Functional characterization of the formate dehydrogenase orthologue and hydrogenases in organohalide respiration by Dehalococcoides mccartyi
Final Report Abstract
Wichtigste Ergebnisse: Demonstration of interphylum complementarity of the Hyp NiFe-hydrogenase maturation machinery between E. coli and Chloroflexi Dehalococcus mccartyi. - Identification of a 250 kDa protein complex comprising a Hup hydrogenase, a formate dehydrogenase-like molybdoenzyme and a reductive dehalogenase with dehalogenating activity. - The demonstration that this protein complex catalyzes H2-dependent reduction of 1,2,3- trichlorobenzene (1,2,3-TCB), even when solubilized from the cytoplasmic membrane. - The H2-oxidizing, 1,2,3-TCB-reducing complex is composed of proteins from minimally three different operons, hup ome and rdh. - A central protein in the H2-oxidizing, 1,2,3-TCB-reducing complex is HupX, which is a predicted electron-transfer protein with four [4Fe-4S] clusters and which is encoded in the hup operon. HupX tightly associates with the molybdoenzyme OmeA (organohalide molybdoenzyme) and is predicted to be key in electron transfer within the complex. - The lack of quinone involvement indicates that the H2-oxidizing, 1,2,3-TCB-reducing complex is likely energy-conserving. - The H2-oxidizing, 1,2,3-TCB-reducing complex has been meanwhile characterized in D. mccartyi strains CBDB1 and DCMB5, suggesting its universality in this genus. The complex is also involved in reduction of 1,2,4-TCB, apparently using a different terminal reductive dehalogenase. - H2-oxidizing activity of the HupXSL complex in the heterologous host E. coli proved to be highly redox-sensitive and was also dependent on interaction specifically with one of the host’s formate dehydrogenase, Fdh-H. Enzyme activity was recovered by deletion of the gene encoding the electron-transfer protein HupX. This suggests that in the absence of Fdh-H, HupX couples electron transfer from H2 via HupLS to other enzyme host complexes, thus inactivating the redox-sensitive HupLS large-small subunit heterodimer.
Publications
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2015. Heterologous complementation studies in Escherichia coli with the Hyp accessory protein machinery from Chloroflexi provide insight into [NiFe]-hydrogenase large subunit recognition by the HypC protein family. Microbiology 161:2204-2219
Hartwig S, Thomas C, Krumova N, Quitzke V, Türkowsky D, Jehmlich N, Adrian L, Sawers RG
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2017. A H2-oxidizing, 1,2,3-trichlorobenzene-reducing multienzyme complex isolated from the obligately organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1. Environ Microbiol Reports 9:618-625
Hartwig S, Dragomirova N, Kublik A, Türkowsky D, von Bergen M, Lechner U, Adrian L, Sawers RG
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2018. Insights into the redox sensitivity of Chloroflexi Hup-hydrogenase derived from studies in Escherichia coli: merits and pitfalls of heterologous NiFe-hydrogenase synthesis. Frontiers Microbiol. 9: 2837
Dragomirova N, Rothe P, Schwoch S, Hartwig S, Pinske C, Sawers RG
