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

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https://ir.library.oregonstate.edu/concern/articles/0r967441j

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  • 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 sMMO
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  • Cooley, R. B., Dubbels B. L., Sayavedra-Soto, L. A., Bottomley, P. J., Arp, D. J. (2009). Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly ‘Pseudomonas butanovora’. Microbiology, 155, 286-2096
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  • 155
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  • 6
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  • We thank Michael Schimerlik for his helpful comments and suggestions during the course of this study, and Christine Lastovica and Lisa Roberton for culturing and harvesting of cells. We are grateful for the research support from the National Institutes of Health, grant number 5RO1 GM56128-06
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