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Journal of Bacteriology, July 2006, p. 4962-4969, Vol. 188, No. 13
0021-9193/06/$08.00+0     doi:10.1128/JB.00280-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Site-Directed Amino Acid Substitutions in the Hydroxylase {alpha} Subunit of Butane Monooxygenase from Pseudomonas butanovora: Implications for Substrates Knocking at the Gate

Kimberly H. Halsey,1 Luis A. Sayavedra-Soto,2 Peter J. Bottomley,3 and Daniel J. Arp2*

Molecular and Cellular Biology Program,1 Department of Botany and Plant Pathology,2 Department of Microbiology, Oregon State University, Cordley 2082, Corvallis, Oregon 97331-29023

Received 22 February 2006/ Accepted 22 April 2006

Butane monooxygenase (BMO) from Pseudomonas butanovora has high homology to soluble methane monooxygenase (sMMO), and both oxidize a wide range of hydrocarbons; yet previous studies have not demonstrated methane oxidation by BMO. Studies to understand the basis for this difference were initiated by making single-amino-acid substitutions in the hydroxylase {alpha} subunit of butane monooxygenase (BMOH-{alpha}) in P. butanovora. Residues likely to be within hydrophobic cavities, adjacent to the diiron center, and on the surface of BMOH-{alpha} were altered to the corresponding residues from the {alpha} subunit of sMMO. In vivo studies of five site-directed mutants were carried out to initiate mechanistic investigations of BMO. Growth rates of mutant strains G113N and L279F on butane were dramatically slower than the rate seen with the control P. butanovora wild-type strain (Rev WT). The specific activities of BMO in these strains were sevenfold lower than those of Rev WT. Strains G113N and L279F also showed 277- and 5.5-fold increases in the ratio of the rates of 2-butanol production to 1-butanol production compared to Rev WT. Propane oxidation by strain G113N was exclusively subterminal and led to accumulation of acetone, which P. butanovora could not further metabolize. Methane oxidation was measurable for all strains, although accumulation of 23 µM methanol led to complete inhibition of methane oxidation in strain Rev WT. In contrast, methane oxidation by strain G113N was not completely inhibited until the methanol concentration reached 83 µM. The structural significance of the results obtained in this study is discussed using a three-dimensional model of BMOH-{alpha}.

* Corresponding author. Mailing address: Department of Botany and Plant Pathology, Cordley 2082, Oregon State University, Corvallis, OR 97331. Phone: (541) 737-1294. Fax: (541) 737-5310. E-mail: arpd{at}science.oregonstate.edu.

Journal of Bacteriology, July 2006, p. 4962-4969, Vol. 188, No. 13
0021-9193/06/$08.00+0     doi:10.1128/JB.00280-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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