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

Abstract

Soluble butane monooxygenase (sBMO), a three-component di-iron monooxygenase complex expressed by the C2–C9alkane-utilizing bacteriumThauera butanivorans, was kinetically characterized by measuring substrate specificities for C1–C5alkanes 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 bykcat : Kmratios). Relative rates of oxidation for C1–C5alkanes differed minimally, implying that substrate specificity is heavily influenced by differences in substrateKmvalues. The low micromolarKmfor linear C2–C5alkanes and the millimolarKmfor methane demonstrate that sBMO is two to three orders of magnitude more specific for physiologically relevant substrates ofT. butanivorans. Methanol, the product of methane oxidation and also a substrate itself, was found to have similarKmandkcatvalues to those of methane. This inability to kinetically discriminate between the C1alkane and C1alcohol 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–C5do 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–C5alkanes 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 kineticin vitrocharacterization of the closest known homologue of sMMO.