Involvement of a Mate Chaperone (TorD) in the Maturation Pathway of Molybdoenzyme TorA

Ilbert, Marianne; Méjean, Vincent; Giudici‐Orticoni, Marie‐Thérèse; Samama, Jean‐Pierre; Iobbi‐Nivol, Chantal

doi:10.1074/jbc.m302730200

Cited by 87 publications

(85 citation statements)

References 38 publications

(26 reference statements)

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“…The strains were grown in Luria Broth medium, and, when necessary, ampicillin (50 g⅐ml Ϫ1 ) was added to maintain plasmid selection. Plasmid pTorA allowing His 6 -tagged apoTorA production was previously described (10). To construct plasmid pBD, which allows the synthesis of His 6 -tagged TorD, the same cloning strategy as previously described for pTorD (11) was used except that the torD coding sequence was cloned into the EcoRI-HindIII cloning sites of pBAD24 (20).…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant TorD protein was purified from the soluble extract of strain LCB515/pBD grown aerobically at 37°C in the presence of arabinose (0.2%). Both purifications were performed by HiTrap Chelating HP chromatography (Amersham Biosciences) as described previously (10). After purification, apoTorA and TorD were dialyzed against phosphate buffer pH 7 (20 mM).…”

Section: Methodsmentioning

confidence: 99%

“…TorD interacts with apoTorA and allows it to become competent to receive the MoCo. Using in vitro assays containing purified apoTorA and a source of MoCo, we showed that TorD alone was sufficient to allow an optimal maturation of the enzyme (10).…”

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TorD, an Essential Chaperone for TorA Molybdoenzyme Maturation at High Temperature

Genest¹,

Ilbert

Méjean

et al. 2005

Journal of Biological Chemistry

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TorD has been recognized as an accessory protein that improves maturation of TorA, the molybdenum cofactor-containing trimethylamine oxide reductase of Escherichia coli. In this study, we show that at 42°C and in the absence of TorD TorA is poorly matured and almost completely degraded. Strikingly, TorD restores TorA maturation to the same level whatever the growth temperature. In vitro experiments in which apoTorA was incubated with or without TorD at various temperatures confirm that TorD is an essential chaperone for TorA at elevated temperatures preventing apoTorA misfolding before cofactor insertion.The understanding of the mechanism of metalloprotein maturation is a challenge, because the insertion of a metal ion into a protein often requires complex pathways involving auxiliary proteins (for a review see Ref. 1). Maturation of molybdenum cofactor-containing proteins is a good example of this complexity, because it can involve cofactor escort proteins as well as specific chaperones (2, 3). Depending on the molybdoenzymes, the molybdenum cofactor (MoCo) 1 can be a molybdopterin (MPT-Mo), a molybdopterin guanine dinucleotide (MGD), or a bis(MGD)Mo (4). This latest form was found in the large Me 2 SO reductase family of bacterial molybdoenzymes (5).In Escherichia coli, TorA, a member of the Me 2 SO reductase family, is the main respiratory enzyme responsible for the TMAO reduction when the cells are grown anaerobically in the presence of TMAO (6). TorA is located in the periplasm and receives electrons from TorC, a pentahemic c-type cytochrome (7). TorA and TorC are encoded by the torCAD operon, which is induced in the presence of TMAO (6). TorA crosses the inner membrane by the TAT machinery in a folded state, meaning that the molybdenum cofactor is inserted into the apoprotein in the cytoplasm before translocation (8). TorA has been used as a model protein for the study of both cofactor insertion and translocation. Recently, we established that TorA maturation is improved two to three times by the presence of the cytoplasmic TorD protein that proved to be the specific chaperone of TorA (9 -11). TorD interacts with apoTorA and allows it to become competent to receive the MoCo. Using in vitro assays containing purified apoTorA and a source of MoCo, we showed that TorD alone was sufficient to allow an optimal maturation of the enzyme (10).A second role was also attributed to TorD during the translocation process of TorA by the TAT translocon to prevent export of immature TorA (12,13). During this proofreading mechanism, TorD binds the signal peptide of TorA and exhibits a quality control activity to ensure that only matured TorA is addressed to the TAT translocase. These two functions of TorD appeared independent, because it was clearly demonstrated that the action of TorD during TorA maturation is independent of the presence of the signal peptide (11). Thus, TorD also binds to a second region of TorA in addition to the signal peptide (9, 13).TorD is a member of a large family of homologous proteins associat...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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TorD, an Essential Chaperone for TorA Molybdoenzyme Maturation at High Temperature

Genest¹,

Ilbert

Méjean

et al. 2005

Journal of Biological Chemistry

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“…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).…”

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“…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).…”

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confidence: 99%

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

Hatzixanthis

Clarke

Oubrie

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

116

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The twin-arginine transport (Tat) system is a protein-targeting pathway of prokaryotes and chloroplasts. Most Escherichia coli Tat substrates are complex metalloenzymes that must be correctly folded and assembled before transport, and a preexport chaperone-mediated ''proofreading'' process is therefore in operation. The paradigm proofreading chaperone is TorD, which coordinates maturation and export of the key respiratory enzyme trimethylamine N-oxide reductase (TorA). It is demonstrated here that purified TorD binds tightly and with exquisite specificity to the TorA twin-arginine signal peptide in vitro. It is also reported that the TorD family constitutes a hitherto unexpected class of nucleotide-binding proteins. The affinity of TorD for GTP is enhanced by initial signal peptide binding, and it is proposed that GTP governs signal peptide binding-and-release cycles during Tat proofreading.ligand binding ͉ molecular chaperone

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Metallocenter Biosynthesis & Assembly

Mulrooney

Hausinger

2005

Encyclopedia of Inorganic Chemistry

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Metalloproteins have been characterized intensively for decades, yet only recently has progress been made in characterizing the mechanisms of biological metallocenter biosynthesis and assembly. Here, we describe how cellular synthesis of metal sites makes use of several recurring themes, often with multiple themes combined into a single pathway. In the simplest situation, binding of a metal ion to a biological ligand occurs by reversible thermodynamic control. However, the prevalence of metallocenters deeply buried within macromolecules, the exceedingly low concentrations of free metal ions within cells, and the sophisticated structures of many metal‐containing active sites and cofactors provide evidence that alternative and more complex approaches also must exist. In some cases, metallochaperones are used to deliver the metal of interest to an apoprotein. In other cases the target protein undergoes posttranslational modification at the metal‐binding site, either prior to or after the metal is bound. Many metallocenters contain additional components that are added along with the metal ion or used to form a preassembled metal‐containing cofactor. Scaffolding proteins may be used to provide a framework for construction of the cofactor. Electron transfer reactions involving the metal, an added compound, or the protein ligand can participate in activation. Finally, molecular chaperones that bind to and alter the conformation of the target apoprotein may be utilized. In many cases, the function of the molecular chaperone is coupled to nucleotide triphosphate hydrolysis.

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Involvement of a Mate Chaperone (TorD) in the Maturation Pathway of Molybdoenzyme TorA

Cited by 87 publications

References 38 publications

TorD, an Essential Chaperone for TorA Molybdoenzyme Maturation at High Temperature

TorD, an Essential Chaperone for TorA Molybdoenzyme Maturation at High Temperature

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

Metallocenter Biosynthesis & Assembly

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