Structure-Function Analysis of Nitrobenzene 1, 2-Dioxygenase

Nitroaromatic compounds are toxic, synthetic chemicals commonly used in the production of pesticides, dyes, plastics, and explosives. Only a few microorganisms have been able to adapt and evolve new metabolic pathways that utilize these man-made compounds as their sole carbon, nitrogen, and energy sources.  One such microbe is Comamonas sp. JS765, a strain capable of completely mineralizing the toxic nitroaromatic compound nitrobenzene to carbon dioxide and nitrite. Nitrobenzene 1, 2-dioxygenase (NBDO) is the initial enzyme in the nitrobenzene degradation pathway. Little information is known about how Rieske dioxygenases interact in order to catalyze these unique reactions. NBDO is a model system for structure function analysis of Rieske dioxygenases because all components are soluble and the structure for the catalytic enzyme has been determined. Site-directed mutagenesis was used to make amino acid substitutions in the oxygenase and ferredoxin components at sites near the Rieske clusters predicted to interfere with electron transfer. Using whole cell biotransformation assays and kinetic analysis with purified mutant and wild-type components, specific amino acid substitutions in the junction of adjacent oxygenase subunits and amino acid substitutions on the acidic region of the ferredoxin resulted in severely reduced enzymatic activity. These studies identify residues on the surface of the oxygenase and ferredoxin that are involved in protein-protein interactions and electron transfer. Chimeric dioxygenases and dioxygenase active site mutants were also analyzed in an effort to characterize NBDO’s unique specificity with nitroaromatic substrates.