Functional Heterologous Production of Reductive Dehalogenases from Desulfitobacterium hafniense Strains
Functional Heterologous Production of Reductive Dehalogenases from Desulfitobacterium hafniense Strains
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DOI：
10.1128/aem.00881-14
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发表时间：
2014-07-01
影响因子：
4.4
通讯作者：
Schubert, Torsten
中科院分区：
文献类型：
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作者：
Mac Nelly, Anita;Kai, Marco;Schubert, Torsten
有机卤化物的厌氧脱卤作用由系统发育多样化细菌中产生的还原脱卤酶 (RdhA) 催化。这些酶在活性位点含有一个钴酰胺辅因子和两个铁硫簇。在这项研究中，革兰氏阳性脱硫杆菌 hafniense 菌株 Y51 的四氯乙烯 (PCE) 还原脱卤酶 (PceA) 在非脱氯、产生钴酰胺的细菌 Shimwellia blattae (ATCC 33430)（一种革兰氏阴性伽马变形菌）中以催化活性形式产生。当共同产生其专用的 PceT 分子伴侣并且将 5,6-二甲基苯并咪唑和羟钴胺添加到 S. blattae 培养物中时，重组催化活性 PceA 酶的形成显着增强。该实验扩展到 D. hafniense DCB-2，这是一种含有多个 rdhA 基因的还原脱卤细菌。为了阐明该生物体 rdhA3 基因产物的底物谱，测试了重组酶对产 RdhA3 的 S. blattae 菌株粗提物中不同二氯酚 (DCP) 的转化。重组 RdhA3 使 3,5-DCP、2,3-DCP 和 2,4-DCP 还原脱氯，但 2,6-DCP 和 3,4-DCP 不发生还原脱氯。此外，该酶以较低的速率将四氯乙烯脱氯成三氯乙烯。
The anaerobic dehalogenation of organohalides is catalyzed by the reductive dehalogenase (RdhA) enzymes produced in phylogenetically diverse bacteria. These enzymes contain a cobamide cofactor at the active site and two iron-sulfur clusters. In this study, the tetrachloroethene (PCE) reductive dehalogenase (PceA) of the Gram-positive Desulfitobacterium hafniense strain Y51 was produced in a catalytically active form in the nondechlorinating, cobamide-producing bacterium Shimwellia blattae (ATCC 33430), a Gram-negative gammaproteobacterium. The formation of recombinant catalytically active PceA enzyme was significantly enhanced when its dedicated PceT chaperone was coproduced and when 5,6-dimethylbenzimidazole and hydroxocobalamin were added to the S. blattae cultures. The experiments were extended to D. hafniense DCB-2, a reductively dehalogenating bacterium harboring multiple rdhA genes. To elucidate the substrate spectrum of the rdhA3 gene product of this organism, the recombinant enzyme was tested for the conversion of different dichlorophenols (DCP) in crude extracts of an RdhA3-producing S. blattae strain. 3,5-DCP, 2,3-DCP, and 2,4-DCP, but not 2,6-DCP and 3,4-DCP, were reductively dechlorinated by the recombinant RdhA3. In addition, this enzyme dechlorinated PCE to trichloroethene at low rates.