The major objective of this proposal is to elucidate the mechanisms used by bacteria to initiate the degradation of aromatic hydrocarbons. These studies will continue to provide, basic health-related information on oxygen fixation, the biodegradation of environmental pollutants and the development of new procedures for the synthesis of pharmaceutical products such as prostaglandins, inositol phosphates and anti tumor compounds. Pseudomonas putida F1 and Pseudomonas sp. NCIB 9816 both utilize multicomponent enzyme systems to incorporate molecular oxygen into toluene and naphthalene respectively. Toluene dioxygenase consists of three components that participate in the transfer of electrons from NADH to the terminal dioxygenase which oxidizes toluene to enantiomerically-pure (+)- cis-(1S,2R)-dihydroxy-3-methylcyclohexa-3,5-diene. Naphthalene dioxygenase utilizes an analogous system to oxidize naphthalene to (+)-cis-(1R,2S)- dihydroxy-1,2-dihydronaphthalene. The properties of the two enzyme systems are different. The project involves the use of recombinant strains of E. coli, to provide significant quantities of individual proteins for studies on: 1. The protein-protein interactions and mechanisms of electron transport from NADH to the terminal dioxygenases. 2. Electron transfer, substrate binding and subunit interactions in the terminal dioxygenase components of toluene and naphthalene dioxygenases. The procedures involved include chemical coupling, spectrophotometric and electron paramagnetic resonance (EPR) studies in the presence and absence of monoclonal antibodies raised against the individual components of both dioxygenases. 3. The oxidation of indan by the cloned naphthalene dioxygenase system. Products will be isolated by high pressure liquid chromatography and the enantiomeric composition of chiral products will be determined by conventional chemical techniques. 4. The biophysical properties of the iron-sulfur clusters and iron in the molecular events leading to the stereospecific incorporation of molecular oxygen into toluene and naphthalene. These will be collaborative studies with scientists who are acknowledged experts in the following techniques: EPR, magnetic circular dichroism, resonance Ramon, Mossbauer, ENDOR and EXAFS spectroscopies. Additional collaborative studies will focus on the crystallization and use of X-ray diffraction technology to determine the structural properties of the individual components of toluene and naphthalene dioxygenases.
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