Reductive dehalogenases (RDases) of organohalide-respiring bacteria are cobamide-containing iron-sulfur proteins that catalyze different reductive dehalogenation reactions. Here, we report a functional analysis of two recombinant RDases, the tetrachloroethene (PCE) reductive dehalogenase (PceA) of Desulfitobacterium hafniense Y51 and the 1,2-dichloroethane (1,2-DCA) reductive dehalogenase (DcaA) of Desulfitobacterium dichloroeliminans DCA1. Both enzymes share 88% protein sequence identity, but appeared to have divergent mechanisms. In this study, the heterologously produced DcaA converted 1,2-DCA and 1,1,2-trichloroethane (1,1,2-TCA) via dihaloelimination to ethene and vinyl chloride, respectively. In addition, halogen substitution at PCE, trichloroethene (TCE) and tribromoethene (TBE) was observed, but only at low rates. In contrast, recombinant PceA exclusively converted halogenated ethenes and showed no dihaloelimination activity. In silico structural analysis of both RDases revealed similar architectures of their active site cavities. Exchange of the highly conserved Tyr298 to Phe led to a complete loss of the PCE, TCE and TBE conversion by both RDases, strengthening the assumption that Tyr298 functions as proton donor in the course of halogen substitution. The exchange did not affect the ability of DcaA to convert 1,2-DCA and 1,1,2-TCA. This result makes the involvement of a proton transfer in the dihaloelimination reaction unlikely and allows for a clear differentiation between two mechanisms working in DcaA and PceA. The analysis of the role of the active site structure for RDase function was extended to the mutations W118F that had a negative effect on DcaA function and W432F or T294V that caused alterations in the substrate specificity of the enzyme.
Enzymes: Tetrachloroethene reductive dehalogenase (EC 1.21.99.5), DCA -RDase.
Keywords: cobamide cofactor; hydrogenolysis; proton transfer; reductive dehalogenation; vicinal reduction.
© 2017 Federation of European Biochemical Societies.