A Spectroscopically Validated Computational Investigation of Viable Reaction Intermediates in the Catalytic Cycle of the Reductive Dehalogenase PceA.

Organisms that produce reductive dehalogenases utilize halogenated aromatic and aliphatic substances as terminal electron acceptors in a process termed organohalide respiration. These organisms can couple the reduction of halogenated substances with the production of ATP. Tetrachloroethylene reductive dehalogenase (PceA) catalyzes the reductive dehalogenation of per- and trichloroethylenes (PCE and TCE, respectively) to primarily cis-dichloroethylene (DCE). The enzymatic conversion of PCE to TCE (and subsequently DCE) could potentially proceed via a mechanism in which the first step involves a single-electron transfer, nucleophilic addition followed by chloride elimination or protonation, or direct attack at the halogen. Difficulties with producing adequate quantities of PceA have greatly hampered direct experimental studies of the reaction mechanism. To overcome these challenges, we have generated computational models of resting and TCE-bound PceA using quantum mechanics/molecular mechanics (QM/MM) calculations and validated these models on the basis of experimental data. Notably, the norpseudo-cob(II)alamin [Co(II)Cbl*] cofactor remains five-coordinate upon binding of the substrate to the enzyme, retaining a loosely bound water on the lower face. Thus, the mechanism for the thermodynamically challenging Co(II) → Co(I)Cbl* reduction used by PceA differs fundamentally from that utilized by adenosyltransferases, which generate four-coordinate Co(II)Cbl species to facilitate access to the Co(I) oxidation state. The same QM/MM computational methodology was then applied to viable reaction intermediates in the catalytic cycle of PceA. The intermediate predicted to possess the lowest energy is that resulting from electron transfer from Co(I)Cbl* to the substrate to yield Co(II)Cbl*, a chloride ion, and a vinylic radical.

Guided cobamide biosynthesis for heterologous production of reductive dehalogenases.

Cobamides (Cbas) are essential cofactors of reductive dehalogenases (RDases) in organohalide-respiring bacteria (OHRB). Changes in the Cba structure can influence RDase function. Here, we report on the cofactor versatility or selectivity of Desulfitobacterium RDases produced either in the native organism or heterologously. The susceptibility of Desulfitobacterium hafniense strain DCB-2 to guided Cba biosynthesis (i.e. incorporation of exogenous Cba lower ligand base precursors) was analysed. Exogenous benzimidazoles, azabenzimidazoles and 4,5-dimethylimidazole were incorporated by the organism into Cbas. When the type of Cba changed, no effect on the turnover rate of the 3-chloro-4-hydroxy-phenylacetate-converting enzyme RdhA6 and the 3,5-dichlorophenol-dehalogenating enzyme RdhA3 was observed. The impact of the amendment of Cba lower ligand precursors on RDase function was also investigated in Shimwellia blattae, the Cba producer used for the heterologous production of Desulfitobacterium RDases. The recombinant tetrachloroethene RDase (PceAY51 ) appeared to be non-selective towards different Cbas. However, the functional production of the 1,2-dichloroethane-dihaloeliminating enzyme (DcaA) of Desulfitobacterium dichloroeliminans was completely prevented in cells producing 5,6-dimethylbenzimidazolyl-Cba, but substantially enhanced in cells that incorporated 5-methoxybenzimidazole into the Cba cofactor. The results of the study indicate the utilization of a range of different Cbas by Desulfitobacterium RDases with selected representatives apparently preferring distinct Cbas.

Selective Utilization of Benzimidazolyl-Norcobamides as Cofactors by the Tetrachloroethene Reductive Dehalogenase of Sulfurospirillum multivorans.

The organohalide-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B12, which serves as cofactor of the tetrachloroethene (PCE) reductive dehalogenase (PceA). As previously reported, a replacement of the adeninyl moiety, the lower base of the cofactor, by exogenously applied 5,6-dimethylbenzimidazole led to inactive PceA. To explore the general effect of benzimidazoles on the PCE metabolism, the susceptibility of the organism for guided biosynthesis of various singly substituted benzimidazolyl-norcobamides was investigated, and their use as cofactor by PceA was analyzed. Exogenously applied 5-methylbenzimidazole (5-MeBza), 5-hydroxybenzimidazole (5-OHBza), and 5-methoxybenzimidazole (5-OMeBza) were found to be efficiently incorporated as lower bases into norcobamides (NCbas). Structural analysis of the NCbas by nuclear magnetic resonance spectroscopy uncovered a regioselectivity in the utilization of these precursors for NCba biosynthesis. When 5-MeBza was added, a mixture of 5-MeBza-norcobamide and 6-MeBza-norcobamide was formed, and the PceA enzyme activity was affected. In the presence of 5-OHBza, almost exclusively 6-OHBza-norcobamide was produced, while in the presence of 5-OMeBza, predominantly 5-OMeBza-norcobamide was detected. Both NCbas were incorporated into PceA, and no negative effect on the PceA activity was observed. In crystal structures of PceA, both NCbas were bound in the base-off mode with the 6-OHBza and 5-OMeBza lower bases accommodated by the same solvent-exposed hydrophilic pocket that harbors the adenine as the lower base of authentic norpseudo-B12 In this study, a selective production of different norcobamide isomers containing singly substituted benzimidazoles as lower bases is shown, and unique structural insights into their utilization as cofactors by a cobamide-containing enzyme are provided.IMPORTANCE Guided biosynthesis of norcobamides containing singly substituted benzimidazoles as lower bases by the organohalide-respiring epsilonproteobacterium Sulfurospirillum multivorans is reported. An unprecedented specificity in the formation of norcobamide isomers containing hydroxylated or methoxylated benzimidazoles was observed that implicated a strict regioselectivity of the norcobamide biosynthesis in the organism. In contrast to 5,6-dimethylbenzimidazolyl-norcobamide, the incorporation of singly substituted benzimidazolyl-norcobamides as a cofactor into the tetrachloroethene reductive dehalogenase was not impaired. The enzyme was found to be functional with different isomers and not limited to the use of adeninyl-norcobamide. Structural analysis of the enzyme equipped with either adeninyl- or benzimidazolyl-norcobamide cofactors visualized for the first time structurally different cobamides bound in base-off conformation to the cofactor-binding site of a cobamide-containing enzyme.

Subtle changes in the active site architecture untangled overlapping substrate ranges and mechanistic differences of two reductive dehalogenases.

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.

Cobamide-mediated enzymatic reductive dehalogenation via long-range electron transfer.

The capacity of metal-containing porphyrinoids to mediate reductive dehalogenation is implemented in cobamide-containing reductive dehalogenases (RDases), which serve as terminal reductases in organohalide-respiring microbes. RDases allow for the exploitation of halogenated compounds as electron acceptors. Their reaction mechanism is under debate. Here we report on substrate-enzyme interactions in a tetrachloroethene RDase (PceA) that also converts aryl halides. The shape of PceA's highly apolar active site directs binding of bromophenols at some distance from the cobalt and with the hydroxyl substituent towards the metal. A close cobalt-substrate interaction is not observed by electron paramagnetic resonance spectroscopy. Nonetheless, a halogen substituent para to the hydroxyl group is reductively eliminated and the path of the leaving halide is traced in the structure. Based on these findings, an enzymatic mechanism relying on a long-range electron transfer is concluded, which is without parallel in vitamin B12-dependent biochemistry and represents an effective mode of RDase catalysis.

Structural basis for organohalide respiration.

Organohalide-respiring microorganisms can use a variety of persistent pollutants, including trichloroethene (TCE), as terminal electron acceptors. The final two-electron transfer step in organohalide respiration is catalyzed by reductive dehalogenases. Here we report the x-ray crystal structure of PceA, an archetypal dehalogenase from Sulfurospirillum multivorans, as well as structures of PceA in complex with TCE and product analogs. The active site harbors a deeply buried norpseudo-B12 cofactor within a nitroreductase fold, also found in a mammalian B12 chaperone. The structures of PceA reveal how a cobalamin supports a reductive haloelimination exploiting a conserved B12-binding scaffold capped by a highly variable substrate-capturing region.

Exogenous 5,6-dimethylbenzimidazole caused production of a non-functional tetrachloroethene reductive dehalogenase in Sulfurospirillum multivorans.

Corrinoid-dependent reductive dehalogenation is mediated by phylogenetically diverse anaerobic bacteria that either synthesize corrinoids de novo or are dependent on corrinoid salvaging from the environment. The tetrachloroethene (PCE) reductive dehalogenase (PceA) of the Gram-negative Epsilonproteobacterium Sulfurospirillum multivorans harbours a norpseudo-B12 as corrinoid cofactor. Norpseudo-B12 differs from coenzyme B12 in the nucleotide loop structure. Adenine instead of 5,6-dimethylbenzimidazole (DMB) serves as lower ligand base of the central cobalt ion, and the nucleotide loop of norpseudo-B12 lacks a methyl group at position 176. In this study, S. multivorans was grown anaerobically with PCE in the presence of DMB. At a DMB concentration of 25 µM, the adenine moiety in the nucleotide loop of norpseudo-B12 was quantitatively replaced by DMB. The formation of the DMB-containing nor-B12 severely affected PCE-dependent growth and the PceA activity. In DMB-treated cells processing of the cytoplasmic PceA precursor was impeded, a result pointing to retarded cofactor incorporation. PceA enriched from cells cultivated with DMB contained nor-B12 . Nor-B12 purified from cells grown in the presence of DMB mediated the abiotic reductive dehalogenation of trichloroacetate to dichloroacetate at a 25-fold lower rate in comparison with norpseudo-B12 , a fact underpinning the relevance of norpseudo-B12 as efficient catalyst for reductive dehalogenation in general.