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The Escherichia coli nap operon and constructs used in this study to investigate NapA–NapD interactions. (A) A cartoon representing the structure of the napFDAGHBC operon located at 46.5 min on the E. coli chromosome. The names of the protein products of the genes are given above the arrows. (B) An overexpression vector based on pQE80 (Qiagen) encoding full-length NapA and NapD with an N-terminal hexa His-tag (NapDNHis). The natural transcriptional and translational coupling between napD and napA is maintained. (C) A pQE80-based expression vector encoding NapDNHis and NapA lacking its entire Tat signal peptide comprising Lys2-Ala31 (NapAΔsp). (D) A pET15b-based expression vector (Novagen/Merck, Darmstadt, Germany) encoding a fusion between NapDNHis and NapA. The amino acid sequence of the linker is shown. (E) An overexpression vector based on pQE70 (Qiagen) encoding a C-terminally hexa His-tagged NapD (NapDCHis). (F) An overexpression vector based on pQE60 (Qiagen) encoding the mature sequence of maltose binding protein (MalE), fused to the NapA signal peptide via a polyasparagine linker and a factor Xa recognition sequence, and a C-terminal hexa His-tag.
Source publication
Escherichia coli is a Gram-negative bacterium that can use nitrate during anaerobic respiration. The catalytic subunit of the periplasmic nitrate reductase NapA contains two types of redox cofactor and is exported across the cytoplasmic membrane by the twin-arginine protein transport pathway. NapD is a small cytoplasmic protein that is essential fo...
Contexts in source publication
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... is a 90 kDa protein that binds a [4Fe-4S] cluster in addition to the molybdenum cofactor [7][8][9]. NapA is encoded, along with its periplasmic di-heme c-type cyto- chrome redox partner NapB, in the seven gene nap operon (Fig. 1A). NapA is exported to the periplasm in a folded form by the twin-arginine protein transport (Tat) pathway [10,11], which is a translocation system dedicated to the export of fully folded proteins. In E. coli approximately two-thirds of the 28 known Tat substrates bind one or more redox cofactors ...
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... for Nap activity. It has been shown to bind with a nanomolar dissociation constant to the isolated NapA Tat signal peptide [22,23]. Site-directed mutagenesis has indicated that NapD recognizes an epitope that straddles the n-and h-regions of the NapA signal peptide, including Arg6 and Lys10 that form part of the twin-arginine consen- sus motif (Fig. 1A) [23]. However, the precise role of NapD in the assembly of NapA remains unclear. To shed light on this process, in this work we have iso- lated and characterized a complex of the two proteins. Our results indicate that the proteins are present in 1 : 1 stoichiometry and that the NapA precursor has a substantial degree of folding ...
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... NapA signal peptide can be produced in isola- tion when genetically fused to maltose binding protein (MalE) [22,23]. Here, this system was modified such that serine residues at positions 4 and 24 of the NapA signal peptide were substituted for cysteine (Figs 2A and S1). Following purification, the resultant MalE- NapA SP S4/24C chimera was site-specifically labelled with S-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methylmethanesulfonothioate (MTSL), a thiol-specific labelling reagent that contains a nitroxide radical. ...
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... known about its interaction with the entire full-length NapA precursor protein. To this end, the two proteins were co-overpro- duced from a pQE80 expression vector. The cloning strategy maintained the natural translational coupling between the napDA genes whilst supplying the NapD protein with an N-terminal hexa-histidine affinity tag, NapD NHis (Fig. ...
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... The most likely explanation for this is that the larger complex is a dimeric form of the smal- ler NapD NHis /NapA complex. To corroborate this analysis, an artificial fusion protein was constructed where the C-terminus of NapD was genetically fused through an RSNLGIEGRPG linker sequence to the extreme N-terminus of the NapA Tat signal peptide (Fig. 1D). The N-terminus of NapD was supplied with a hexa-histidine tag to facilitate purification. The crude cell extract containing the overproduced fusion protein was applied to an IMAC column and the fusion protein was observed to elute as a broad peak (Fig. 4A,B). To estimate the solution molecular mass of this fusion pro- tein, SV AUC was ...
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... without its signal peptide does not stably interact with NapD To ascertain whether NapD is able to interact with NapA in the absence of the NapA twin-arginine signal peptide, a construct was designed where N-terminally His-tagged NapD could be co-overproduced with NapA lacking amino acids 2-31 of its signal peptide (hereafter termed NapA Dsp ; Fig. ...
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... supporting information may be found in the online version of this article at the publisher's web site: Fig. S1. The positions of the spin labels introduced into the NapD signal peptide. Fig. S2. PELDOR data of spin labelled MalE-NapA SP (S4R1, S24R1) in the absence and presence of NapD. Fig. S3. Comparison of the Tikhonov-derived distance distribution with a synthetic distance distribution gen- erated by molecular dynamics simulations on each ...
Citations
... The role of these genes in Campylobacter-related diseases pathogenesis include lpxA, a gene associated with lipid and glycolipid metabolism; napA, is a protein involved in the reduction of nitrate to nitrite; glnA, is the structural gene for glutamine synthetase, a protein responsible for adhesion and colonization; flaA gene, a marker for flagella formation, adhesion, motility, and enterocyte colonization [39,[47][48][49] ( Table 4). ...
... The presence of specific genes, such as tetO, cmeB, gyrA, 23 s rRNA, and genes encoding OXA beta-lactamase enzymes, was attributed to this. Mutations, genetic exchange, and drug abuse are considered the primary causes of emergence of Table 4 The role of virulence genes in Campylobacter diseases pathogenicity in Nigeria [39,47,48] S/N Virulence Genes Role in Disease Pathogenicity i lpxA This is associated with lipids and glycolipids metabolism ii napA It is a protein involve in the reduction of nitrate to nitrite ii glnA This is a structural gene for Glutamine Synthetase, a protein responsible for adhesion and colonization iv flaA It is a marker for flagella formation, adhesion, motility and enterocytes Colonization ...
Purpose of Review
Infectious diseases are serious threats to public health globally. Therefore, regional understanding of pathogens in Nigeria, their sources, common species, genetic diversity, and presence of resistance genes as in the genus Campylobacter which causes campylobacteriosis and related illnesses is important for strategizing control and prevention for public health improvement.
Recent Findings
Campylobacter species of common occurrence in Nigeria are Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Campylobacter lari, Campylobacter upsalienses, and Campylobacter hyointestinalis. They are found in poultry, pets, underground water, and humans. Some strains have genetic diversity with the formation of clones and sequence types (STs). Virulence genes such as lpxA, napA, glnA, and flaA have been reported among some species in the country. Antibiotic resistance among some of the species is due to tetO, cmeB, gyrA, and genes encoding OXA β-lactamase types (OXA-193, OXA-449, OXA-61, OXA-783 OXA-785, OXA-786, OXA-184, and OXA-461).
Summary
Regional control measures should be adopted in the control of Campylobacter species. Treatment of Campylobacter-related diseases in Nigeria should rely on antibiotic susceptibility tests to help with drug selection (reliable option) for successful treatment.
... In E. coli, most molybdoenzymes of the DMSO reductase family harbor a specific chaperone for Moco insertion such as NarJ for NarGHI (Blasco et al., 1998), NarW for NARZYV (Blasco et al., 1992), NapD for NapA (Dow et al., 2014), TorD for TorA (Genest et al., 2005(Genest et al., , 2006, and FdhD for FdhF (Thome et al., 2012), as well as FdnGHI and FdoGHI. Moco insertion into a target apo-protein has been extensively studied for the TMAO reductase enzyme in E. coli, where TorD functions as a chaperone for apo-TorA maturation (Genest et al., 2005). ...
Iron-sulfur clusters are essential enzyme cofactors. The most common and stable clusters are [2Fe-2S] and [4Fe-4S] that are found in nature. They are involved in crucial biological processes like respiration, gene regulation, protein translation, replication and DNA repair in prokaryotes and eukaryotes. In Escherichia coli, Fe-S clusters are essential for molybdenum cofactor (Moco) biosynthesis, which is a ubiquitous and highly conserved pathway. The first step of Moco biosynthesis is catalyzed by the MoaA protein to produce cyclic pyranopterin monophosphate (cPMP) from 5’GTP. MoaA is a [4Fe-4S] cluster containing radical S-adenosyl-L-methionine (SAM) enzyme. The focus of this study was to investigate Fe-S cluster insertion into MoaA under nitrate and TMAO respiratory conditions using E. coli as a model organism. Nitrate and TMAO respiration usually occur under anaerobic conditions, where oxygen is depleted. Under these conditions, E. coli uses nitrate and TMAO as terminal electron. Previous studies revealed that Fe-S cluster insertion is performed by Fe-S cluster carrier proteins. In E. coli, these proteins are known as A-type carrier proteins (ATC) by phylogenomic and genetic studies. So far, three of them have been characterized in detail in E. coli, namely IscA, SufA, and ErpA. This study shows that ErpA and IscA are involved in Fe-S cluster insertion into MoaA under nitrate and TMAO respiratory conditions. ErpA and IscA can partially replace each other in their role to provide [4Fe-4S] clusters for MoaA. SufA is not able to replace the functions of IscA or ErpA under nitrate respiratory conditions. Nitrate reductase is a molybdoenzyme that coordinates Moco and Fe-S clusters. Under nitrate respiratory conditions, the expression of nitrate reductase is significantly increased in E. coli. Nitrate reductase is encoded in narGHJI genes, the expression of which is regulated by the transcriptional regulator, fumarate and nitrate reduction (FNR). The activation of FNR under conditions of nitrate respiration requires one [4Fe-4S] cluster. In this part of the study, we analyzed the insertion of Fe-S cluster into FNR for the expression of narGHJI genes in E. coli. The results indicate that ErpA is essential for the FNR-dependent expression of the narGHJI genes, a role that can be replaced partially by IscA and SufA when they are produced sufficiently under the conditions tested. This observation suggests that ErpA is indirectly regulating nitrate reductase expression via inserting Fe-S clusters into FNR. Most molybdoenzymes are complex multi-subunit and multi-cofactor-containing enzymes that coordinate Fe-S clusters, which are functioning as electron transfer chains for catalysis. In E. coli, periplasmic aldehyde oxidoreductase (PaoAC) is a heterotrimeric molybdoenzyme that consists of flavin, two [2Fe-2S], one [4Fe-4S] cluster and Moco. In the last part of this study, we investigated the insertion of Fe-S clusters into E. coli periplasmic aldehyde oxidoreductase (PaoAC). The results show that SufA and ErpA are involved in inserting [4Fe-4S] and [2Fe-2S] clusters into PaoABC, respectively under aerobic respiratory conditions.
... If the above analysis is correct, then this surface, on the face of the C-terminal cap domain that interfaces with the body of the enzyme, is buried in the structure of the holoenzyme. In support of this model, the C-terminal cap domains of the apo forms of both E. coli trimethylamine-N-oxide reductase TorA [17] and E. coli periplasmic nitrate reductase NapA [18] in complex with their respective chaperones TorD and NapD (that recognize the proteins' N-terminal twin-Arg signal sequence that targets them to the periplasm) have been reported to assume an open position in readiness to accept the mature molybdenum cofactor. This indicates that the C-terminal cap domain is indeed able to adopt the type of open configuration that is proposed here. ...
Here, we report recent progress our laboratories have made in understanding the maturation and reaction mechanism of the cytosolic and NAD+-dependent formate dehydrogenase from Cupriavidus necator. Our recent work has established that the enzyme is fully capable of catalyzing the reverse of the physiological reaction, namely, the reduction of CO2 to formate using NADH as a source of reducing equivalents. The steady-state kinetic parameters in the forward and reverse directions are consistent with the expected Haldane relationship. The addition of an NADH-regenerating system consisting of glucose and glucose dehydrogenase increases the yield of formate approximately 10-fold. This work points to possible ways of optimizing the reverse of the enzyme’s physiological reaction with commercial potential as an effective means of CO2 remediation. New insight into the maturation of the enzyme comes from the recently reported structure of the FdhD sulfurase. In E. coli, FdhD transfers a catalytically essential sulfur to the maturing molybdenum cofactor prior to insertion into the apoenzyme of formate dehydrogenase FdhF, which has high sequence similarity to the molybdenum-containing domain of the C. necator FdsA. The FdhD structure suggests that the molybdenum cofactor may first be transferred from the sulfurase to the C-terminal cap domain of apo formate dehydrogenase, rather than being transferred directly to the body of the apoenzyme. Closing of the cap domain over the body of the enzymes delivers the Mo-cofactor into the active site, completing the maturation of formate dehydrogenase. The structural and kinetic characterization of the NADH reduction of the FdsBG subcomplex of the enzyme provides further insights in reversing of the formate dehydrogenase reaction. Most notably, we observe the transient formation of a neutral semiquinone FMNH·, a species that has not been observed previously with holoenzyme. After initial reduction of the FMN of FdsB by NADH to the hydroquinone (with a kred of 680 s−1 and Kd of 190 µM), one electron is rapidly transferred to the Fe2S2 cluster of FdsG, leaving FMNH·. The Fe4S4 cluster of FdsB does not become reduced in the process. These results provide insight into the function not only of the C. necator formate dehydrogenase but also of other members of the NADH dehydrogenase superfamily of enzymes to which it belongs.
... Interestingly, some complex Tat substrates have signal peptides that contain greatly extended n-regions prior to the twin-arginine motif (37). Such extensions are almost invariably found on substrates that bind redox cofactors and/or partner proteins prior to export, and they appear to serve as binding sites for dedicated chaperones that coordinate folding and assembly (38)(39)(40)(41)(42). FecR is distinct from these Tat substrates since it does not contain any redox cofactor, and its signal sequence n-region is considerably longer than other Tat signal peptide n-regions. ...
In Escherichia coli, citrate-mediated iron transport is a key non-heme pathway for the acquisition of iron. Binding of ferric citrate to the outer membrane protein FecA induces a signal cascade that ultimately activates the cytoplasmic sigma factor FecI, resulting in transcription of the fecABCDE ferric citrate transport genes. Central to this process is signal transduction mediated by the inner membrane protein, FecR. FecR spans the inner membrane through a single transmembrane helix, which is flanked by cytoplasmic and periplasmic-orientated moieties at the N- and C- terminus. The transmembrane helix of FecR resembles a twin-arginine signal sequence, and substitution of the paired arginine residues of the consensus motif decouples the FecR-FecI signal cascade, rendering the cells unable to activate transcription of the fec operon when grown on ferric citrate. Furthermore, fusion of beta-lactamase C-terminal to the FecR transmembrane helix results in translocation of the C-terminal domain that is dependent on the twin-arginine translocation (Tat) system. Our findings demonstrate that FecR belongs to a select group of bitopic inner membrane proteins that contain an internal twin arginine signal sequence.
Importance Iron is essential for nearly all living organisms due to its role in metabolic processes and as a cofactor for many enzymes. The FecRI signal transduction pathway regulates citrate-mediated iron import in many Gram-negative bacteria, including Escherichia coli . The interaction of FecR to outer membrane protein, FecA, and cytoplasmic anti-sigma factor, FecI, has been extensively studied. However, the mechanism by which FecR inserts into the membrane has not previously been reported. In this study, we demonstrate that targeting of FecR to the cytoplasmic membrane is dependent on the Tat system. As such, FecR represents a new class of bitopic Tat-dependent membrane proteins with an internal twin arginine signal sequence.
... These biochemical results are consistent with the GFP patterns in the protoplasts (Figures 2B, C). To corroborate this finding, we evaluated another TAT signal sequence of NapA (nitrate reductase) (Dow et al., 2014). The N-terminal 34 amino acids of the FA presequence were replaced with NapA[ Figure S1A). ...
Plants have two endosymbiotic organelles, chloroplast and mitochondrion. Although they have their own genomes, proteome assembly in these organelles depends on the import of proteins encoded by the nuclear genome. Previously, we elucidated the general design principles of chloroplast and mitochondrial targeting signals, transit peptide, and presequence, respectively, which are highly diverse in primary structure. Both targeting signals are composed of N-terminal specificity domain and C-terminal translocation domain. Especially, the N-terminal specificity domain of mitochondrial presequences contains multiple arginine residues and hydrophobic sequence motif. In this study we investigated whether the design principles of plant mitochondrial presequences can be applied to those in other eukaryotic species. We provide evidence that both presequences and import mechanisms are remarkably conserved throughout the species. In addition, we present evidence that the N-terminal specificity domain of presequence might have evolved from the bacterial TAT (twin-arginine translocation) signal sequence.
... Directly upstream of tatA2 in the same operon is cj0785 encoding a TAT chaperone or redox enzyme maturation protein (REMP; Turner et al., 2004) homologous to NapD in E. coli and other bacteria. REMPs are small cytoplasmic proteins that bind tightly to TAT signal peptides and which serve to co-ordinate cofactor insertion with translocation through the TAT system, thus preventing premature export (Dow et al., 2014;Jack et al., 2004). Despite using many different cofactor-containing TAT-dependent electron transport enzymes, C. jejuni appears to possess only one other REMP, namely FdhM, a TorD homologue encoded by cj1514c upstream of the formate dehydrogenase (fdh) operon, which we have shown is only required for Fdh activity (Hitchcock et al., 2010). ...
The food-borne zoonotic pathogen Campylobacter jejuni has complex electron transport chains required for growth in the host, many of which contain cofactored periplasmic enzymes localised by the twin-arginine translocase (TAT). We report here the identification of two paralogues of the TatA translocase component in strain NCTC 11168, encoded by cj1176 (tatA1) and cj0786 (tatA2). Deletion mutants constructed in either or both of the tatA1 and tatA2 genes displayed distinct growth and enzyme activity phenotypes. For sulphite oxidase (SorAB), the multicopper oxidase (CueO) and alkaline phosphatase (PhoX), complete dependency on TatA1 for correct periplasmic activity was observed. However, the activities of nitrate reductase (NapA), formate dehydrogenase (FdhA) and TMAO reductase (TorA), were significantly reduced in the tatA2 mutant. In contrast, the specific rate of fumarate reduction catalysed by the flavoprotein subunit of the methyl menaquinone fumarate reductase (MfrA) was similar in periplasmic fractions of both the tatA1 and tatA2 mutants and only the deletion of both genes abolished activity. Nevertheless, unprocessed MfrA accumulated in the periplasm of the tatA1 (but not tatA2) mutant, indicating aberrant signal peptide cleavage. Surprisingly, TatA2 lacks two conserved residues (Gln8 and Phe39) known to be essential in E. coli TatA and we suggest it is unable to function correctly in the absence of TatA1. Finally, only two TAT chaperones (FdhM and NapD) are encoded in strain NCTC 11168, which mutant studies confirmed are highly specific for formate dehydrogenase and nitrate reductase assembly respectively. Thus, other TAT substrates must use general chaperones in their biogenesis.
In prokaryotes, the role of Mo/W enzymes in physiology and bioenergetics is widely recognized. It is worth noting that the most diverse family of Mo/W enzymes is exclusive to prokaryotes, with the probable existence of several of them from the earliest forms of life on Earth. The structural organization of these enzymes, which often include additional redox centers, is as diverse as ever, as is their cellular localization. The most notable observation is the involvement of dedicated chaperones assisting with the assembly and acquisition of the metal centers, including Mo/W-bisPGD, one of the largest organic cofactors in nature. This review seeks to provide a new understanding and a unified model of Mo/W enzyme maturation.
Nitrous oxide reductase (N2OR) is the only known enzyme that can reduce the powerful greenhouse gas nitrous oxide (N2O) to harmless nitrogen at the final step of bacterial denitrification. To alleviate the N2O emission, emerging approaches aim at microbiome biotechnology. In this study, the genome sequence of facultative anaerobic bacteria Pseudomonas citronellolis WXP-4, which efficiently degrades N2O, was obtained by de novo sequencing for the first time, and then, four key reductase structure coding genes related to complete denitrification were identified. The single structural encoding gene nosZ with a length of 1914 bp from strain WXP-4 was cloned in Escherichia coli BL21(DE3), and the N2OR protein (76 kDa) was relatively highly efficiently expressed under the optimal inducing conditions of 1.0 mM IPTG, 5 h, and 30 °C. Denitrification experiment results confirmed that recombinant E. coli had strong denitrification ability and reduced 10 mg L-1 of N2O to N2 within 15 h under the optimal conditions of pH 7.0 and 40 °C, its corresponding N2O reduction rate was almost 2.3 times that of Alcaligenes denitrificans strain TB, but only 80% of that of wild strain WXP-4, meaning that nos gene cluster auxiliary gene deletion decreased the activity of N2OR. The 3D structure of N2OR predicted on the basis of sequence homology found that electron transfer center CuA had only five amino acid ligands, and the S2 of the catalytically active center CuZ only bound one CuI atom. The unique 3D structure was different from previous reports and may be closely related to the strong N2O reduction ability of strain WXP-4 and recombinant E. coli. The findings show a potential application of recombinant E. coli in alleviating the greenhouse effect and provide a new perspective for researching the relationship between structure and function of N2OR.
Bacteria which use nitrate as respiratory terminal acceptor convert it to ammonia via nitrite by the action of dissimilatory nitrate and nitrite reductases. E. coli has three nitrate reductases and two major nitrite reductases. NO3⁻ and NO2⁻ are transported across the cell membrane via specific membrane transporters. In this chapter, a two-component system regulating the expression of these enzymes and transporters has been discussed. Molecular basis of switching to other respiratory pathways using alternate terminal acceptors like fumarate, DMSO, etc., in absence of nitrate and nitrite has also been described.
