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Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly ‘Pseudomonas butanovora’

¹ Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA
² Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA
³ Department of Microbiology, Oregon State University, Corvallis, OR 97331, USA

Soluble butane monooxygenase (sBMO), a three-component di-iron monooxygenase complex expressed by the C₂–C₉ alkane-utilizing bacterium Thauera butanivorans, was kinetically characterized by measuring substrate specificities for C₁–C₅ 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 C₁–C₅ alkanes differed minimally, implying that substrate specificity is heavily influenced by differences in substrate K_m values. The low micromolar K_m for linear C₂–C₅ 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 C₁ alkane and C₁ 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 C₂–C₅ 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 C₃–C₅ alkanes and the release of their respective alcohol products. Additionally, sBMO is particularly efficient at preventing methane oxidation during growth on linear alkanes 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.

Correspondence
Daniel J. Arp
arpd{at}science.oregonstate.edu