Site-directed amino acid substitutions in the hydroxylase alpha subunit of butane monooxygenase from Pseudomonas butanovora: Implications for substrates knocking at the gate.
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 microM 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 microM. The structural significance of the results obtained in this study is discussed using a three-dimensional model of BMOH-alpha.
Halsey
Kimberly H
KH
Molecular and Cellular Biology Program, Oregon State University, Corvallis, OR 97331, USA.
Sayavedra-Soto
Luis A
LA
Bottomley
Peter J
PJ
Arp
Daniel J
DJ
D019943:Amino Acid Substitution; D002073:Butanes; D000440:Butanols / Q000378:metabolism; D003470:Culture Media; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008697:Methane / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000201:enzymology* / Q000254:growth & development
D002073:Butanes; D000440:Butanols; D003470:Culture Media; C046888:butane; D006899:Mixed Function Oxygenases; D006867:Hydrolases; D008697:Methane