Functional and structural analysis of members of the TorD family, a large chaperone family dedicated to molybdoproteins

Marianne Ilbert, Vincent Méjean and Chantal Iobbi-Nivol

Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et Microbiologie, Centre National de la Recherche Scientifique, 31, chemin Joseph Aiguier, 13402 Marseille Cedex 20, France

Correspondence
Chantal Iobbi-Nivol
iobbi{at}ibsm.cnrs-mrs.fr


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The trimethylamine N-oxide (TMAO) reductase TorA, a DMSO reductase family member, is a periplasmic molybdoenzyme of Escherichia coli. The cytoplasmic protein TorD acts as a chaperone for TorA, allowing the efficient insertion of the molybdenum cofactor into the apoform of the enzyme prior to its secretion. This paper demonstrates that TorD is a member of a large family of prokaryotic proteins that are structurally related. Moreover, their genes generally belong to operons also encoding molybdoenzymes of the DMSO reductase family. Both the TorD and the DMSO reductase families present a similar phylogenetic organization, suggesting a co-evolution of these two families of proteins. This hypothesis is also supported by the fact that the TorD and DmsD chaperones cannot replace each other and thus appear dedicated to specific molybdopartners. Interestingly, it was found that the positive effect of TorD on TorA maturation, and the partial inhibitory effect of DmsD and homologues, are independent of the TorA signal sequence.


Abbreviations: bis(MGD)Mo, bis(molybdopterine guanine dinucleotide)molybdenum; CD, circular dichroism; TMAO, trimethylamine N-oxide


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Molybdoenzymes comprise a wide range of redox enzymes found in most organisms, from bacteria to plants and humans. They are classified into four families according to their molybdenum cofactor composition; among them is the DMSO reductase family, containing only prokaryotic enzymes, mainly involved in anaerobic respiration (Hille, 1996; Richardson, 2000). Enzymes of this family contain a common form of molybdenum cofactor, bis(molybdopterine guanine dinucleotide)molybdenum [bis(MGD)Mo], and they share a similar overall structure (Rajagopalan & Johnson, 1992; Schindelin et al., 1996; Schneider et al., 1996; McAlpine et al., 1997; Czjzek et al., 1998). They are compact molecules organized in four domains surrounding the molybdenum cofactor (Schindelin et al., 1996; Schneider et al., 1996; McAlpine et al., 1997; Czjzek et al., 1998). Based on sequence similarities, the DMSO reductase family has been divided into three types (McDevitt et al., 2002). Furthermore, enzymes of these three types can be distinguished by their first domain. Type I enzymes, exemplified by the soluble nitrate reductase (NapA) and the formate dehydrogenase (FdhA), contain a [4Fe–4S] iron–sulfur cluster at the N-terminus of the first domain, whereas the type II enzymes, such as the membranous DMSO reductase (DmsA) and the recently characterized DMSO dehydrogenase (DdhA), still conserve a cysteine-rich motif but are thought not to bind an [Fe–S] cluster. Finally, this conserved motif is absent in the first domain of type III proteins, comprising monomeric enzymes such as TorA and DorA (McDevitt et al., 2002).

In Escherichia coli, TorA is the terminal reductase of the Tor system, the main anaerobic respiratory pathway responsible for reduction of trimethylamine N-oxide (TMAO) to trimethylamine (Méjean et al., 1994). TorA is translocated into the periplasm by the Tat system (Weiner et al., 1998; Santini et al., 1998; Berks et al., 2000) and it receives the electrons from the membrane anchored c-type cytochrome TorC (Gon et al., 2001). The genes encoding TorC and TorA are part of the torCAD operon, which is induced in the presence of TMAO or related compounds by the two-component regulatory system TorS/TorR (Méjean et al., 1994; Iobbi-Nivol et al., 1996; Jourlin et al., 1997). The reduction of TMAO in E. coli is also catalysed by the DMSO reductase complex (made up of three subunits: DmsA, DmsB and DmsC) synthesized in anaerobiosis (Weiner et al., 1992). As described above, DmsA and TorA belong to type II and III, respectively, of the DMSO reductase family of molybdoenzymes (McDevitt et al., 2002). They also differ in their substrate specificity, since DmsA reduces a wide range of S- and N-oxide compounds, whereas TorA only reduces TMAO (Simala-Grant & Weiner, 1996; Iobbi-Nivol et al., 1996).

TorD, the product of the last gene of the torCAD operon, does not participate in the electron transfer pathway but it is the specific chaperone of TorA (Pommier et al., 1998; Ilbert et al., 2003). In vivo, the lack of TorD leads to a significant decrease of the final amount of TorA; however, specific activity and biochemical properties of the enzyme are not altered (Pommier et al., 1998). Recently, we demonstrated, by an in vitro approach, the involvement of TorD during the first step of the maturation process of TorA (Ilbert et al., 2003). When an appropriate molybdenum cofactor source is provided to the apoform of TorA, the addition of TorD increases fourfold the level of apoTorA maturation and leads to the maturation of most of the apoenzyme. We have also shown that TorD binds to apoTorA and probably modifies its conformation to a competent state, facilitating its maturation (Ilbert et al., 2003).

Based on sequence similarity and on the results described below, DmsD seems to belong to the TorD specific chaperones, dedicated to the maturation of TMAO/DMSO reductases (Sargent et al., 2002; Tranier et al., 2003). Indeed, recent work has shown that DmsA processing requires the presence of the DmsD chaperone (Oresnik et al., 2001). The absence of DmsD leads to the loss of anaerobic growth of the bacteria on DMSO-containing medium, indicating that DmsD is required for the DMSO reductase biogenesis (Ray et al., 2003). Furthermore, DmsD was described as binding to the signal peptide of preDmsA and preTorA, targeting these proteins to the Tat translocation system (Oresnik et al., 2001). However, this point seems debatable since the fusion of the signal sequence of each enzyme to green fluorescent protein (GFP) indicated that DmsD was not required for the functioning of these signal peptides (Ray et al., 2003). Although the role of DmsD is not yet clearly defined, a recent report indicates that, under certain conditions of bacterial growth, DmsD is associated with the inner membrane and that the TatB and TatC subunits could be important for this interaction (Papish et al., 2003).

In a previous study, we showed that the TorD family comprised eleven proteins from various bacteria such as E. coli, Shewanella and Rhodobacter species, Vibrio cholerae, Salmonella typhimurium and Haemophilus influenzae (Tranier et al., 2003). The TorD family contains proteins of approximately 200 amino acids and shares sequence similarities with the E. coli TorD (TorDec) (Sargent et al., 2002; Tranier et al., 2003). The location of their genes is often found in operons also encoding molybdoenzymes of the DMSO reductase family. Among the members of the TorD family, a dimer of TorD from Shewanella massilia (TorDsm) was crystallized and its three-dimensional structure was solved at 2·4 Å resolution (Tranier et al., 2002, 2003). The X-ray structure of TorDsm revealed extensive domain swapping between the two subunits and an all-helical architecture of the globular domains, showing no similarity with other known protein structures (Tranier et al., 2003).

This report presents an extension and classification of the TorD family members made possible by the increasing availability of sequenced bacterial genomes. Moreover, a structural and functional study of various members of the TorD family indicates that although they are structurally related, they seem to be highly specific. This analysis thus reveals a possible co-evolution of the molybdoenzymes of the DMSO reductase family and of their dedicated chaperones of the TorD family.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growth conditions.
The bacterial strains and plasmids used in this work are listed in Table 1. The strains were grown in Luria Broth (LB) medium at 37 °C. To maintain plasmid selection, antibiotics were added at the following concentrations: 50 µg ampicillin ml–1, 10 µg kanamycin ml–1.


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Table 1. Bacterial strains and plasmids used in this study

 
For the biochemical study, strains were grown anaerobically in LB medium. When necessary, IPTG (1 mM) was added to induce expression of the cloned genes and TMAO (20 mM) was added to induce chromosomal torA expression.

For the growth kinetics studies, anaerobic growth was performed in full-capped cuvettes at 37 °C with a minimal salt medium (MSM), derived from that described by Gon et al. (2000). The MSM was supplemented with 0·5 % (v/v) glycerol, as a nonfermentable carbon source, and with DMSO (28 mM). The MSM was inoculated at a dilution of 1 % with cells grown overnight in LB medium supplemented with ampicillin (50 µl ml–1) when necessary, centrifuged and resuspended in the same volume of MSM. Growth was monitored in the same full-capped cuvettes at 600 nm. Data are typical of at least three independent experiments.

Construction of mutant strains.
Construction of strain LCB514, with the entire dmsD gene deleted, was performed according to the method of Datsenko & Wanner (2000). The dmsD gene was replaced by a chloramphenicol-resistance gene, generated by PCR. The primers were 60 nts long and included 40-nt sequence extensions, corresponding to the 5' region upstream of the start codon of dmsD gene, or to the 3' extremity of the dmsD gene and 20 nt priming sequences that hybridized to the resistance gene flanking sequence of plasmid pKD3. The PCR mixture was treated with DpnI and used to transform strain MC4100 carrying the {lambda} Red helper plasmid pKD46. The mutation of strain LCB514 ({Delta}dmsD) was transferred to LCB641 (torD) by P1 transduction, resulting in strain LCB515 (torD{Delta}dmsD).

Construction of plasmids.
Plasmids pTorDsm and pTorDec, allowing the synthesis of His6-tagged-TorDsm and -TorDec, respectively, were constructed previously (Tranier et al., 2002; Ilbert et al., 2003). Plasmids pDmsD, pYcdY and pTorDso, allowing the synthesis of His6-tagged-DmsD, -YcdY and -TorDso, respectively, were constructed by cloning DNA fragments encoding each tagged protein preceded by a consensus Shine–Dalgarno motif into the XbaI and HindIII sites of the expression vector pET28 under the control of T7 promoter. In each construction, the His6-tag coding sequence comes from the reverse primers. The PCR amplification of the coding sequences were performed with E. coli or Shewanella oneidensis chromosomal DNA, according to the origin of the protein of interest, and appropriate primers, available upon request. All PCR products and fusion sites were confirmed by sequencing. The recombinant vectors pDmsD, pYcdY and pTorDso were introduced into strain BL21(DE3) according to the method of Chung & Miller (1988).

Plasmids pJFTorD, pJFDmsD, pJFYcdY were constructed as described above except that the PCR products were cloned into the corresponding sites of expression vector pJF119EH containing the PTAC promoter.

Plasmid p'TorA was constructed as for pTorA (Ilbert et al., 2003) except that the coding sequence of torA was deleted from nucleotide 4 to117, encoding the signal sequence of the protein. This construction leads to the production of a TorA protein devoid of its signal sequence.

Purification of recombinant proteins.
Recombinant TorD-like proteins were purified by a HiTrap chelating Ni2+ column (Amersham Pharmacia Biotech) as described previously for TorDec and TorDsm (Tranier et al., 2002; Ilbert et al., 2003). As often happens during overproduction, the proteins presented several oligomeric states, which were separated during the elution from the affinity column. In all the experiments carried out in this study, only the fractions containing mainly monomeric forms of the TorD-like proteins were used (see Fig. 2a). After purification, TorDec, TorDsm, DmsD, YcdY and TorDso were loaded on a desalting PD-10 column (Amersham Biosciences) equilibrated with 20 mM phosphate buffer (pH 7). The proteins were purified near to homogeneity as checked by SDS/15 % PAGE and Coomassie staining of the gel (data not shown). His6-tagged apoTorA and apo'TorA were overproduced in a molybdenum-cofactor-deficient strain harbouring plasmid pTorA or p'TorA, respectively, and purified from the soluble fraction as described previously (Ilbert et al., 2003).



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Fig. 2. (a) Coomassie blue stained native 15 % PAGE of monomeric and dimeric forms of TorDsm (1), and purified monomeric forms of TorDsm (2), TorDec (3), DmsD (4), YcdY (5) and TorDso (6). (b) CD spectra of purified monomeric forms of TorDsm, TorDec, DmsD, YcdY and TorDso, recorded by using 1 µM of each protein in 20 mM phosphate buffer, pH 7·0.

 
In vitro activation of apoTorA and apo'TorA.
The cofactor source (SN504) corresponded to the soluble fraction of strain LCB504 grown anaerobically. It was kept at a protein concentration of 40 mg ml–1 in phosphate buffer (20 mM, pH 7) and at 4 °C under nitrogen atmosphere as previously described by Ilbert et al. (2003). ApoTorA (0·55 µM) or apo'TorA (0·55 µM) were mixed with 100 µl of SN504 in presence or absence of TorDec (2·1 µM) or homologous monomeric proteins (2·1 µM). For the assays containing both TorDec and a homologous protein, their concentrations were 2·1 µM and 5 µM, respectively. The mixture was incubated for 180 min at 37 °C under nitrogen atmosphere. TMAO reductase activities were given as a percentage of the activity of holoTorA (0·55 µM). As a control, an assay containing TorDec and BSA (20 µM) was performed.

Analytical procedure.
The soluble and membrane extracts were prepared in Tris buffer (40 mM, pH 7·6). Membranes (4 mg ml–1) were solubilized with Triton X-100 (4 %, 30 min at 4 °C) in the same buffer. 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. SDS-PAGE was performed using 10 or 15 % polyacrylamide gels as indicated in the legends. DMSO reductase activity in the polyacrylamide gel was revealed as described for TMAO reductase activity, except that methyl viologen and DMSO were used as electron donor and acceptor, respectively. The amount of TorA present in the extracts was determined by rocket immunoelectrophoresis using a polyclonal antiserum specific for TorA (Pommier et al., 1998).

Circular dichroism (CD) spectra.
Far UV CD spectra of purified monomeric forms of proteins were monitored from 190 to 260 nm. The spectra were recorded on a Jasco J-715 instrument equipped with a Peltier-type temperature control system (model PTC-348WI) with a 2 mm pathlength and a scan rate of 5 nm min–1 at 20 °C. Each spectrum was a mean of three scans. Baselines were corrected by subtracting the buffer spectrum.

Phylogenetic analysis.
Amino acid sequences of the TorD homologues were retrieved via the NCBI server (http://www.ncbi.nlm.nih.gov/). Multiple alignments were performed using CLUSTALX and the phylogenetic relationships were calculated using the neighbour-joining approach (CLUSTALX) (Altschul et al., 1990).


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The TorD family is a large family that can be divided into four groups
Using the primary sequence of TorD from E. coli (TorDec), a new search in the sequence databases allowed the identification of 33 prokaryotic proteins of approximately 200 amino acids as potential members of the TorD family. The level of identity between TorDec and these proteins is rather low, ranging from 20 to 38 %, except for TorD of S. typhimurium (TorDst), which shares 64 % identity with TorDec. This last point is not surprising because a high level of identity is found for most of the proteins of these two bacteria, indicating a close phylogenetic relationship of the parent species. The primary sequences of the TorD homologues were aligned and an unrooted phylogenic tree was constructed (Fig. 1a). The overall topology of the tree suggested that the proteins can be divided into four distinct groups. Three of them can be related to the phylogenetic groups of the DMSO reductase family (McDevitt et al., 2002). One type, exemplified by TorDec, comprises proteins such as TorDsm, TorDso, DorDrc and DmsBrs, encoded by genes which are part of operons homologous to the E. coli torCAD transcriptional unit and encoding molybdoenzymes of type III (Méjean et al., 1994; Mouncey et al., 1997; Dos Santos et al., 1998; Shaw et al., 1999). In line with the molybdoenzyme classification, this type of TorD homologue was therefore named type III. According to the phylogenetic tree, DmsD belongs to a class distinct from that of TorDec. Recent reports indicated that DmsD could be required for maturation and export of DmsA, the catalytic subunit of the membranous DMSO reductase complex that is part of the type II enzymes (Oresnik et al., 2001; Ray et al., 2003). DmsD and other proteins of the same type are encoded by genes organized in operons similar to the E. coli dmsABC operon encoding a type II DMSO reductase family enzyme, an [Fe–S] cluster containing subunit and a membrane anchor subunit (Weiner et al., 1992). This group of TorD homologues was correspondingly named type II. Unlike types II and III, type I does not contain any E. coli proteins. Genes encoding TorD homologues of type I are often part of operons also encoding molybdoenzymes, but homologous to the formate dehydrogenase (FDH) complex which are classified as type I in the DMSO reductase family of enzymes (McDevitt et al., 2002). In addition to these three types of TorD homologues, a fourth type was identified, exemplified by the E. coli protein YcdY. This clade presents a closer relationship to type II than to the other two types of TorD homologues. It was named type IV and is made up of proteins whose genes seem to be isolated on the bacterial genomes. Thus, type IV proteins cannot be obviously related to any specific molybdoenzyme family. From this study, it appears that the proteins of the TorD family can be divided into four types. Based on their gene organization, the classification of the first three types of the TorD homologues clearly correlates with that of the DMSO reductase family enzymes. This point supports the idea that both partners, the molybdoenzyme and the chaperone, have evolved in a coordinate manner.



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Fig. 1. (a) Unrooted phylogenetic tree of the TorD homologues. Bootstrap values indicated in phylograms correspond to the frequency (%) of occurrence of node in 1000 bootstrap replicates. Attributions to the four types defined in the text are shown by arcs. Proteins encoded by genes which belong to loci also encoding molybdoenzymes of the same type as that described by McDevitt et al. (2002) are shown in bold. The abbreviations used for protein names are listed as follows: TorDec, gi|16128964|, YcdY, gi|16128998| and DmsD, gi|3183478| of E. coli; ActPl, gi|32034681| of Actinobacillus pleuropneumoniae; HaeIn1, gi|1175398| and HaeIn2, gi|1175887| of Haemophilus influenzae; HaeSo, gi|23467672| of Haemophilus somnus; MagMa, gi|23007820| of Magnetospirillum magnetotacticum; PasMu1, gi|15603659|, PasMu2, gi|15603622| and PasMu3, gi|15602751| of Pasteurella multocida; RalSo, gi|17547094| of Ralstonia solanacearum; DorDrc, gi|1769459| of Rhodobacter capsulatus; DmsBrs, gi|1711288| of Rhodobacter sphaeroides; TorDst, gi|16504806|, SalTy2, gi|16502662|, SalTy3, gi|16767558| and SalTy4, gi|29142215| of Salmonella typhimurium; TorDsm, gi|31615594| of Shewanella massilia; TorDso, gi|24372812|, SheOn1, gi|24375984|, SheOn2, gi|24375843| and SheOn3, gi|24373009| of Shewanella oneidensis; ShiFl1, gi|24112436| and ShiFl2, gi|30063106| of Shigella flexneri; VibCh1, gi|15641724| and VibCh2, gi|15641523| of Vibrio cholerae; VibPa1, gi|28897805| and VibPa2, gi|28898285| of Vibrio parahaemolyticus; YerPe1, gi|16123475|, YerPe2, gi|16123149| and YerPe3, gi|16122277| of Yersinia pestis; and YerPK, gi|22125413| of Y. pestis KIM. (b) The TorD family signature. The three motifs contain amino acids conserved in at least 80 % of the aligned sequences used for the phylogenetic tree. Positions of the residues correspond to the TorDec primary sequence and residues in brackets can be found at the same position.

 
Related structures of TorD family proteins
Although the proteins of the TorD family present a low level of identity along their entire sequences, the alignment of the four types of TorD homologues reveals three highly conserved motifs (Fig. 1b). We propose that these motifs can be taken as the signature of the TorD protein family. The structure of a dimer of the type III TorDsm was recently solved. This structure revealed a novel all-helical architecture of the globular domains of the protein, and suggested that the few invariant or highly conserved hydrophobic residues present in the conserved motifs constitute the hydrophobic core of the protein (Tranier et al., 2003). To check whether the secondary fold of the TorD homologues was conserved, several members were purified, mainly as monomers, and analysed by CD (Fig. 2). The CD spectrum obtained for the monomeric form of TorDsm, used as a control, indicated a secondary structure with high {alpha}-helix content and no {beta}-sheet, in agreement with the solved X-ray structure (Tranier et al., 2002, 2003). Strikingly, the CD spectra obtained from four additional members of the TorD family (TorDec and TorDso of type III, DmsD of type II and YcdY of type IV) were almost identical to each other and similar to that of TorDsm, indicating that these proteins probably possess a similar secondary fold. The spectra were dominated by minima close to those observed with TorDsm (208–222 nm) and characteristic of {alpha}-helices (Tranier et al., 2003; Adler et al., 1973). We thus propose that the members of the TorD family are structurally related and contain a high level of {alpha}-helices.

Specificity of the TorD homologues
It was recently shown by an in vitro assay that TorDec plays a key role during the maturation of apoTorA, its presence leading to a fourfold increase in the apoenzyme maturation (Ilbert et al., 2003). This activation test was used to determine whether other types of TorD homologues, and in particular those from E. coli, could substitute for TorDec. In the assay, apoTorA and a source of molybdenum cofactor were mixed either with DmsD or YcdY. None of the proteins increased significantly the level of TMAO reductase activity recovered in the assay after 180 min incubation, indicating that neither DmsD nor YcdY could substitute for TorDec during apoTorA maturation (Fig. 3). One possibility is that DmsD and YcdY are unable to replace TorDec because they do not belong to the same subfamily, and thus are quite distinct from TorDec. A similar test was therefore performed with TorDsm, a type III TorD homologue sharing 34 % identity with TorDec. Again, no complementation was observed, showing that TorDsm cannot replace TorDec either (Fig. 3). From these experiments, it seems clear that TorDec is specifically devoted to apoTorA and cannot be replaced by any of the other proteins tested.



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Fig. 3. In vitro effect of TorD homologues on TorA maturation. ApoTorA or apo'TorA (0·55 µM) were mixed with 100 µl of cofactor source SN504 (40 mg ml–1) and, where indicated, with either TorDec (2·1 µM) and homologous proteins (5 µM) or homologous proteins (2·1 µM) alone. The mixtures were incubated for 180 min at 37 °C under a nitrogen atmosphere and TMAO reductase activities were measured and expressed as a percentage of the activity of holoTorA (0·55 µM). As a control, an assay containing TorDec and BSA (20 µM) was performed. Error bars corresponding to standard deviations are indicated.

 
These results were confirmed by an in vivo approach. The E. coli proteins TorDec, DmsD and YcdY were overproduced in the torD strain LCB641 (harbouring plasmids pJFTorD, pJFDmsD and pJFYcdY respectively) grown anaerobically in rich media supplemented with TMAO. The TMAO reductase activity in the soluble extracts was then measured. The production of DmsD or YcdY in this strain did not lead to an increase of the TMAO reductase activity level, whereas production of TorDec, from a plasmid complementing the mutation, increased by 2·5-fold the final amount of active TorA (Fig. 4a). For each sample, the activity level was correlated to the amount of TorA protein as shown by rocket immunoelectrophoresis. This indicates that neither DmsD nor YcdY can increase the final amount of TorA in a torD strain, and thus that both proteins are probably not involved during TorA maturation process as is TorDec (Fig. 4b). Moreover, as a possible interaction has been described between DmsD and TorA (Oresnik et al., 2001), we investigated this in a torD mutant where the dmsD gene was deleted. The absence of DmsD in this strain (LCB515) did not decrease the level of TorA, indicating that DmsD did not partially complement the absence of TorDec and thus was not responsible for the remaining TorA maturation in a torD background (Fig. 4). These results strongly suggest that although these proteins are part of the same family of chaperones, they possess a high specificity toward their partners.



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Fig. 4. In vivo effect of TorD homologues on apoTorA maturation. (a) The TMAO reductase activity was measured in the soluble fraction of strains LCB641, LCB515 and LCB641 harbouring either pJFTorD, pJFDmsD or pJFYcdY. The enzymic activities were expressed as a percentage of that observed in strain LCB641 harbouring plasmid pJFTorD. Error bars corresponding to standard deviations are indicated. (b) Quantification of protein TorA in the various strains. To determine the amount of TMAO reductase present in the extracts, the same fractions as in (a) (60 µg in each well) were submitted to rocket immunoelectrophoresis on an agarose plate containing TorA antibodies.

 
Although TorD homologues did not enhance apoTorA maturation, we wondered whether members of the same family could interfere with TorDec, lowering its activity. To test this, in vitro assays were performed by mixing TorDec (2·1 µM) with DmsD, YcdY or TorDsm (5 µM). In all three samples, the TMAO reductase activity decreased slightly but significantly compared to that measured in a sample containing TorDec alone as a chaperone (Fig. 3). As a control, BSA was added with TorDec in the sample; no change of the TMAO reductase activity level was observed. Therefore, DmsD, YcdY and TorDsm, which belong to three distinct types of the TorD family, interfere with TorDec, hampering its action. These results, together with the fact that DmsD is involved in the biogenesis of DmsA and that TorDsm interacts with TorAsm, suggest a similar mechanism of action of the members of the TorD family (Ray et al., 2003; Tranier et al., 2002).

The effect of TorDec is independent of the signal sequence of TorA
To determine if the signal sequence of apoTorA plays a role during the maturation process of the apoenzyme mediated by TorDec, we constructed a plasmid allowing the production of 'TorA (or apo'TorA in a mo mutant strain), a TorA form devoid of the signal sequence. The N-terminal extremity of purified apo'TorA (AQAAT) was checked by sequencing. The activation of apo'TorA in the presence of TorDec and of the molybdenum cofactor source clearly shows that the absence of the signal sequence does not hamper the action of TorDec during TorA maturation. Indeed, no significant difference was measured in the level of activation of apoTorA and apo'TorA (Fig. 3). DmsD was reported to bind to the signal sequence of TorA to target it to the Tat machinery (Oresnick et al., 2001; Ray et al. 2003). To test whether DmsD still interferes during the in vitro maturation of apo'TorA in the presence of TorDec, we performed an assay containing apo'TorA, the molybdenum cofactor source, TorDec (2·1 µM) and DmsD (5 µM). The maturation level was lower in the presence of DmsD than in its absence, showing that the inhibitory effect of DmsD is independent of the TorA signal sequence. The same conclusion could be drawn when the experiment was performed with YcdY or TorDsm.

TorDec cannot substitute for DmsD
To determine whether the high level of specificity between TorDec and TorA was a common feature of the TorD family proteins toward their cognate enzymes, we tested whether TorDec could replace DmsD during the maturation of DmsA. It was recently shown that a dmsD mutant strain cannot grow anaerobically on glycerol minimal medium supplemented with DMSO (Ray et al., 2003). As expected, the LCB514 strain ({Delta}dmsD) was unable to grow on DMSO-containing medium (Fig. 5a). Moreover, the presence of the dmsD gene in trans (LCB514/pJFDmsD) restored the wild-type phenotype. In contrast, when TorDec was produced in this strain (LCB514/pJFTorD), no growth increase was observed compared to the mutant strain (LCB514) (Fig. 5a). The absence of complementation by TorDec was confirmed using extracts of the same strains grown anaerobically on rich medium. After electrophoresis of the samples, the gel was stained for DMSO reductase activity; this was observed only in the lanes of DmsD-producing extracts (Fig. 5b). This point indicates that the action of DmsD toward the DMSO reductase complex cannot be carried out by TorDec.



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Fig. 5. TorD plays no role in DMSO reductase biogenesis. (a) Anaerobic growth profiles of E. coli strains MC4100 (black squares), LCB514 (black triangles) and LCB514 carrying either plasmid pJFDmsD (white squares) or plasmid pJFTorD (white triangles). Strains were grown in MSM with DMSO (28 mM). Growth was monitored at 600 nm. Data are typical of at least three independent experiments. (b) In-gel DMSO reductase activity staining. The membrane fractions (40 µg) of the same strains as in (a) but grown anaerobically in LB medium, were loaded on an SDS/10 % polyacrylamide gel. After electrophoresis, the bands possessing DMSO reductase activity were revealed using reduced methyl viologen and DMSO.

 
Conclusion
From this study, it appears that there is a large family of TorD homologous proteins which are structurally related. The members of this family can be classified into four types, where three appear genetically connected to the three types of the molybdoenzymes of the DMSO reductase family. The fact that both the DMSO reductase family and the TorD chaperone family present a similar phylogenetic organization strongly supports the idea of a co-evolution of the molybdoenzymes and of their partner chaperones. This might also explain the high specificity of the chaperones that are devoted to the maturation of their partner, and thus cannot replace each other. It will be important to confirm the direct involvement of the homologues of TorD in the appropriate folding of their partners and in the molybdenum cofactor insertion, and to define the role of the members of type IV, whose encoding genes appear to be isolated on the bacterial genomes.


   ACKNOWLEDGEMENTS
 
We gratefully thank M. Ansaldi, M.-T. Guidici-Orticoni, C. Jourlin-Castelli and W. Nitschke for fruitful discussions. We also thank Kevin Copp for reviewing the manuscript, L. Théraulaz and D. Redelberger for technical assistance, and R. Lebrun for N-terminal sequencing. This work was supported by grants from the Centre National de la Recherche Scientifique and the Université de la Méditerranée. M . I. was supported by an MRT fellowship.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Adler, A. J., Greenfield, N. J. & Fasman, G. D. (1973). Circular dichroism and optical rotatory dispersion of proteins and polypeptides. Methods Enzymol 27, 675–735.[Medline]

Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef][Medline]

Berks, B. C., Sargent, F. & Palmer, T. (2000). The Tat protein export pathway. Mol Microbiol 35, 260–274.[CrossRef][Medline]

Chung, C. T. & Miller, R. H. (1988). A rapid and convenient method for the preparation and storage of competent bacterial cells. Nucleic Acids Res 16, 3580.[Medline]

Czjzek, M., Dos Santos, J. P., Pommier, J., Giordano, G., Méjean, V. & Haser, R. (1998). Crystal structure of oxidized trimethylamine N-oxide reductase from Shewanella massilia at 2·5 Å resolution. J Mol Biol 284, 435–447.[CrossRef][Medline]

Datsenko, K. A. & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 6640–6645.[Abstract/Free Full Text]

Dos Santos, J. P., Iobbi-Nivol, C., Couillault, C., Giordano, G. & Méjean, V. (1998). Molecular analysis of the trimethylamine N-oxide (TMAO) reductase respiratory system from a Shewanella species. J Mol Biol 284, 421–433.[CrossRef][Medline]

Fürste, J. P., Pansegrau, W., Frank, R., Blocker, H., Scholz, P., Bagdasarian, M. & Lanka, E. (1986). Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene 48, 119–131.[CrossRef][Medline]

Gon, S., Patte, J. C., Méjean, V. & Iobbi-Nivol, C. (2000). The torYZ (yecKbisZ) operon encodes a third respiratory trimethylamine N-oxide (TMAO) reductase in Escherichia coli. J Bacteriol 182, 5779–5786.[Abstract/Free Full Text]

Gon, S., Giudici-Orticoni, M. T., Méjean, V. & Iobbi-Nivol, C. (2001). Electron transfer and binding of the c-type cytochrome TorC to the trimethylamine N-oxide reductase in Escherichia coli. J Biol Chem 276, 11545–11551.[Abstract/Free Full Text]

Hille, R. (1996). The mononuclear molybdenum enzymes. Chemical Reviews 96, 2757–2816.[CrossRef][Medline]

Ilbert, M., Méjean, V., Giudici-Orticoni, M. T., Samama, J. P. & Iobbi-Nivol, C. (2003). Involvement of a mate chaperone (TorD) in the maturation pathway of molybdoenzyme TorA. J Biol Chem 278, 28787–28792.[Abstract/Free Full Text]

Iobbi-Nivol, C., Pommier, J., Simala-Grant, J., Méjean, V. & Giordano, G. (1996). High substrate specificity and induction characteristics of trimethylamine-N-oxide reductase of Escherichia coli. Biochim Biophys Acta 1294, 77–82.[Medline]

Jourlin, C., Ansaldi, M. & Méjean, V. (1997). Transphosphorylation of the TorR response regulator requires the three phosphorylation sites of the TorS unorthodox sensor in Escherichia coli. J Mol Biol 267, 770–777.[CrossRef][Medline]

McAlpine, A. S., McEwan, A. G., Shaw, A. L. & Bailey, S. (1997). Molybdenum active centre of DMSO reductase from Rhodobacter capsulatus: crystal structure of the oxidized enzyme at 1·82 Å resolution. J Biol Inorg Chem 2, 690–701.[CrossRef]

McDevitt, C. A., Hugenholtz, P., Hanson, G. R. & McEwan, A. G. (2002). Molecular analysis of dimethyl sulphide dehydrogenase from Rhodovulum sulfidophilum: its place in the dimethyl sulphoxide reductase family of microbial molybdopterin-containing enzymes. Mol Microbiol 44, 1575–1587.[CrossRef][Medline]

Méjean, V., Iobbi-Nivol, C., Lepelletier, M., Giordano, G., Chippaux, M. & Pascal, M. C. (1994). TMAO anaerobic respiration in Escherichia coli: involvement of the tor operon. Mol Microbiol 11, 1169–1179.[Medline]

Mouncey, N. J., Choudhary, M. & Kaplan, S. (1997). Characterization of genes encoding dimethyl sulfoxide reductase of Rhodobacter sphaeroides 2.4.1T: an essential metabolic gene function encoded on chromosome II. J Bacteriol 179, 7617–7624.[Abstract]

Oresnik, I. J., Ladner, C. L. & Turner, R. J. (2001). Identification of a twin-arginine leader-binding protein. Mol Microbiol 40, 323–331.[CrossRef][Medline]

Papish, A. L., Ladner, C. L. & Turner, R. J. (2003). The twin-arginine leader-binding protein, DmsD, interacts with the TatB and TatC subunits of the Escherichia coli twin-arginine translocase. J Biol Chem 278, 32501–32506.[Abstract/Free Full Text]

Pommier, J., Méjean, V., Giordano, G. & Iobbi-Nivol, C. (1998). TorD, a cytoplasmic chaperone that interacts with the unfolded trimethylamine N-oxide reductase enzyme (TorA) in Escherichia coli. J Biol Chem 273, 16615–16620.[Abstract/Free Full Text]

Rajagopalan, K. V. & Johnson, J. L. (1992). The pterin molybdenum cofactors. J Biol Chem 267, 10199–10202.[Free Full Text]

Ray, N., Oates, J., Turner, R. J. & Robinson, C. (2003). DmsD is required for the biogenesis of DMSO reductase in Escherichia coli but not for the interaction of the DmsA signal peptide with the Tat apparatus. FEBS Lett 534, 156–160.[CrossRef][Medline]

Richardson, D. J. (2000). Bacterial respiration: a flexible process for a changing environment. Microbiology 146, 551–571.[Free Full Text]

Santini, C. L., Ize, B., Chanal, A., Muller, M., Giordano, G. & Wu, L. F. (1998). A novel sec-independent periplasmic protein translocation pathway in Escherichia coli. EMBO J 17, 101–112.[Abstract/Free Full Text]

Sargent, F., Berks, B. C. & Palmer, T. (2002). Assembly of membrane-bound respiratory complexes by the Tat protein-transport system. Arch Microbiol 178, 77–84.[CrossRef][Medline]

Schindelin, H., Kisker, C., Hilton, J., Rajagopalan, K. V. & Rees, D. C. (1996). Crystal structure of DMSO reductase: redox-linked changes in molybdopterin coordination. Science 272, 1615–1621.[Abstract]

Schneider, F., Lowe, J., Huber, R., Schindelin, H., Kisker, C. & Knablein, J. (1996). Crystal structure of dimethyl sulfoxide reductase from Rhodobacter capsulatus at 1·88 Å resolution. J Mol Biol 263, 53–69.[CrossRef][Medline]

Shaw, A. L., Leimkuhler, S., Klipp, W., Hanson, G. R. & McEwan, A. G. (1999). Mutational analysis of the dimethylsulfoxide respiratory (dor) operon of Rhodobacter capsulatus. Microbiology 145, 1409–1420.[Abstract]

Simala-Grant, J. L. & Weiner, J. H. (1996). Kinetic analysis and substrate specificity of Escherichia coli dimethyl sulfoxide reductase. Microbiology 142, 3231–3239.[Abstract]

Tranier, S., Mortier-Barrière, I., Ilbert, M., Birck, C., Iobbi-Nivol, C., Méjean, V. & Samama, J. P. (2002). Characterization and multiple molecular forms of TorD from Shewanella massilia, the putative chaperone of the molybdoenzyme TorA. Protein Sci 11, 2148–2157.[Abstract/Free Full Text]

Tranier, S., Iobbi-Nivol, C., Mortier-Barrière, I., Birck, C., Ilbert, M., Méjean, V. & Samama, J. P. (2003). A novel protein fold and extreme domain swapping in the dimeric TorD chaperone from Shewanella massilia. Structure 11, 165–174.[CrossRef][Medline]

Weiner, J. H., Rothery, R. A., Sambasivarao, D. & Trieber, C. A. (1992). Molecular analysis of dimethylsulfoxide reductase: a complex iron-sulfur molybdoenzyme of Escherichia coli. Biochim Biophys Acta 1102, 1–18.[Medline]

Weiner, J. H., Bilous, P. T., Shaw, G. M., Lubitz, S. P., Frost, L., Thomas, G. H., Cole, J. A. & Turner, R. J. (1998). A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins. Cell 93, 93–101.[Medline]

Received 13 November 2004; revised 7 January 2004; accepted 7 January 2004.



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