Collins Fraser A, Fisher Karl, Payne Karl A P, Gaytan Mondragon Samantha, Rigby Stephen E J, Leys David
Manchester Institute of Biotechnology , University of Manchester , 131 Princess Street , Manchester M1 7DN , U.K.
Biochemistry. 2018 Jun 26;57(25):3493-3502. doi: 10.1021/acs.biochem.8b00255. Epub 2018 Jun 13.
Reductive dehalogenases are corrinoid and iron-sulfur cluster-dependent enzymes that mostly act as the terminal oxidoreductases in the bacterial organohalide respiration process. This process often leads to detoxification of recalcitrant organohalide pollutants. While low cell yields and oxygen sensitivity hamper the study of many reductive dehalogenases, this is not the case for the nonrespiratory reductive dehalogenase NpRdhA from Nitratireductor pacificus. We here report in vitro and in vivo reconstitution of an NADPH-dependent reducing system for NpRdhA. Surprisingly, NpRdhA mediated organohalide reduction could not be supported using N. pacificus ferredoxin-NAD(P)H oxidoreductase and associated ferredoxins. Instead, we found a nonphysiological system comprised of the Escherichia coli flavodoxin reductase (EcFldr) in combination with spinach ferredoxin (SpFd) was able to support NADPH-dependent organohalide reduction by NpRdhA. Using this system, organohalide reduction can be performed under both anaerobic and aerobic conditions, with 1.1 ± 0.1 and 3.5 ± 0.3 equiv of NADPH consumed per product produced, respectively. No significant enzyme inactivation under aerobic conditions was observed, suggesting a Co(I) species is unlikely to be present under steady state conditions. Furthermore, reduction of the Co(II) resting state was not observed in the absence of substrate. Only the coexpression of EcFldr, SpFd, and NpRdhA in Bacillus megaterium conferred the latter with the ability to reduce brominated NpRdhA substrates in vivo, in agreement with our in vitro observations. Our work provides new insights into biological reductive dehalogenase reduction and establishes a blueprint for the minimal functional organohalide reduction module required for bioremediation in situ.
还原性脱卤酶是一类依赖钴胺素和铁硫簇的酶，在细菌有机卤化物呼吸过程中大多作为末端氧化还原酶发挥作用。这一过程通常会使难降解的有机卤化物污染物解毒。虽然低细胞产量和对氧气的敏感性阻碍了许多还原性脱卤酶的研究，但来自太平洋硝酸盐还原菌的非呼吸性还原性脱卤酶NpRdhA并非如此。我们在此报告了NpRdhA的NADPH依赖性还原系统的体外和体内重构。令人惊讶的是，使用太平洋硝酸盐还原菌铁氧化还原蛋白-NAD(P)H氧化还原酶及相关铁氧化还原蛋白无法支持NpRdhA介导的有机卤化物还原。相反，我们发现由大肠杆菌黄素氧化还原蛋白还原酶（EcFldr）与菠菜铁氧化还原蛋白（SpFd）组成的非生理性系统能够支持NpRdhA进行NADPH依赖性有机卤化物还原。使用该系统，有机卤化物还原可在厌氧和好氧条件下进行，每产生一个产物分别消耗1.1±0.1和3.5±0.3当量的NADPH。在好氧条件下未观察到明显的酶失活，这表明在稳态条件下不太可能存在Co(I)物种。此外，在没有底物的情况下未观察到Co(II)静止状态的还原。只有在巨大芽孢杆菌中共表达EcFldr、SpFd和NpRdhA才能使后者在体内具有还原溴化NpRdhA底物的能力，这与我们的体外观察结果一致。我们的工作为生物还原性脱卤酶还原提供了新的见解，并为原位生物修复所需的最小功能性有机卤化物还原模块建立了蓝图。
