Purification and Characterization of the Isoprene Monooxygenase from Rhodococcus sp. Strain AD45.
Purification and Characterization of the Isoprene Monooxygenase from Rhodococcus sp. Strain AD45.
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DOI：
10.1128/aem.00029-22
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发表时间：
2022-04-12
影响因子：
4.4
通讯作者：
Murrell, J. Colin
中科院分区：
文献类型：
--
作者：
Sims, Leanne P.;Lockwood, Colin W. J.;Crombie, Andrew T.;Bradley, Justin M.;Le Brun, Nick E.;Murrell, J. Colin
异戊二烯（2-甲基-1，3-丁二烯）是一种大量释放到大气中的气候活性气体，其数量与甲烷相当。已经分离出几种可以以异戊二烯作为唯一碳源和能源生长的细菌，但是关于这些细菌在生物化学水平上降解异戊二烯的信息很少。异戊二烯的利用取决于多步途径，第一步是异戊二烯氧化为环氧-异戊二烯。这是由四组分可溶性二铁单加氧酶，异戊二烯单加氧酶（IsoMO）催化的。IsoMO是一种六蛋白复合物，包含含有二铁活性位点的加氧酶（IsoABE）、Rieske型铁氧还蛋白（IsoC）、NADH还原酶（IsoF）和偶联/效应蛋白（IsoD），与可溶性甲烷单加氧酶和烯烃/芳香族单加氧酶同源。在这里，我们描述了从红球菌属AD45的IsoMO组分的纯化和体外异戊二烯氧化活性的重建。从同源宿主Rhodococcus sp.AD45-ID中表达并纯化了一些IsoMO组分，Rhodococcus sp.AD45-ID是一种缺乏含有异戊二烯代谢基因簇的大质粒的Rhodococcus sp.AD45菌株。其他的在大肠杆菌中表达并纯化为融合蛋白。我们描述了这些纯化组分的表征，并证明了它们的活性与红球菌属AD45细胞裂解物相结合时。IsoMO体外活性的证明为进一步表征这种新型可溶性二铁中心单加氧酶的生物化学和生物物理特性提供了平台，促进了对异戊二烯细菌降解的酶基础的新认识。异戊二烯是一种非常丰富的气候活性气体，也是一些细菌的碳源。编码异戊二烯单加氧酶（IsoMO）的基因的分析表明，这种酶是一种可溶性二铁中心单加氧酶，与可溶性甲烷单加氧酶、烯烃单加氧酶和甲苯单加氧酶属于同一个加氧酶家族。我们报告了红球菌属的IsoMO的初步生物化学表征，这是第一个来自任何细菌的IsoMO，描述了其四种组分的体外活性的具有挑战性的纯化和重建。本研究为进一步深入研究全球异戊二烯循环中的关键酶IsoMO的作用机制奠定了基础。
Isoprene (2-methyl-1,3-butadiene) is a climate-active gas released to the atmosphere in large quantities, comparable to methane in magnitude. Several bacteria have been isolated which can grow on isoprene as a sole carbon and energy source, but very little information is available about the degradation of isoprene by these bacteria at the biochemical level. Isoprene utilization is dependent on a multistep pathway, with the first step being the oxidation of isoprene to epoxy-isoprene. This is catalyzed by a four-component soluble diiron monooxygenase, isoprene monooxygenase (IsoMO). IsoMO is a six-protein complex comprising an oxygenase (IsoABE), containing the di-iron active site, a Rieske-type ferredoxin (IsoC), a NADH reductase (IsoF), and a coupling/effector protein (IsoD), homologous to the soluble methane monooxygenase and alkene/aromatic monooxygenases. Here, we describe the purification of the IsoMO components from Rhodococcus sp. AD45 and reconstitution of isoprene-oxidation activity in vitro. Some IsoMO components were expressed and purified from the homologous host Rhodococcus sp. AD45-ID, a Rhodococcus sp. AD45 strain lacking the megaplasmid which contains the isoprene metabolic gene cluster. Others were expressed in Escherichia coli and purified as fusion proteins. We describe the characterization of these purified components and demonstrate their activity when combined with Rhodococcus sp. AD45 cell lysate. Demonstration of IsoMO activity in vitro provides a platform for further biochemical and biophysical characterization of this novel soluble diiron center monooxygenase, facilitating new insights into the enzymatic basis for the bacterial degradation of isoprene. IMPORTANCE Isoprene is a highly abundant climate-active gas and a carbon source for some bacteria. Analyses of the genes encoding isoprene monooxygenase (IsoMO) indicate this enzyme is a soluble diiron center monooxygenase in the same family of oxygenases as soluble methane monooxygenase, alkene monooxygenase, and toluene monooxygenase. We report the initial biochemical characterization of IsoMO from Rhodococcus, the first from any bacterium, describing the challenging purification and reconstitution of in vitro activity of its four components. This study lays the foundation for future detailed mechanistic studies of IsoMO, a key enzyme in the global isoprene cycle.
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DOI：
10.1073/pnas.1812668115
发表时间：
2018-12-18
影响因子：
11.1
作者：
Crombie AT;Larke-Mejia NL;Emery H;Dawson R;Pratscher J;Murphy GP;McGenity TJ;Murrell JC
通讯作者：
Murrell JC
DOI：
10.1007/s10482-008-9227-1
发表时间：
2008-06-01
影响因子：
2.6
作者：
Holmes, Andrew J.;Coleman, Nicholas V.
通讯作者：
Coleman, Nicholas V.
影响因子：
2.9
作者：
Anderson, LJ;Richardson, DJ;Butt, JN
通讯作者：
Butt, JN
影响因子：
5.1
作者：
El Khawand, Myriam;Crombie, Andrew T.;Murrell, J. Colin
通讯作者：
Murrell, J. Colin
影响因子：
4.5
作者：
Dawson, Robin A.;Larke-Mejia, Nasmille L.;Murrell, J. Colin
通讯作者：
Murrell, J. Colin