Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly 'Pseudomonas butanovora'
Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly 'Pseudomonas butanovora'
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DOI：
10.1099/mic.0.028175-0
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发表时间：
2009-06-01
期刊：
影响因子：
2.8
通讯作者：
Arp, Daniel J.
中科院分区：
文献类型：
--
作者：
Cooley, Richard B.;Dubbel, Bradley L.;Arp, Daniel J.
通过测定底物对C-1-C-5烷烃的特异性和产物抑制谱，对利用C-2-C-9烷烃的细菌Thauera butanivorans表达的三组分二铁单加氧酶（sBMO）进行了动力学表征。sBMO与可溶性甲烷单加氧酶（sMMO）具有高度的序列同源性，并且具有相似的底物范围，包括气态和液态烷烃、芳烃、烯烃和卤化异种生物。结果表明，丁烷是首选底物（由k（cat）定义为：k -m比）。C-1-C-5烷烃的相对氧化速率差异很小，这意味着底物特异性受到底物K-m值差异的严重影响。线性C-2-C-5烷烃的低微摩尔K-m和甲烷的毫摩尔K-m表明，sBMO对丁烷酸T生理相关底物的特异性要高两到三个数量级。甲醇是甲烷氧化的产物，也是一种底物，其k -m和k（cat）值与甲烷相似。在sBMO将甲烷两步氧化为甲醛的过程中，甲醇的稳态浓度不能从动力学上区分C-1烷烃和C-1醇。与甲醇不同，链长为C-2-C-5的醇不能与其各自的烷烃底物有效竞争。产物抑制实验结果表明，活性位点的几何形状对长度为4 - 5个碳的线性分子进行了优化，并受到调节蛋白组分B（丁烷单加氧酶调节组分；BMOB）的影响。数据表明，sBMO对烷烃的氧化高度专一于C-3-C-5烷烃的转化和它们各自的醇产物的释放。此外，sBMO在>= C-2链烷烃生长过程中特别有效地防止甲烷氧化，尽管其序列与sMMO高度同源。据我们所知，这些结果代表了sMMO最接近的已知同源物的第一个体外动力学表征。
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) at: 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.