Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly 'Pseudomonas butanovora'.
Soluble butane monooxygenase (sBMO), a three-component di-iron monooxygenase complex expressed by the C(2)-C(9) alkane-utilizing bacterium Thauera butanivorans, was kinetically characterized by measuring substrate specificities for C(1)-C(5) alkanes and product inhibition profiles. sBMO has high sequence homology with soluble methane monooxygenase (sMMO) and shares a similar substrate range, including gaseous and liquid alkanes, aromatics, alkenes and halogenated xenobiotics. Results indicated that butane was the preferred substrate (defined by k(cat) : K(m) ratios). Relative rates of oxidation for C(1)-C(5) alkanes differed minimally, implying that substrate specificity is heavily influenced by differences in substrate K(m) values. The low micromolar K(m) for linear C(2)-C(5) alkanes and the millimolar K(m) for methane demonstrate that sBMO is two to three orders of magnitude more specific for physiologically relevant substrates of T. butanivorans. Methanol, the product of methane oxidation and also a substrate itself, was found to have similar K(m) and k(cat) values to those of methane. This inability to kinetically discriminate between the C(1) alkane and C(1) alcohol is observed as a steady-state concentration of methanol during the two-step oxidation of methane to formaldehyde by sBMO. Unlike methanol, alcohols with chain length C(2)-C(5) do not compete effectively with their respective alkane substrates. Results from product inhibition experiments suggest that the geometry of the active site is optimized for linear molecules four to five carbons in length and is influenced by the regulatory protein component B (butane monooxygenase regulatory component; BMOB). The data suggest that alkane oxidation by sBMO is highly specialized for the turnover of C(3)-C(5) alkanes and the release of their respective alcohol products. Additionally, sBMO is particularly efficient at preventing methane oxidation during growth on linear alkanes > or =C(2,) despite its high sequence homology with sMMO. These results represent, to the best of our knowledge, the first kinetic in vitro characterization of the closest known homologue of sMMO.
Cooley
Richard B
RB
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA.
Dubbels
Bradley L
BL
Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA.
Sayavedra-Soto
Luis A
LA
Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA.
Bottomley
Peter J
PJ
Department of Microbiology, Oregon State University, Corvallis, OR 97331, USA.
Arp
Daniel J
DJ
Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA.
D002073:Butanes / Q000378:metabolism*; D020134:Catalytic Domain; D007700:Kinetics; D008697:Methane / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D020628:Thauera / Q000201:enzymology*
D002073:Butanes; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C021555:methane monooxygenase; D008697:Methane