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 involves the terminal molybdoreductase TorA, located in the periplasm, and the membrane anchored c type cytochrome TorC. In this study, the role of the TorD protein, encoded by the third gene of torCAD operon, is investigated. Construction of a mutant, in which the torD gene is interrupted, showed that the absence of TorD protein leads to a two times decrease of the final amount of TorA enzyme. However, specific activity and biochemical properties of TorA enzyme were similar to those of the enzyme produced in the wild type. Excess of TorD protein restores the normal level of TorA enzyme, and also, leads to the appearance of a new cytoplasmic form of TorA on SDS-polyacrylamide gel electrophoresis using gentle conditions. This probably indicates a new folding state of the cytoplasmic TorA protein when TorD is overexpressed. BIAcore techniques demonstrated direct specific interaction between the TorA and TorD proteins. This interaction was enhanced when TorA was previously unfolded by heating. Finally, as TorA is a molybdoenzyme, we demonstrated that TorD can interact with TorA before the molybdenum cofactor has been inserted. As TorD homologue encoding genes are found in various TMAO reductase loci, we propose that TorD is a chaperone protein specific for the TorA enzyme. It belongs to a family of TorD-like chaperones present in several bacteria, and, probably, involved in TMAO reductase folding.
Pommier
J
J
Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et Microbiologie, Centre National de la Recherche Scientifique, 31, chemin Joseph Aiguier, BP71, 13402 Marseille Cedex 20, France.
Méjean
V
V
Giordano
G
G
Iobbi-Nivol
C
C
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006418:Heme / Q000378:metabolism; D008744:Methylamines / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D017510:Protein Folding; D016415:Sequence Alignment; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008744:Methylamines; D018832:Molecular Chaperones; C088083:TorD protein, E coli; D006418:Heme; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase; C005855:trimethyloxamine