A molecular chaperone dedicated to folding and translocation of reductive dehalogenases

Reductive dehalogenases (rdhA, RDases) are key enzymes involved in anaerobic organohalide respiration (OHR), during which bacteria are able to use chlorinated compounds as terminal electron acceptors. RDases are redox enzymes containing FeS clusters and a corrinoid as cofactors, and are translocated across the cytoplasmic membrane by the Twin-arginine translocation (Tat) system. In members of Dehalobacter and Desulfitobacterium spp., the product of an accompanying gene, generally named rdhT, was recently proposed to play a role as molecular chaperone in the folding of the reductive dehalogenase (1,2). Recently, this finding was applied to heterologously produce active RDases (3). However, the mechanism by which the molecular chaperone acts on the maturation of RDases is not yet solved. In this study, we investigate the diversity of RdhT chaperones in Dehalobacter restrictus and their interaction with the Tat signal peptide of their cognate redox component, as well as their specificity or cross-reactivity towards alternative signal peptides. To this respect, both in vivo and in vitro experimental approaches are conducted and will be presented. References (1) Morita et al., 2009. Appl. Microbiol. Biotechnol. 83:775. (2) Maillard et al., 2011. Microbiol. 157:2410. (3) Mac Nelly et al., 2014. Appl. Environ. Microbiol. 80:4313

FOLDING OF COMPLEX REDOX PROTEINS BY DEDICATED MOLECULAR CHAPERONES

Chlorinated compounds (so-called organohalides) are widespread soil and groundwater pollutants. Only few bacteria have the ability to degrade these compounds via organohalide respiration (OHR). Reductive dehalogenases (RDases) are complex redox enzymes involved in the reduction of organohalides, and contribute to the biodegradation of these pollutants. RDases need to be folded and loaded with iron-sulfur centers and a corrinoid cofactor prior to their transport across the cytoplasmic membrane via the Twin-arginine translocation (Tat) pathway. A new family of Tat molecular chaperones, named RdhT, was recently shown to participate in the maturation of RDases (1-2), and successfully applied for heterologous production of these complex redox enzymes (3). The present study focuses on the interaction of RdhT molecular chaperones with their cognate RDases. PceT (the paradigmatic member of the RdhT family) interacts as a dimer with the Tat signal peptide of PceA, its cognate RDase, as shown by isothermal titration calorimetry. When recombinant pceT and pceAHis genes are heterologously expressed in E. coli, both proteins co-purify on Ni-NTA chromatography which indicates that PceT binds to PceA also in vivo. Although recombinant PceA is not functional in E. coli, it is produced in a soluble form when pceT is co-expressed and represent the basis for reconstitution experiments. Currently, in vivo strategies are developed in E. coli to allow a rapid screening of interacting RdhT chaperones with the Tat signal peptides of RDases. This will further help evaluating the cross-reactivity of RdhT chaperones towards Tat signal peptides, and help identifying specific amino acids of the chaperones that are involved in the interaction event. References: 1. Morita Y, Futagami T, Goto M, Furukawa K. Appl Microbiol Biotechnol 2009;83:775-81. 2. Maillard J, Genevaux P, Holliger C. Microbiology 2011;157:2410-21. 3. Mac Nelly A, Kai M, Svatoš A, Diekert G, Schubert T. Appl Environ Microbiol 2014;80:4313-22