Manchester Institute of Biotechnology, University of Manchester, Manchester, UK.
Manchester Institute of Biotechnology, University of Manchester, Manchester, UK.
J Biol Chem. 2023 Sep;299(9):105086. doi: 10.1016/j.jbc.2023.105086. Epub 2023 Jul 25.
Reductive dehalogenases are corrinoid and iron-sulfur cluster-containing enzymes that catalyze the reductive removal of a halogen atom. The oxygen-sensitive and membrane-associated nature of the respiratory reductive dehalogenases has hindered their detailed kinetic study. In contrast, the evolutionarily related catabolic reductive dehalogenases are oxygen tolerant, with those that are naturally fused to a reductase domain with similarity to phthalate dioxygenase presenting attractive targets for further study. We present efficient heterologous expression of a self-sufficient catabolic reductive dehalogenase from Jhaorihella thermophila in Escherichia coli. Combining the use of maltose-binding protein as a solubility-enhancing tag with the btuCEDFB cobalamin uptake system affords up to 40% cobalamin occupancy and a full complement of iron-sulfur clusters. The enzyme is able to efficiently perform NADPH-dependent dehalogenation of brominated and iodinated phenolic compounds, including the flame retardant tetrabromobisphenol, under both anaerobic and aerobic conditions. NADPH consumption is tightly coupled to product formation. Surprisingly, corresponding chlorinated compounds only act as competitive inhibitors. Electron paramagnetic resonance spectroscopy reveals loss of the Co(II) signal observed in the resting state of the enzyme under steady-state conditions, suggesting accumulation of Co(I)/(III) species prior to the rate-limiting step. In vivo reductive debromination activity is readily observed, and when the enzyme is expressed in E. coli strain W, supports growth on 3-bromo-4-hydroxyphenylacetic as a sole carbon source. This demonstrates the potential for catabolic reductive dehalogenases for future application in bioremediation.
还原脱卤酶是一类含有钴胺素和铁硫簇的酶，能够催化卤原子的还原去除。呼吸还原脱卤酶对氧气敏感且与膜相关，这阻碍了对其详细动力学研究。相比之下，进化上相关的代谢还原脱卤酶对氧气具有耐受性，那些天然融合有与邻苯二甲酸二加氧酶具有相似性的还原酶结构域的代谢还原脱卤酶是进一步研究的有吸引力的目标。我们在大肠杆菌中高效异源表达了来自 Jhaorihella thermophila 的自足代谢还原脱卤酶。将麦芽糖结合蛋白用作增加可溶性的标签与 btuCEDFB 钴胺素摄取系统结合使用，可使钴胺素占有率达到 40%，并使铁硫簇得到完全补充。该酶能够在厌氧和需氧条件下有效进行溴代和碘代酚类化合物的 NADPH 依赖性脱卤反应，包括阻燃剂四溴双酚 A。NADPH 的消耗与产物形成紧密偶联。令人惊讶的是，相应的氯化化合物仅作为竞争性抑制剂起作用。电子顺磁共振波谱揭示了在稳态条件下观察到的酶的静止状态下 Co(II)信号的丢失，表明在限速步骤之前积累了 Co(I)/(III)物种。在体内观察到还原脱溴活性，当该酶在大肠杆菌 W 菌株中表达时，支持以 3-溴-4-羟基苯乙酸作为唯一碳源进行生长。这证明了代谢还原脱卤酶在生物修复中的应用潜力。
