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

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 mis-folding before cofactor insertion. 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 mis-folding 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. 1Kuchar J. Haussinger R.P. Chem. Rev. 2004; 104: 509-525Crossref PubMed Scopus (99) Google Scholar). 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 (2Palmer T. Santini C.L. Iobbi-Nivol C. Eaves D.J. Boxer D. Giordano G. Mol. Microbiol. 1996; 20: 875-884Crossref PubMed Scopus (146) Google Scholar, 3Vergnes A. Gouffi-Belhabich K. Blasco F. Giordano G. Magalon A. J. Biol. Chem. 2004; 279: 41398-41403Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Depending on the molybdoenzymes, the molybdenum cofactor (MoCo) 1The abbreviations used are: MoCo, molybdenum cofactor; TMAO, trimethylamine oxide; bis(MGD)Mo, bis(molybdopterin guanine dinucleotide)molybdenum; MPT-Mo, molybdopterin-molybdenum; ATPγS, adenosine 5′-O-(thiotriphosphate). can be a molybdopterin (MPT-Mo), a molybdopterin guanine dinucleotide (MGD), or a bis(MGD)Mo (4Mendel R.R. Schwarz G. Met. Ions Biol. Syst. 2002; 39: 317-368PubMed Google Scholar). This latest form was found in the large Me2SO reductase family of bacterial molybdoenzymes (5Hille R. Holzenburg A. Scrutton N.S. Subcellular Biochemistry: Enzyme-Catalyzed Electron and Radical Transfer. Kluwer Academic/Plenum Publishers, New York2000: 445-485Google Scholar). In Escherichia coli, TorA, a member of the Me2SO reductase family, is the main respiratory enzyme responsible for the TMAO reduction when the cells are grown anaerobically in the presence of TMAO (6Méjean V. Iobbi-Nivol C. Lepelletier M. Giordano G. Chippaux M. Pascal M.C. Mol. Microbiol. 1994; 11: 1169-1179Crossref PubMed Scopus (221) Google Scholar). TorA is located in the periplasm and receives electrons from TorC, a pentahemic c-type cytochrome (7Gon S. Giudici-Orticoni M.T. Méjean V. Iobbi-Nivol C. J. Biol. Chem. 2001; 276: 11545-11551Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). TorA and TorC are encoded by the torCAD operon, which is induced in the presence of TMAO (6Méjean V. Iobbi-Nivol C. Lepelletier M. Giordano G. Chippaux M. Pascal M.C. Mol. Microbiol. 1994; 11: 1169-1179Crossref PubMed Scopus (221) Google Scholar). 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 (8Santini C.L. Ize B. Chanal A. Muller M. Giordano G. Wu L.F. EMBO J. 1998; 17: 101-112Crossref PubMed Scopus (290) Google Scholar). 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 (9Pommier J. Méjean V. Giordano G. Iobbi-Nivol C. J. Biol. Chem. 1998; 273: 16615-16620Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 10Ilbert M. Méjean V. Giudici-Orticoni M.T. Samama J.P. Iobbi-Nivol C. J. Biol. Chem. 2003; 278: 28787-28792Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 11Ilbert M. Méjean V. Iobbi-Nivol C. Microbiology. 2004; 150: 935-943Crossref PubMed Scopus (60) Google Scholar). 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 (10Ilbert M. Méjean V. Giudici-Orticoni M.T. Samama J.P. Iobbi-Nivol C. J. Biol. Chem. 2003; 278: 28787-28792Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). A second role was also attributed to TorD during the translocation process of TorA by the TAT translocon to prevent export of immature TorA (12Oresnik I.J. Ladner C.L. Turner R.J. Mol. Microbiol. 2001; 40: 323-331Crossref PubMed Scopus (145) Google Scholar, 13Jack R.L. Buchanan G. Dubini A. Hatzixanthis K. Palmer T. Sargent F. EMBO J. 2004; 23: 3962-3972Crossref PubMed Scopus (166) Google Scholar). 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 (11Ilbert M. Méjean V. Iobbi-Nivol C. Microbiology. 2004; 150: 935-943Crossref PubMed Scopus (60) Google Scholar). Thus, TorD also binds to a second region of TorA in addition to the signal peptide (9Pommier J. Méjean V. Giordano G. Iobbi-Nivol C. J. Biol. Chem. 1998; 273: 16615-16620Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 13Jack R.L. Buchanan G. Dubini A. Hatzixanthis K. Palmer T. Sargent F. EMBO J. 2004; 23: 3962-3972Crossref PubMed Scopus (166) Google Scholar). TorD is a member of a large family of homologous proteins associated to molybdoenzymes of the Me2SO reductase family (11Ilbert M. Méjean V. Iobbi-Nivol C. Microbiology. 2004; 150: 935-943Crossref PubMed Scopus (60) Google Scholar, 14Turner R.J. Papish A.L. Sargent F. Can. J. Microbiol. 2004; 50: 225-238Crossref PubMed Scopus (90) Google Scholar). Among them, TorD from Shewanella massilia was crystallized, and its three-dimensional structure was solved revealing an all-helical architecture showing no similarity with other known protein structures (15Tranier S. Mortier-Barrière I. Ilbert M. Birck C. Iobbi-Nivol C. Méjean V. Samama J.P. Protein Sci. 2002; 11: 2148-2157Crossref PubMed Scopus (47) Google Scholar, 16Tranier S. Iobbi-Nivol C. Birck C. Ilbert M. Mortier-Barrière I. Méjean V. Samama J.P. Structure (Lond.). 2003; 11: 165-174Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). We and others have also shown that members of the TorD family are structurally related and contain mainly α-helices (11Ilbert M. Méjean V. Iobbi-Nivol C. Microbiology. 2004; 150: 935-943Crossref PubMed Scopus (60) Google Scholar, 17Sarfo K. Winstone T.L. Papish A.L. Howell J.M. Kadir H. Vogel H.J. Turner R.J. Biochem. Biophys. Res. Commun. 2004; 315: 397-403Crossref PubMed Scopus (30) Google Scholar). Although these proteins are part of the same family of chaperones, they probably possess a high specificity toward their partner. For instance, DmsD, a TorD homologue in E. coli, is necessary for the activity of molybdoenzyme DmsA (11Ilbert M. Méjean V. Iobbi-Nivol C. Microbiology. 2004; 150: 935-943Crossref PubMed Scopus (60) Google Scholar, 18Ray N. Oates J. Turner R.J. Robinson C. FEBS Lett. 2003; 534: 156-160Crossref PubMed Scopus (72) Google Scholar) but cannot replace TorD during TorA maturation, and, conversely, TorD cannot play the role of DmsD toward DmsA (11Ilbert M. Méjean V. Iobbi-Nivol C. Microbiology. 2004; 150: 935-943Crossref PubMed Scopus (60) Google Scholar). In this study we show that although TorD is an accessory protein at 37 °C, it becomes essential for TorA maturation at elevated temperatures. This study also reveals that misfolded apoTorA is degraded in vivo and that TorD not only avoids apoTorA degradation but also plays a key role in the first step of TorA maturation. Bacterial Strains, Plasmids, and Growth Conditions—The E. coli strains used in this work are LCB514 (MC4100 ΔdmsD Kmr) (11Ilbert M. Méjean V. Iobbi-Nivol C. Microbiology. 2004; 150: 935-943Crossref PubMed Scopus (60) Google Scholar), LCB515 (MC4100 torD::Ω Spcr ΔdmsD Kmr) (11Ilbert M. Méjean V. Iobbi-Nivol C. Microbiology. 2004; 150: 935-943Crossref PubMed Scopus (60) Google Scholar), RK5208 (araD139 Δ(lacIPOZYA-argF) U169 rpsL gyrA mobA207::Mucts) (19Stewart V. MacGregor C.H. J. Bacteriol. 1982; 151: 788-799Crossref PubMed Google Scholar), LCB440 (MC4100 Δ(torSTRCAD) Δ(dmsABC) Kmr, this study), and LCB620 (MC4100 torA8::MudII 1734 (torA-lacZ) (6Méjean V. Iobbi-Nivol C. Lepelletier M. Giordano G. Chippaux M. Pascal M.C. Mol. Microbiol. 1994; 11: 1169-1179Crossref PubMed Scopus (221) Google Scholar). The strains were grown in Luria Broth medium, and, when necessary, ampicillin (50 μg·ml–1) was added to maintain plasmid selection. Plasmid pTorA allowing His6-tagged apoTorA production was previously described (10Ilbert M. Méjean V. Giudici-Orticoni M.T. Samama J.P. Iobbi-Nivol C. J. Biol. Chem. 2003; 278: 28787-28792Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). To construct plasmid pBD, which allows the synthesis of His6-tagged TorD, the same cloning strategy as previously described for pTorD (11Ilbert M. Méjean V. Iobbi-Nivol C. Microbiology. 2004; 150: 935-943Crossref PubMed Scopus (60) Google Scholar) was used except that the torD coding sequence was cloned into the EcoRI-HindIII cloning sites of pBAD24 (20Guzman L.M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3978) Google Scholar). The absence of mutation in the DNA insert of the recombinant plasmid was checked by sequencing. Transformations were carried out according to the method of Chung and Miller (21Chung C.T. Miller R.H. Nucleic Acids Res. 1988; 16: 3580Crossref PubMed Scopus (218) Google Scholar). Preparation of the Soluble Fractions—Strains LCB514, LCB515, LCB515/pBAD24, and LCB515/pBD were grown overnight anaerobically at 37 and 42 °C with TMAO (0.2%) to induce chromosomal tor operon expression. The cells were resuspended in 40 mm Tris-HCl, pH 7.6, washed twice, and disrupted by a French press. The extracts were centrifuged at 15,000 rpm, with the recovered supernatants then being centrifuged at 45,000 rpm to obtain the soluble fractions. The periplasmic and cytoplasmic fractions of strains LCB514 and LCB515 were prepared according to the sucrose-lysozyme-EDTA procedure as described previously (9Pommier J. Méjean V. Giordano G. Iobbi-Nivol C. J. Biol. Chem. 1998; 273: 16615-16620Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Analytical Procedure—Protein concentrations were measured by the technique of Lowry. TMAO reductase activity was measured spectrophotometrically at 37 °C by following the oxidation of reduced benzyl viologen at 600 nm coupled to the reduction of TMAO (9Pommier J. Méjean V. Giordano G. Iobbi-Nivol C. J. Biol. Chem. 1998; 273: 16615-16620Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The amount of TorA present in the extracts was determined by rocket immunoelectrophoresis. Samples were submitted to electrophoresis at 2 mA overnight in 4 × 4-cm (1%, w/v) agarose plates buffered with 20 mm sodium barbital (pH 8.6) containing TorA antibodies (20 μl). β-Galactosidase activities were measured by the method of Miller (22Miller J. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar) from strain LCB620 grown overnight anaerobically with TMAO (0.1%) at 25, 37, and 42 °C. Purification of Recombinant Proteins—Recombinant apoTorA protein was purified from the soluble extract of strain RK5208/pTorA grown aerobically at 30 °C in the presence of isopropyl 1-thio-β-d-galactopyranoside (1 mm). 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 (10Ilbert M. Méjean V. Giudici-Orticoni M.T. Samama J.P. Iobbi-Nivol C. J. Biol. Chem. 2003; 278: 28787-28792Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). After purification, apoTorA and TorD were dialyzed against phosphate buffer pH 7 (20 mm). In Vitro Maturation System—ApoTorA (3 μm, 25 μl) and phosphate buffer, pH 7 (20 mm, 25 μl), or apoTorA (3 μm, 25 μl) and TorD (60 μm, 25 μl) mixed together were incubated for 30 min at 37, 42, and 47 °C before the addition of 100 μl of the MoCo source. The MoCo source corresponds to the soluble fraction of strain LCB440 grown anaerobically at 37 °C, prepared in 20 mm phosphate buffer, pH 7, at a protein concentration of 40 mg·ml–1 (10Ilbert M. Méjean V. Giudici-Orticoni M.T. Samama J.P. Iobbi-Nivol C. J. Biol. Chem. 2003; 278: 28787-28792Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). When ATP (1 mm) or ATPγS (1 mm and 5 mm) and MgCl2 (1 mm) were tested, they were added during the 30-min incubation of apoTorA in the presence or absence of TorD at various temperatures. The MoCo source is then added as mentioned above. All solutions were oxygen-depleted. After an incubation of 60 min at 37 °C the TMAO reductase activity was measured. Units corresponded to μmol TMAO reduced/min/μg of apoTorA. Defined in Vitro Assays—After the incubation of apoTorA (3 μm, 25 μl) or apoTorA (3 μm, 25 μl) and TorD (60 μm, 25 μl) for 30 min at 37, 42, and 47 °C as described above, the bis(MGD)Mo source was added. It was made up of MobA (0.4 μm), GTP (1 mm), MgCl2 (1 mm), and 96 μlof a solution containing MPT-Mo from the °C, 5 of cells (10Ilbert M. Méjean V. Giudici-Orticoni M.T. Samama J.P. Iobbi-Nivol C. J. Biol. Chem. 2003; 278: 28787-28792Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). All these were a After a at 37 °C the TMAO reductase activity was measured. Units corresponded to μmol of TMAO reduced/min/μg of apoTorA. TorD for TorA Maturation at in TorA protein is when cells are grown in in the presence of In an study, we showed that the of TorD not TorA maturation but its level (9Pommier J. Méjean V. Giordano G. Iobbi-Nivol C. J. Biol. Chem. 1998; 273: 16615-16620Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). of the TMAO reductase activity was measured in a strain with a strain when both were grown anaerobically in the presence of the were performed at 30 or 37 °C. To the role of TorD as a specific chaperone of TorA, we both the torD and strains at an elevated temperature. After overnight growth at 42 °C, soluble extracts were and the TMAO reductase activities were measured. shown in the level of TMAO reductase activity measured in the soluble extract of strain LCB514 was when cells were grown at 37 and at 42 °C and that the high of growth not TMAO reductase activity in the presence of In when cells of strain LCB515 were grown at 42 °C, the TorA activity was because it was times that measured in the strain grown in the same of for growth at 37 °C, the absence of TorD to times TMAO reductase activity with in the presence of To that the is only because of the absence of TorD in strain LCB515, the torD mutation was by a plasmid the torD LCB515 containing and the control strain were grown in at 37 and 42 °C. A was because for growth at 42 °C, times TMAO reductase activity was measured when TorD was in at 37 °C, times activity was in the presence of This that TorD is responsible for the of TorA maturation at elevated temperatures of We also performed experiments at 25 °C. In this growth the of TorD only two times TMAO reductase activity not We also checked the absence of TorD the of TorA at high temperature. We that in the presence or in the absence of TorD, at of the TMAO reductase activity was found in the periplasmic fraction of cells grown at 42 °C. This that at high and in the absence of TorD, the activity is mainly by a TorA, which has into the the absence of TorD not the translocation of TorA at high temperature. These experiments clearly established that TorD is for TorA maturation when cells are grown in In this the of TorD almost TorA The essential role of TorD was by the of strain LCB515 to anaerobically at elevated in with as the source and TMAO as the only not these that TorD plays an essential role at elevated temperatures of growth and improves TorA maturation at temperatures. because it is established that TorD is during TorA maturation (10Ilbert M. Méjean V. Giudici-Orticoni M.T. Samama J.P. Iobbi-Nivol C. J. Biol. Chem. 2003; 278: 28787-28792Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), TorD induce or the competent form of apoTorA at high temperature. of TorD on of TMAO reductase activity for cells grown at 42 °C with 37 °C is to a of the amount of TorA or to the of TorA To the two we checked the amount of TorA protein from cells grown at 37 and 42 °C, and we also the of the on the To the of a chromosomal was were performed in at 25, 37, and 42 °C. In the three of the of activity measured were Miller meaning that the of from 25 to 42 °C not the level of torCAD expression. This that the in the TorA activity not from The amount of TorA present in the extracts was by rocket antibodies against TorA When strains LCB514 and LCB515 were grown at 37 °C, of TorA protein was recovered in the soluble extract of the strain with the strain and a amount of TorA was found in extracts of LCB514 grown at 37 or 42 °C with Strikingly, in the soluble extract of strain LCB515 grown at 42 °C, only a amount of TorA was and the was at times that in both for the strain 4 with or TorA is degraded at 42 °C in the absence of This with the of the TMAO reductase activity and that TorD the immature form of TorA or is that the presence of TorD apoTorA to prevent misfolded induced by the high and to of on the in Vitro Maturation of the of the on apoTorA and the role of TorD in we incubated apoTorA at various temperatures for 30 min in the presence or in the absence of We then added a MoCo source and incubated the for at 37 °C to the of apoTorA. The TMAO reductase which corresponds to the level of TorA maturation, the amount of competent apoTorA in the In a first of apoTorA was incubated alone at 37, 42, and 47 °C for 30 The MoCo source was then added to for at 37 °C. a activity when apoTorA was incubated at 47 °C and an when apoTorA was incubated at 42 °C with the level of activity measured in the incubated at 37 °C. These that high temperatures also the in vitro maturation of TorA and that apoTorA is misfolded in these to a of its in vitro In a second of TorD was added to apoTorA during the incubation at 37, 42, and 47 °C. After 30 min of the MoCo source was added for at 37 °C, and the TMAO reductase activity was then measured. In to was when apoTorA was incubated in the presence of TorD the level of TMAO reductase activity at 37 and 42 °C and at 47 °C a when the the activity measured in the presence or in the absence of TorD from 2 at 37 °C and 5 at 42 °C, up to at 47 °C. These that TorD the of elevated on apoTorA probably by it in a competent for maturation. Thus, in vivo and in vitro experiments that TorD an essential role as a chaperone when apoTorA elevated temperatures. For their chaperones ATP as an source. To the action of TorD toward apoTorA be by (1 mm), or its ATPγS (1 or 5 mm) were added during the 30 of apoTorA and TorD at 42 and 47 °C. We that the activities measured the in vitro were not by the presence of ATP or ATPγS and in the same of as described in not a a was performed by apoTorA at 42 and 47 °C for 30 min in presence of (1 mm) or ATPγS (1 or 5 mm), and no was These that TorD not ATP to as a chaperone toward apoTorA and to maintain it in a competent rocket TorA we have the amount of and immature TorA present in the various for TMAO reductase When apoTorA was incubated in the presence of TorD, the amount of protein was whatever the incubation temperatures were When apoTorA was incubated the amount of protein when the This clearly that misfolded apoTorA is poorly degraded in in vitro assays in to was in that the role of TorD is not to apoTorA against but is to allow its Maturation of TorA in an in Vitro Defined a we have previously shown that TorD alone is sufficient to allow an maturation of apoTorA at 37 °C (10Ilbert M. Méjean V. Giudici-Orticoni M.T. Samama J.P. Iobbi-Nivol C. J. Biol. Chem. 2003; 278: 28787-28792Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In this apoTorA or apoTorA and TorD were mixed with MPT-Mo from a °C, 5 GTP and the purified MobA which the of MPT-Mo to After 30 min of incubation of apoTorA or apoTorA and TorD at 37, 42, and 47 °C, we added the MoCo source for 2 at 37 °C to allow the synthesis and the of the The shown in that incubation of apoTorA alone at 42 and 47 °C to a and with the activity measured in the apoTorA incubated at 37 °C. This that at high apoTorA is In when apoTorA was first incubated with TorD, the activity recovered was at 37 and 42 °C and at 47 °C The of the activity recovered in the presence or in the absence of TorD from 4 at 37 °C and at 42 °C up to 25 at 47 °C. These confirm the key role of TorD at high but the of are with the with that with the fraction with This probably from the amount of bis(MGD)Mo in the (10Ilbert M. Méjean V. Giudici-Orticoni M.T. Samama J.P. Iobbi-Nivol C. J. Biol. Chem. 2003; 278: 28787-28792Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In the we demonstrated that TorD alone is sufficient to maintain apoTorA in a competent and to the MoCo insertion into the as MobA was also present in the we cannot a role of MobA during the MoCo insertion in addition to its in the step of MoCo study reveals the essential role of TorD during TorA maturation at high and to the model shown in temperatures of which not insert the MoCo and becomes to in TorD this by or the competent of the which then the MoCo. apoTorA to be in an a competent and an and the TorD chaperone this toward the competent state, and, as a TorD also to the of apoTorA that competent apoTorA is not degraded. This study is the first of the essential role of a chaperone elevated and this be used as a to study in TorD in both TorA maturation and translocation. It be also to study this of TorD is by other members of this family of We M. and C. for We also for the for and A. Magalon for the of MobA

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