Alkane monooxygenases initiate the oxidation of alkanes as growth-supporting substrates for bacteria. Monooxygenases which oxidize C2-C5 alkanes are not well studied, in contrast to C1 alkane particulate and soluble methane monooxygenases and C6-C12 alkane hydroxylase. The proposed research will focus on butane monooxygenases as representatives of C2-C5 alkane utilizers. An unusual level of diversity with regard to butane monooxygenases in three bacteria (Pseudomonas butanovora, isolate CF8, and Mycobacterium vaccae) that grow on butane will be characterized. Knowledge of monooxygenases in general will be enhanced and the results could lead to new model systems for the study of monooxygenases in health and disease. A working hypothesis is that the butane monooxygenases of P. butanovora contains a P450 prosthetic group, the butane monooxygenase of isolate CF8 contains copper, and the butane monooxygenase of M. vaccae contains a diiron center.
The specific aims of the proposal are: 1. Characterization of diversity in butane oxidation at the physiological level in intact cells. Alkane substrate ranges, products, kinetic constants, inactivator profiles, and protein patterns associated with alkane oxidation will be examined. 2. Purification and characterization of butane monooxygenase. Butane monooxygenase and associated reductase components will be purified from the targeted bacteria. Physical and catalytic properties of the purified proteins will be examined. 3. Characterization of the genes associated with butane oxidation. The genes coding for each butane monooxygenase will be isolated and sequenced for comparison to known monooxygenases, Additional genes (e.g. for reductase components) will also be isolated. The organization of the genes will be examined. Gene expression experiments will be performed. Our long-range plans are to continue to develop the knowledge of butane oxidation. Topics of interest outside the scope of this proposal include characterization of additional enzymes in the butane oxidation pathway, examination of other butane oxidizing bacteria, examination of the control of regulation of genes encoding the ability to utilize butane, and detailed physical (spectroscopic and structural) and kinetic characterizations of the purified enzymes.
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