Gaseous and liquid alkanes can serve as bacterial growth substrates. Alkane monooxygenases initiate the metabolism of alkanes by catalyzing the oxidation of the alkanes to alcohols. While metabolism of methane and liquid alkanes (e.g., octane) is well studied, much less is known about the oxidation of short-chain, gaseous alkanes. The applicant's research has focused on BMOs as representatives of this understudied group of alkane utilizers and on the complete pathway of butane metabolism. Previous results with three butane-utilizing bacteria indicated a surprising level of diversity among the enzymes that harvest butane as well as those involved in the subsequent metabolism of the alcohols. The investigators now propose to focus on just one of these bacteria, Pseudomonas butanovora.
The specific aims of the project are: 1. Characterize the diiron containing BMO, which is similar to soluble MMO, but does not oxidize methane. 2. The applicants discovered two clusters of genes, each coding for a butanol dehydrogenase and a putative butyraldehyde dehydrogenase, which use parallel pathways of electron transport. The applicant now proposes to identify the components of these parallel pathways. 3. Previous studies indicated independent regulation of the production of BMO, butanol dehydrogenases and butyraldehyde dehydrogenases by butane and intermediates in the pathway. The investigators will examine the molecular basis of the regulation of induction of the butane pathway in P. butanovora. The proposed experiments may provide unique insights to the basis of substrate specificity in the broad class of diiron monooxygenases that oxidize nonpolar molecules. Studies of parallel pathways will be relevant to other metabolic pathways where toxic metabolites are necessarily produced as part of the metabolism of a growth supporting substrate.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056128-06
Application #
6735713
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Ikeda, Richard A
Project Start
2003-05-01
Project End
2007-04-30
Budget Start
2004-05-01
Budget End
2005-04-30
Support Year
6
Fiscal Year
2004
Total Cost
$248,757
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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