The major objective is to elucidate the mechanism of action of the enzyme systems from two different strains of Pseudomonas that are responsible for the oxidation of toluene and naphthalene to (+)-1(S),2(R)-dihydroxy-3-methyl-3,5-cyclohexadiene and (+)-1(R),2(S)-dihydroxy-1,2-dihydronaphthalene respectively. These unique dioxygenases have no counterparts in eucaryotic organisms and cis-hydroxylation seems to be an important mechanism used by bacteria to degrade aromatic hydrocarbons without forming reactive electrophilic intermediates such as arene oxides. Toluene dioxygenase consists of three protein components that participate in the transfer of electrons to the terminal dioxygenase which forms cis-toluene dihydrodiol. The mechanism of action of this enzyme system will be investigated by determining the nature of the protein interactions between the three components by chemical coupling and spectrophotometric techniques. The nature of electron transfer, substrate binding and oxygen activation in ISPTOL will be determined by spectrophotometric and electron paramagnetic resonance techniques. The structural genes for all three components of toluene dioxygenase will be identified by hybridization with synthetic oligonucleotide probes and cloned into appropriate expression vectors. The nucleotide sequence of each structural gene will be determined by the dideoxy method. High expression vectors containing the cloned genes will be used to overproduce large amounts of each protein in strains of E. coli. These proteins will be used in reconstitution experiments to assemble an active toluene dioxygenase complex with identical properties to the system obtained from P. putida F1. Chemical studies with indan and indene as substrates will be used to investigate the monooxygenase activity of toluene dioxygenase. Naphthalene dioxygenase also consists of three protein components. The properties of these proteins are different to those described for toluene dioxygenase. The mechanism of action of this enzyme will be investigated using similar techniques to those mentioned above for toluene dioxygenase. Particular attention will be paid to the mechanisms of oxidation of indan and indene by naphthalene dioxygenase since preliminary results indicate that desaturase, monoxygenase and dioxygenase reactions are catalyzed by the purified enzyme system.
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