TorD, A Cytoplasmic Chaperone That Interacts with the Unfolded Trimethylamine N-Oxide Reductase Enzyme (TorA) in Escherichia coli

Abstract

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.In Escherichia coli, the main anaerobic respiratory pathway responsible for reduction of trimethylamine N-oxyde (TMAO) 1 to TMA (trimethylamine) requires the products of the torCAD operon which, in anaerobiosis, is induced in the presence of TMAO or related compounds via the two-component regulatory system, TorS/TorR (1, 2).TMAO reductase (TorA), the terminal reductase of this system, is a 97-kDa molybdoprotein encoded by the torA gene (3, 4). Based on sequence homologies, TorA belongs to the Me 2 SO/ TMAO reductase family which includes the M 2 SO/TMAO reductases from Rhodobacter capsulatus and Rhodobacter sphaeroides (5) and TMAO reductase of Shewanella species.2 These enzymes share several properties: (i) they are all molybdoenzymes located in the periplasm of the bacterium and (ii) in each case, it has been proposed that a membrane anchored pentahemic c type cytochrome feeds electrons to the terminal enzyme (6, 7). In E. coli, this cytochrome, TorC, is encoded by torC, the first gene of the torCAD operon (4,8). While the role of the TorC and TorA proteins is well documented, the role of the third protein, TorD, predicted in the torCAD operon is not (4). However, the presence of TorD homologue proteins, not only in R. capsulatus and R. sphaeroides Tor systems (6, 7), but also in Shewanella species 2 is intriguing and suggests a similar role for this protein in these systems. As TorD contains two small hydrophobic segments, at its amino and carboxyl ends, respectively, it was proposed to be a membranous b type cytochrome involved in the electron transfer path...

Signal peptide–chaperone interactions on the twin-arginine protein transport pathway

“…TorD was initially not expected to bind the twin-arginine signal peptide directly, however genetic experiments suggested E. coli TorD could indeed recognize the TorA signal peptide during the preexport proofreading process (10). Thus, TorD performs dual roles by assisting cofactor insertion into the TorA enzyme (10,(12)(13)(14) and facilitating Tat proofreading by recognition of the TorA twin-arginine signal peptide (10).…”

“…TorA is the archetypal model Tat substrate, its twin-arginine signal peptide being the most heavily studied and exploited in the field. Loading of the cofactor is an essential prerequisite to TorA transport (11) and the TorD protein was originally identified as a cytoplasmic accessory protein required for efficient cofactor loading into the TorA apoenzyme (12)(13)(14). The 3D structure of a TorD homolog from Shewanella massilia showed that this family of proteins comprise two separate ␣-helical domains connected by a short hinge region (15).…”

Assembly pathway of a bacterial complex iron sulfur molybdoenzyme

“…It has been suggested that GTP binding by REMP proteins themselves may play a role in the REMP/substrate maturation process (33). Accordingly, the domain-swapper dimer of TorD, showed an increase in GTP affinity following TorA ligand binding (27,117,118), and it was suggested to in fact be binding to the mature molybdopterin-guanine dinucleotide (MGD) form of Moco as a step in the cofactor insertion event (49). Recent investigation has illustrated strong cooperativity in the DmsD binding to both GTP and the RR-leader, suggesting that GTP binding at one site on DmsD alters affinity at additional binding sites (other protomers of a multimeric state of DmsD).…”

“…Twinarginine leader peptide binding is a canonical feature of REMPs, and as such, binding of DmsD to the DmsA RRleader peptide is an absolute prerequisite for DMSO reductase biogenesis (26,27). It is clear that DmsD binds the DmsA RR-leader at 1:1 stoichiometry and that the entire hydrophobic region is required for binding (46) although specificity is more difficult to define (47).…”

“…Of special interest to CISM maturation are system specific chaperones that help mediate the complete final folding and assembly (23,24). Such chaperones were termed redox enzyme maturation proteins (REMPs) and are essential for the proper assembly of CISMs albeit absent in the final assembled holoenzyme (25)(26)(27)(28).…”