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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056128-04
Application #
6181210
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Ikeda, Richard A
Project Start
1997-09-01
Project End
2002-08-31
Budget Start
2000-09-01
Budget End
2002-08-31
Support Year
4
Fiscal Year
2000
Total Cost
$167,509
Indirect Cost
Name
Oregon State University
Department
Other Basic Sciences
Type
Schools of Arts and Sciences
DUNS #
053599908
City
Corvallis
State
OR
Country
United States
Zip Code
97339
Cooley, Richard B; Dubbels, Bradley L; Sayavedra-Soto, Luis A et al. (2009) Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly 'Pseudomonas butanovora'. Microbiology 155:2086-96
Dubbels, Bradley L; Sayavedra-Soto, Luis A; Bottomley, Peter J et al. (2009) Thauera butanivorans sp. nov., a C2-C9 alkane-oxidizing bacterium previously referred to as 'Pseudomonas butanovora'. Int J Syst Evol Microbiol 59:1576-8
Kurth, Elizabeth G; Doughty, David M; Bottomley, Peter J et al. (2008) Involvement of BmoR and BmoG in n-alkane metabolism in 'Pseudomonas butanovora'. Microbiology 154:139-47
Doughty, D M; Kurth, E G; Sayavedra-Soto, L A et al. (2008) Evidence for involvement of copper ions and redox state in regulation of butane monooxygenase in Pseudomonas butanovora. J Bacteriol 190:2933-8
Halsey, Kimberly H; Doughty, David M; Sayavedra-Soto, Luis A et al. (2007) Evidence for modified mechanisms of chloroethene oxidation in Pseudomonas butanovora mutants containing single amino acid substitutions in the hydroxylase alpha-subunit of butane monooxygenase. J Bacteriol 189:5068-74
Doughty, D M; Halsey, K H; Vieville, C J et al. (2007) Propionate inactivation of butane monooxygenase activity in 'Pseudomonas butanovora': biochemical and physiological implications. Microbiology 153:3722-9
Dubbels, Bradley L; Sayavedra-Soto, Luis A; Arp, Daniel J (2007) Butane monooxygenase of 'Pseudomonas butanovora': purification and biochemical characterization of a terminal-alkane hydroxylating diiron monooxygenase. Microbiology 153:1808-16
Halsey, Kimberly H; Sayavedra-Soto, Luis A; Bottomley, Peter J et al. (2006) Site-directed amino acid substitutions in the hydroxylase alpha subunit of butane monooxygenase from Pseudomonas butanovora: Implications for substrates knocking at the gate. J Bacteriol 188:4962-9
Doughty, D M; Sayavedra-Soto, L A; Arp, D J et al. (2006) Product repression of alkane monooxygenase expression in Pseudomonas butanovora. J Bacteriol 188:2586-92
Doughty, D M; Sayavedra-Soto, L A; Arp, D J et al. (2005) Effects of dichloroethene isomers on the induction and activity of butane monooxygenase in the alkane-oxidizing bacterium ""Pseudomonas butanovora"". Appl Environ Microbiol 71:6054-9

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