The major objective of this research proposal is to elucidate the mechanisms used by bacteria to add both atoms of molecular oxygen to toluene and naphthalene. These studies will continue to provide basic health related information on: the mechanisms of oxygen fixation; the biodegradation of benzene; toluene, ethylbenzene and xylenes (BTEX), which are common ground water pollutants; and the formation of new chiral intermediates for the synthesis of biologically-active compounds, for example, prostaglandins, inositol phosphates and anti-tumor compounds. Pseudomonas putida F1 and Pseudomonas sp. NCIB 9816 both use multicomponent enzyme systems to oxidize toluene and naphthalene to enantiomerically-pure (+)-cis-(1S,2R)-dihydroxy-3-methylcyclohexa-3,5-diene and (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene respectively. Interest in these and other arene cis-diene diols as chiral synthons for the production of new pharmaceutical products can be seen in the synthesis of 180 arene cis-diene diols in the past five years. The project includes the use of recombinant strains of E. coli to produce high levels of the reductase, ferredoxin (Rieske-type) and iron-sulfur oxygenase (alpha and beta subunits) components of toluene and naphthalene dioxygenases. Although the structural organization of both dioxygenases are similar they show fundamental differences in their substrate specificities. This is particularly evident in reactions where the only differences in the reaction products are their enantiomeric compositions, for example, the formation of (1R)-indanol and (1S)-indanol by toluene and naphthalene dioxygenase respectively. Purified proteins and subunits of the oxygenase components will be used to 1) study the protein-protein interactions involved in electron transfer to the terminal oxygenases; 2) study the assembly of the oxygenase components from their purified alpha and beta subunits; 3) study the role of the beta subunits in hybrid oxygenases; and 4) conduct collaborative studies on the nature of the iron-oxygen complex involved in the enantiospecific formation of cis-arene-diene diols. These collaborative studies will be with scientists who are acknowledged experts in the following biophysical techniques: EPR, 2D-NMR, magnetic circular dichroism, resonance Ramon, Mossbauer, ENDOR and EXAFS spectroscopies, and X-ray crystallography.
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