase; (3) initiate the isolation, cloning, and characterization of gene(s) encoding acetone carboxylase; and (4) perform comparative studies of acetone metabolism in the propane-oxidizing bacterium Mycobacteriurn vaccae strain JOB5. Although the pathways and b iochemistry of microbial acetone formation have been studied in detail, there is a lack of information on the pathways and biochemistry of acetone degradation. The research to be conducted is intended to fill this gap in our understanding of biological acetone cycling and provide important fundamental insights into biochemical mechanisms of microbial acetone catabolism. %%% The objective of this project is to investigate metabolic pathways of acetone degradation by microorganisms and the biochemical properties of the enzymes which are required for acetone degradation. Acetone is an important industrial solvent, and is one of many organic compounds of environmental concern due to its toxicity. In addition to its production in industry, acetone is also formed by some microorganisms as a product of fermentation. Acetone is also formed by mammals, including humans, during starvation. In diabetics, acetone is formed as a product of incomplete carbohydrate breakdown, and accumulates to high levels in the blood and urine. The metabolism (breakdown) of acetone has been observed in both mammals and microorganisms, although the details and significance of these processes are not fully understood. This project is an investigation of the metabolism of acetone by bacteria that are able to grow at the expense of acetone as a source of carbon and energy. Our goals are to determine the sequence of reactions involved in acetone degradation and to determine how acetone is degraded at the molecular level. These studies will provide important new information on biological strategies for the degradation of toxic organic compounds. ***
