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
kcat : Km ratios). Relative rates of oxidation for C1–C5 alkanes differed minimally, implying that
substrate specificity is heavily influenced by differences in substrate Km values. The low
micromolar Km for linear C2–C5 alkanes and the millimolar Km 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 Km and kcat 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 ≥C2, 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 sMM
