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 C 2 -C 9 alkane-utilizing bacterium Thauera butanivorans, was kinetically characterized by measuring substrate specificities for C 1 -C 5 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 C 1 -C 5 alkanes differed minimally, implying that substrate specificity is heavily influenced by differences in substrate K m values. The low micromolar K m for linear C 2 -C 5 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 C 1 alkane and C 1 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 C 2 -C 5 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 C 3 -C 5 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.

Identification of Novel Methane-, Ethane-, and Propane-Oxidizing Bacteria at Marine Hydrocarbon Seeps by Stable Isotope Probing

“…Most propane-oxidizing strains can oxidize butane, as well as a range of longer chain n-alkanes, but differ in the ability to oxidize ethane. These strains show little, if any, ability to oxidize methane, and none have been shown to grow with methane as the sole carbon source (13,27,38,45,65). As with methane metabolism, the first step in aerobic ethane and propane metabolism is the oxidation of the alkane to an alcohol (70).…”

“…SIP allows the identification of organisms actively consuming a 13 C-labeled substrate of interest, based on the incorporation of 13 C into biomass, including DNA and lipids (67). We collected sediment from the Coal Oil Point seep field and incubated sediment-seawater slurries with 13 C methane, ethane, or propane.…”

“…SIP allows the identification of organisms actively consuming a 13 C-labeled substrate of interest, based on the incorporation of 13 C into biomass, including DNA and lipids (67). We collected sediment from the Coal Oil Point seep field and incubated sediment-seawater slurries with 13 C methane, ethane, or propane. Samples were removed at three time points, chosen to ensure sufficient 13 C incorporation into DNA while minimizing the spread of 13 C through the community as a result of cross-feeding on metabolic byproducts.…”

Trace-gas metabolic versatility of the facultative methanotroph Methylocella silvestris

“…Short-chain alkane utilizers have been thought to be incapable of methane metabolism, and most are members of the class Actinobacteria 12 . An exception is Thauera butanivorans, which is a betaproteobacterium that grows on butane using a monooxygenase that is two to three orders of magnitude more specific for butane than for methane 17 , despite its sequence similarity (up to 65%) to bona fide methane monooxygenases.…”

Structural and Kinetic Studies of Novel Cytochrome P450 Small-Alkane Hydroxylases

“…36 min -1 on butane. 8 Octane total turnover number (TTN) was found to be even less sensitive to fdrA6 and fdxA6 concentration. For reactions with at least a 5:1 ratio of fdxA6:A6, 500-750 TTN was obtained for all values of fdrA6 tested.…”