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

Oregon State University

ABSTRACT

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 of alkanes C1-C5 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 alkanes C1-C5 differed minimally; implying substrate specificity is heavily influenced by differences in substrate Km's. The low micromolar Km for linear alkanes C2-C5 and millimolar Km for methane demonstrate sBMO is 2-3 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 as 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 no compete effectively with their respective alkane substrates. Results from product inhibition experiments suggest the geometry of the active site is optimized for linear molecules 4-5 carbons in length and is influenced by the regulatory protein component B (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 in preventing methane oxidation during growth on linear alkanes C2 despite its high sequence homology with sMMO. These results represent the first kinetic in vitro characterization of the closest known homolog of sMMO.

¹ E-mail: arpd{at}science.oregonstate.edu