{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Parthasarathy A"],"funding":["Eni","National Institute of General Medical Sciences","NIGMS NIH HHS","National Science Foundation"],"pagination":["3525-32"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC4516053"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["137(10)"],"pubmed_abstract":["Reductive dehalogenases play a critical role in the microbial detoxification of aquifers contaminated with chloroethenes and chlorethanes by catalyzing the reductive elimination of a halogen. We report here the first heterologous production of vinyl chloride reductase VcrA from Dehalococcoides mccartyi strain VS. Heterologously expressed VcrA was reconstituted to its active form by addition of hydroxocobalamin/adenosylcobalamin, Fe(3+), and sulfide in the presence of mercaptoethanol. The kinetic properties of reconstituted VcrA catalyzing vinyl chloride reduction with Ti(III)-citrate as reductant and methyl viologen as mediator were similar to those obtained previously for VcrA as isolated from D. mccartyi strain VS. VcrA was also found to catalyze a novel reaction, the environmentally important dihaloelimination of 1,2-dichloroethane to ethene. Electron paramagnetic resonance (EPR) spectroscopic studies with reconstituted VcrA in the presence of mercaptoethanol revealed the presence of Cob(II)alamin. Addition of Ti(III)-citrate resulted in the appearance of a new signal characteristic of a reduced [4Fe-4S] cluster and the disappearance of the Cob(II)alamin signal. UV-vis absorption spectroscopy of Ti(III)citrate-treated samples revealed the formation of two new absorption maxima characteristic of Cob(I)alamin. No evidence for the presence of a [3Fe-4S] cluster was found. We postulate that during the reaction cycle of VcrA, a reduced [4Fe-4S] cluster reduces Co(II) to Co(I) of the enzyme-bound cobalamin. Vinyl chloride reduction to ethene would be initiated when Cob(I)alamin transfers an electron to the substrate, generating a vinyl radical as a potential reaction intermediate."],"journal":["Journal of the American Chemical Society"],"pubmed_title":["Biochemical and EPR-spectroscopic investigation into heterologously expressed vinyl chloride reductive dehalogenase (VcrA) from Dehalococcoides mccartyi strain VS."],"pmcid":["PMC4516053"],"funding_grant_id":["R01 GM104543","GM-104543"],"pubmed_authors":["Britt RD","Parthasarathy A","Stich TA","Lesnefsky A","Lohner ST","Spormann AM"],"additional_accession":[]},"is_claimable":false,"name":"Biochemical and EPR-spectroscopic investigation into heterologously expressed vinyl chloride reductive dehalogenase (VcrA) from Dehalococcoides mccartyi strain VS.","description":"Reductive dehalogenases play a critical role in the microbial detoxification of aquifers contaminated with chloroethenes and chlorethanes by catalyzing the reductive elimination of a halogen. We report here the first heterologous production of vinyl chloride reductase VcrA from Dehalococcoides mccartyi strain VS. Heterologously expressed VcrA was reconstituted to its active form by addition of hydroxocobalamin/adenosylcobalamin, Fe(3+), and sulfide in the presence of mercaptoethanol. The kinetic properties of reconstituted VcrA catalyzing vinyl chloride reduction with Ti(III)-citrate as reductant and methyl viologen as mediator were similar to those obtained previously for VcrA as isolated from D. mccartyi strain VS. VcrA was also found to catalyze a novel reaction, the environmentally important dihaloelimination of 1,2-dichloroethane to ethene. Electron paramagnetic resonance (EPR) spectroscopic studies with reconstituted VcrA in the presence of mercaptoethanol revealed the presence of Cob(II)alamin. Addition of Ti(III)-citrate resulted in the appearance of a new signal characteristic of a reduced [4Fe-4S] cluster and the disappearance of the Cob(II)alamin signal. UV-vis absorption spectroscopy of Ti(III)citrate-treated samples revealed the formation of two new absorption maxima characteristic of Cob(I)alamin. No evidence for the presence of a [3Fe-4S] cluster was found. We postulate that during the reaction cycle of VcrA, a reduced [4Fe-4S] cluster reduces Co(II) to Co(I) of the enzyme-bound cobalamin. Vinyl chloride reduction to ethene would be initiated when Cob(I)alamin transfers an electron to the substrate, generating a vinyl radical as a potential reaction intermediate.","dates":{"release":"2015-01-01T00:00:00Z","publication":"2015 Mar","modification":"2025-04-05T09:02:16.412Z","creation":"2019-03-27T01:55:52Z"},"accession":"S-EPMC4516053","cross_references":{"pubmed":["25686300"],"doi":["10.1021/ja511653d"]}}