TORD, A CYTOPLASMIC CHAPERONE THAT INTERACTS WITH THE UNFOLDED TRIMETHYLAMINE N-OXIDE REDUCTASE ENZYME (TORA) IN ESCHERICHIA-COLI

Reduction of trimethylamine N-oxide (TMAO) in Escherichia coli involve s the terminal molybdoreductase TorA, located in the periplasm, and th e membrane anchored c type cytochrome TorC. In this study, the role of the Toro protein, encoded by the third gene of torCAD operon, is inve stigated. Construction of a mutant, in which the torD gene is interrup ted, showed that the absence of Toro protein leads to a two times decr ease of the final amount of TorA enzyme. However, specific activity an d biochemical properties of TorA enzyme were similar to those of the e nzyme produced in the wild type. Excess of Toro protein restores the n ormal level of TorA enzyme, and also, leads to the appearance of a new cytoplasmic form of TorA on SDS-polyacrylamide gel electrophoresis us ing gentle conditions. This probably indicates a new folding state of the cytoplasmic TorA protein when Toro is overexpressed. BIAcore techn iques demonstrated direct specific interaction between the TorA and To ro proteins. This interaction was enhanced when TorA was previously un folded by heating, Finally, as TorA is a molybdoenzyme, we demonstrate d that Toro can interact with TorA before the molybdenum cofactor has been inserted. As Toro homologue encoding genes are found in various T MAO reductase loci, we propose that Toro is a chaperone protein specif ic for the TorA enzyme. It belongs to a family of TorD-like chaperones present in several bacteria, and, probably, involved in TMAO reductas e folding.