Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation.

Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides. Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle. Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria, with substrates including polychlorinated biphenyls or dioxins. Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies. Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon-cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies, we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen-cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis.

NADPH-Driven Organohalide Reduction by a Nonrespiratory Reductive Dehalogenase.

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.