Sequence analysis of bacterial redox enzyme maturation proteins (REMPs)

The twin-arginine protein transport (Tat) system is a remarkable molecular machine dedicated to the translocation of fully folded proteins across energy-transducing membranes. Complex cofactor-containing Tat substrates acquire their cofactors prior to export, and substrate proteins actually require to be folded before transport can proceed. Thus, it is very likely that mechanisms exist to prevent wasteful export of immature Tat substrates or to curb competition between immature and mature substrates for the transporter. Here we assess the primary sequence relationships between the accessory proteins implicated in this process during assembly of key respiratory enzymes in the model prokaryote Escherichia coli. For each respiratory enzyme studied, a redox enzyme maturation protein (REMP) was assigned. The main finding from this review was the hitherto unexpected link between the Tat-linked REMP DmsD and the nitrate reductase biosynthetic protein NarJ. The evolutionary link between Tat transport and cofactor insertion processes is discussed.     

Biogenesis of a respiratory complex is orchestrated by a single accessory protein

The biogenesis of respiratory complexes is a multistep process that requires finely tuned coordination of subunit assembly, metal cofactor insertion, and membrane-anchoring events. The dissimilatory nitrate reductase of the bacterial anaerobic respiratory chain is a membrane-bound heterotrimeric complex nitrate reductase A (NarGHI) carrying no less than eight redox centers. Here, we identified different stable folding assembly intermediates of the nitrate reductase complex and analyzed their redox cofactor contents using electron paramagnetic resonance spectroscopy. Upon the absence of the accessory protein NarJ, a global defect in metal incorporation was revealed. In addition to the molybdenum cofactor, we show that NarJ is required for specific insertion of the proximal iron-sulfur cluster (FS0) within the soluble nitrate reductase (NarGH) catalytic dimer. Further, we establish that NarJ ensures complete maturation of the b-type cytochrome subunit NarI by a proper timing for membrane anchoring of the NarGH complex. Our findings demonstrate that NarJ has a multifunctional role by orchestrating both the maturation and the assembly steps.     

NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli

The formation of active membrane-bound nitrate reductase A in Escherichia coli requires the presence of three subunits, NarG, NarH and NarI, as well as a fourth protein, NarJ, that is not part of the active nitrate reductase. In narJ strains, both NarG and NarH subunits are associated in an unstable and inactive NarGH complex. A significant activation of this complex was observed in vitro after adding purified NarJ-6His polypeptide to the cell supernatant of a narJ strain. Once the apo-enzyme NarGHI of a narJ mutant has become anchored to the membrane via the NarI subunit, it cannot be reactivated by NarJ in vitro . NarJ protein specifically recognizes the catalytic NarG subunit. Fluorescence, electron paramagnetic resonance (EPR) spectroscopy and molybdenum quantification based on inductively coupled plasma emission spectroscopy (ICPES) clearly indicate that, in the absence of NarJ, no molybdenum cofactor is present in the NarGH complex. We propose that NarJ is a specific chaperone that binds to NarG and may thus keep it in an appropriate competent-open conformation for the molybdenum cofactor insertion to occur, resulting in a catalytically active enzyme. Upon insertion of the molybdenum cofactor into the apo-nitrate reductase, NarJ is then dissociated from the activated enzyme.     

NarJ chaperone binds on two distinct sites of the aponitrate reductase of Escherichia coli to coordinate molybdenum cofactor insertion and assembly

Understanding when and how metal cofactor insertion occurs into a multisubunit metalloenzyme is of fundamental importance. Molybdenum cofactor insertion is a tightly controlled process that involves specific interactions between the proteins that promote cofactor delivery, enzyme-specific chaperones, and the apoenzyme. In the assembly pathway of the multisubunit molybdoenzyme, membrane-bound nitrate reductase A from Escherichia coli, a NarJ-assisted molybdenum cofactor (Moco) insertion step, must precede membrane anchoring of the apoenzyme. Here, we have shown that the NarJ chaperone interacts at two distinct binding sites of the apoenzyme, one interfering with its membrane anchoring and another one being involved in molybdenum cofactor insertion. The presence of the two NarJ-binding sites within NarG is required to ensure productive formation of active nitrate reductase. Our findings supported the view that enzyme-specific chaperones play a central role in the biogenesis of multisubunit molybdoenzymes by coordinating subunits assembly and molybdenum cofactor insertion.     

Differential effects of a molybdopterin synthase sulfurylase (moeB) mutation on Escherichia coli molybdoenzyme maturation.

We have generated a chromosomal mutant of moeB (moeBA228T) that demonstrates limited molybdenum cofactor (molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD)) availability in Escherichia coli and have characterized its effect on the maturation and physiological function of two well-characterized respiratory molybdoenzymes: the membrane-bound dimethylsulfoxide (DMSO) reductase (DmsABC) and the membrane-bound nitrate reductase A (NarGHI). In the moeBA228T mutant strain, E. coli F36, anaerobic respiratory growth is possible on nitrate but not on DMSO, indicating that cofactor insertion occurs into NarGHI but not into DmsABC. Fluorescence analyses of cofactor availability indicate little detectable cofactor in the moeBA228T mutant compared with the wild-type, suggesting that NarGHI is able to scavenge limiting cofactor, whereas DmsABC is not. MoeB functions to sulfurylate MoaD, and in the structure of the MoeB-MoaD complex, Ala-228 is located in the interface region between the two proteins. This suggests that the moeBA228T mutation disrupts the interaction between MoeB and MoaD. In the case of DmsABC, despite the absence of cofactor, the twin-arginine signal sequence of DmsA is cleaved in the moeBA228T mutant, indicating that maturation of the holoenzyme is not cofactor-insertion dependent.     

Export of complex cofactor-containing proteins by the bacterial Tat pathway

The twin-arginine (Tat) protein translocase is a highly unusual protein transport machine that is dedicated to the movement of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat pathway by means of N-terminal signal peptides harbouring a distinctive twin-arginine motif. In the model organism Escherichia coli, many of the Tat substrates bind redox cofactors that are inserted into apo-proteins before they engage with the Tat machinery. Here we review recent advances in understanding the events involved in the coordination of cofactor insertion with the export process. Current models for Tat protein transport are also discussed.     

‘Come into the fold’: A comparative analysis of bacterial redox enzyme maturation protein members of the NarJ subfamily

Redox enzyme maturation proteins (REMPs) are system-specific chaperones required for the maturation of complex iron sulfur molybdoenzymes that are important for anaerobic respiration in bacteria. Although they perform similar biological roles, REMPs are strikingly different in terms of sequence, structure, systems biology, and type of terminal electron acceptor that it supports for growth. Here we critically dissect current knowledge pertaining to REMPs of the nitrate reductase delta superfamily, specifically recognized in Escherichia coli to include NarJ, NarW, TorD, DmsD, and YcdY, also referred to as the NarJ REMP subfamily. We show that NarJ subfamily members share sequence homology and similar structural features as revealed by alignments performed on structurally characterized REMPs. We include an updated phylogenetic analysis of subfamily members, justifying their classification in this subfamily. The structural and functional roles of each member are presented herein and these discussions suggest that although NarJ subfamily members are related in sequence and structure, each member demonstrates remarkable uniqueness, validating the concept of system-specific chaperones.     

DmsD,a Tat system specific chaperone,interacts with other general chaperones and proteins involved in the molybdenum cofactor biosynthesis

Many bacterial oxidoreductases depend on the Tat translocase for correct cell localization. Substrates for the Tat translocase possess twin-arginine leaders. System specific chaperones or redox enzyme maturation proteins (REMPs) are a group of proteins implicated in oxidoreductase maturation. DmsD is a REMP discovered in Escherichia coli, which interacts with the twin-arginine leader sequence of DmsA, the catalytic subunit of DMSO reductase. In this study, we identified several potential interacting partners of DmsD by using several in vitro protein–protein interaction screening approaches, including affinity chromatography, co-precipitation, and cross-linking. Candidate hits from these in vitro findings were analyzed by in vivo methods of bacterial two-hybrid (BACTH) and bimolecular fluorescence complementation (BiFC). From these data, DmsD was confirmed to interact with the general molecular chaperones DnaK, DnaJ, GrpE, GroEL, Tig and Ef-Tu. In addition, DmsD was also found to interact with proteins involved in the molybdenum cofactor biosynthesis pathway. Our data suggests that DmsD may play a role as a “node” in escorting its substrate through a cascade of chaperone assisted protein-folding maturation events.     

An essential role for the DnaK molecular chaperone in stabilizing over-expressed substrate proteins of the bacterial twin-arginine translocation pathway

All secreted proteins in Escherichia coli must be maintained in an export-competent state before translocation across the inner membrane. In the case of the Sec pathway, this function is carried out by the dedicated SecB chaperone and the general chaperones DnaK-DnaJ-GrpE and GroEL-GroES, whose job collectively is to render substrate proteins partially or entirely unfolded before engagement of the translocon. To determine whether these or other general molecular chaperones are similarly involved in the translocation of folded proteins through the twin-arginine translocation (Tat) system, we screened a collection of E. coli mutant strains for their ability to transport a green fluorescent protein (GFP) reporter through the Tat pathway. We found that the molecular chaperone DnaK was essential for cytoplasmic stability of GFP bearing an N-terminal Tat signal peptide, as well as for numerous other recombinantly expressed endogenous and heterologous Tat substrates. Interestingly, the stability conferred by DnaK did not require a fully functional Tat signal as substrates bearing translocation defective twin lysine substitutions in the consensus Tat motif were equally unstable in the absence of DnaK. These findings were corroborated by crosslinking experiments that revealed an in vivo association between DnaK and a truncated version of the Tat substrate trimethylamine N-oxide reductase (TorA502) bearing an RR or a KK signal peptide. Since TorA502 lacks nine molybdo-cofactor ligands essential for cofactor attachment, the involvement of DnaK is apparently independent of cofactor acquisition. Finally, we show that the stabilizing effects of DnaK can be exploited to increase the expression and translocation of Tat substrates under conditions where the substrate production level exceeds the capacity of the Tat translocase. This latter observation is expected to have important consequences for the use of the Tat system in biotechnology applications where high levels of periplasmic expression are desirable.     

The twin-arginine leader-binding protein,DmsD, interacts with the TatB and TatC subunits of the Escherichia coli twin-arginine translocase

The twin-arginine translocase (Tat) pathway is involved in the targeting and translocation of fully folded proteins to the inner membrane and periplasm of bacteria. Proteins that use this pathway contain a characteristic twin-arginine signal sequence, which interacts with the receptor complex formed by the TatBC subunits. Recently, the DmsD protein was discovered, which binds to the twin-arginine signal sequences of the anaerobic respiratory enzymes dimethylsulfoxide reductase (DmsABC) and trimethylamine N-oxide (TMAO) reductase. In this work, the targeting of DmsD within Escherichia coli was investigated. Using cell fractionation and Western blot analysis, DmsD is found to be associated with the inner membrane of wild-type E. coli and a dmsABC mutant E. coli under anaerobic conditions. In contrast, DmsD is predominantly found in the cytoplasmic fraction of a Delta tatABCDE strain, which suggests that DmsD interacts with the membrane-associated Tat complex. Under aerobic conditions DmsD was also found primarily in the cytoplasmic fraction of wild-type E. coli, suggesting that physiological conditions have a significant effect upon the targeting of DmsD to the inner membrane. Size exclusion chromatography data and membrane washing studies indicate that DmsD is interacting tightly with an integral membrane protein and not with the lipid component of the E. coli inner membrane. Additional investigation into the nature of this interaction revealed that the TatB and TatC subunits of the translocase are important for the interaction of DmsD with the E. coli inner membrane.     

The Rieske protein from Paracoccus denitrificans is inserted into the cytoplasmic membrane by the twin-arginine translocase

The Rieske [2Fe-2S] protein (ISP) is an essential subunit of cytochrome bc(1) complexes in mitochondrial and bacterial respiratory chains. Based on the presence of two consecutive arginines, it was argued that the ISP of Paracoccus denitrificans, a Gram-negative soil bacterium, is inserted into the cytoplasmic membrane via the twin-arginine translocation (Tat) pathway. Here, we provide experimental evidence that membrane integration of the bacterial ISP indeed relies on the Tat translocon. We show that targeting of the ISP depends on the twin-arginine motif. A strict requirement is established particularly for the second arginine residue (R16); conservative replacement of the first arginine (R15K) still permits substantial ISP transport. Comparative sequence analysis reveals characteristics common to Tat signal peptides in several bacterial ISPs; however, there are distinctive features relating to the fact that the presumed ISP Tat signal simultaneously serves as a membrane anchor. These differences include an elevated hydrophobicity of the h-region compared with generic Tat signals and the absence of an otherwise well-conserved '+5'-consensus motif lysine residue. Substitution of the +5 lysine (Y20K) compromises ISP export and/or cytochrome bc(1) stability to some extent and points to a specific role for this deviation from the canonical Tat motif. EPR spectroscopy confirms cytosolic insertion of the [2Fe-2S] cofactor. Mutation of an essential cofactor binding residue (C152S) decreases the ISP membrane levels, possibly indicating that cofactor insertion is a prerequisite for efficient translocation along the Tat pathway.     

Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli

The Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide. The mobB gene is not essential for molybdenum cofactor biosynthesis because a deletion of both mob genes can be fully complemented by just mobA. Inactive nitrate reductase, purified from a mob strain, can be activated in vitro by incubation with protein FA (the mobA gene product), GTP, MgCl₂, and a further protein fraction, factor X. Factor X activity is present in strains that lack MobB, indicating that it is not an essential component of factor X, but over-expression of MobB increases the level of factor X. MobB, therefore, can participate in nitrate reductase activation. The narJ protein is not a component of mature nitrate reductase but narJ mutants cannot express active nitrate reductase A. Extracts from narJ strains are unable to support the in vitro activation of purified mob nitrate reductase: they lack factor X activity. Although the mob gene products are necessary for the biosynthesis of all E. coli molybdoenzymes as a result of their requirement for molybdopterin guanine dinucleotide, NarJ action is specific for nitrate reductase A. The inactive nitrate reductase A derivative in a narJ strain can be activated in vitro following incubation with cell extracts containing the narJ protein. NarJ acts to activate nitrate reductase after molybdenum cofactor biosynthesis is complete.     

Overlapping transport and chaperone-binding functions within a bacterial twin-arginine signal peptide

The twin-arginine translocation (Tat) pathway is a protein targeting system present in many prokaryotes. The physiological role of the Tat pathway is the transmembrane translocation of fully-folded proteins, which are targeted by N-terminal signal peptides bearing conserved SRRxFLK 'twin-arginine' amino acid motifs. In Escherichia coli the majority of Tat targeted proteins bind redox cofactors and it is important that only mature, cofactor-loaded precursors are presented for export. Cellular processes have been unearthed that sequence these events, for example the signal peptide of the periplasmic nitrate reductase (NapA) is bound by a cytoplasmic chaperone (NapD) that is thought to regulate assembly and export of the enzyme. In this work, genetic, biophysical and structural approaches were taken to dissect the interaction between NapD and the NapA signal peptide. A NapD binding epitope was identified towards the N-terminus of the signal peptide, which overlapped significantly with the twin-arginine targeting motif. NMR spectroscopy revealed that the signal peptide adopted a α-helical conformation when bound by NapD, and substitution of single residues within the NapA signal peptide was sufficient to disrupt the interaction. This work provides an increased level of understanding of signal peptide function on the bacterial Tat pathway.     

Identification of residues in DmsD for twin-arginine leader peptide binding, defined through random and bioinformatics-directed mutagenesis

The twin-arginine translocase (Tat) system is used for the targeting and translocation of folded proteins across the cell membrane of most bacteria. Substrates of this system contain a conserved "twin-arginine" (RR) motif within their signal/leader peptide sequence. Many Tat substrates have their own system-specific chaperone called redox enzyme maturation proteins (REMPs). Here, we study the binding of DmsD, the REMP for dimethyl sulfoxide reductase in Escherichia coli, toward the RR-containing leader peptide of the catalytic subunit DmsA. We have used a multipronged approach targeted at the amino acid sequence of DmsD to define residues and regions important for recognition of the DmsA leader sequence. Residues identified through bioinformatics and THEMATICS analysis were mutated using site-directed mutagenesis. These DmsD residue variants were purified and screened with an in vitro dot-blot far-Western assay to analyze the binding to the DmsA leader sequence. Degenerative polymerase chain reaction was also used to produce a bank of random DmsD amino acid mutants, which were then screened by an in vivo bacterial two-hybrid assay. Using this hybrid method, each DmsD variant was classified into one of three groups based on their degree of interaction with the DmsA leader (none, weak, and moderate). The data from both the in vitro and in vivo analyses were then applied to a model structure of DmsD based on the crystal structure of the Salmonella typhimurium homologue. Our results illustrate the positions of important DmsD residues involved in binding the DmsA leader peptide and identify a "hot pocket" of residues important for leader binding on the structure of DmsD.     

Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli

The twin-arginine translocation (Tat) system targets cofactor-containing proteins across the Escherichia coli cytoplasmic membrane via distinct signal peptides bearing a twin-arginine motif. In this study, we have analysed the mechanism and capabilities of the E. coli Tat system using green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). Fractionation studies and fluorescence measurements demonstrate that GFP is exported to the periplasm where it is fully active. Export is almost totally blocked in tat deletion mutants, indicating that the observed export in wild-type cells occurs predominantly, if not exclusively, by the Tat pathway. Imaging studies reveal a halo of fluorescence in wild-type cells corresponding to the exported periplasmic form; the GFP is distributed uniformly throughout the cytoplasm in a tat mutant. Because previous work has shown GFP to be incapable of folding in the periplasm, we propose that GFP is exported in a fully folded, active state. These data also show for the first time that heterologous proteins can be exported in an active form by the Tat pathway.     

A dual functional redox enzyme maturation protein for respiratory and assimilatory nitrate reductases in bacteria

Nitrate is available to microbes in many environments due to sustained use of inorganic fertilizers on agricultural soils and many bacterial and archaeal lineages have the capacity to express respiratory (Nar) and assimilatory (Nas) nitrate reductases to utilize this abundant respiratory substrate and nutrient for growth. Here, we show that in the denitrifying bacterium Paracoccus denitrificans, NarJ serves as a chaperone for both the anaerobic respiratory nitrate reductase (NarG) and the assimilatory nitrate reductase (NasC), the latter of which is active during both aerobic and anaerobic nitrate assimilation. Bioinformatic analysis suggests that the potential for this previously unrecognized role for NarJ in functional maturation of other cytoplasmic molybdenum‐dependent nitrate reductases may be phylogenetically widespread as many bacteria contain both Nar and Nas systems.     

A genetic analysis of in vivo selenate reduction by <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium LT2 and <Emphasis Type="Italic">Escherichia coli</Emphasis> K12

The twin-arginine transport (Tat) system is dedicated to the translocation of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat system by signal peptides containing a twin-arginine motif. In Salmonella enterica serovar Typhimurium and Escherichia coli many Tat substrates are known or predicted to bind a molybdenum cofactor in the cytoplasm prior to export. In the case of N- and S-oxide reductases, co-ordination of molybdenum cofactor insertion with protein export involves a ‘Tat proofreading’ process where chaperones of the TorD family bind the signal peptides, thus preventing premature export. Here, a genetic approach was taken to determine factors required for selenate reductase activity in Salmonella and E. coli. It is reported for both biological systems that an active Tat translocase and a TorD-like chaperone (DmsD) are required for complete in vivo reduction of selenate to elemental red selenium. Further mutagenesis and in vitro biophysical experiments implicate the Salmonella ynfE gene product, and the E. coli YnfE and YnfF proteins, as putative Tat-targeted selenate reductases.     

The narJ gene product is required for biogenesis of respiratory nitrate reductase in Escherichia coli.

Respiratory nitrate reductase purified from the cell membrane of Escherichia coli is composed of three subunits, alpha, beta, and gamma, which are encoded, respectively, by the narG, narH, and narI genes of the narGHJI operon. The product of the narJ gene was deduced previously to be a highly charged, acidic protein which was not found to be associated with any of the purified preparations of the enzyme and which, in studies with putative narJ mutants, did not appear to be absolutely required for formation of the membrane-bound enzyme. To test this latter hypothesis, the narJ gene was disrupted in a plasmid which contained the complete narGHJI operon, and the operon was expressed in a narG::Tn10 insertion mutant. The chromosomal copy of the narJ gene of a wild-type strain was also replaced by the disrupted narJ gene. In both cases, when nar operon expression was induced, the alpha and beta subunits accumulated in a form which expressed only very low activity with either reduced methyl viologen (MVH) or formate as electron donors, although an alpha-beta complex separated from the gamma subunit is known to catalyze full MVH-linked activity but not the formate-linked activity associated with the membrane-bound complex. The low-activity forms of the alpha and beta subunits also accumulated in the absence of the NarJ protein when the gamma subunit (NarI) was provided from a multicopy plasmid, indicating that NarJ is essential for the formation of the active, membrane-bound complex. When both NarJ and NarI were provided from a plasmid in the narJ mutant, fully active, membrane-bound activity was formed. When NarJ only was provided from a plasmid in the narJ mutant, a cytosolic form of the alpha and beta subunits, which expressed significantly increased levels of the MVH-dependent activity, accumulated, and the alpha subunit appeared to be protected from the proteolytic clipping which occurred in the absence of NarJ. We conclude that NarJ is indispensible for the biogenesis of membrane-bound nitrate reductase and is involved either in the maturation of a soluble, active alpha-beta complex or in facilitating the interaction of the complex with the membrane-bound gamma subunit.     

A genetic screen for suppressors of Escherichia coli Tat signal peptide mutations establishes a critical role for the second arginine within the twin-arginine motif

The Escherichia coli Tat protein export pathway transports folded proteins synthesized with N-terminal twin-arginine signal peptides. Twin-arginine signal sequences contain a conserved SRRxFLK "twin-arginine" amino acid sequence motif which is required for protein export by the Tat pathway. The E. coli trimethylamine N-oxide reductase (TorA) is a Tat-dependent periplasmic molybdoenzyme that facilitates anaerobic respiration with trimethylamine N-oxide as terminal electron acceptor. Here, we describe mutant strains constructed with modified TorA twin-arginine signal peptides. Substitution of the second arginine residue of the TorA signal peptide twin-arginine motif with either lysine or aspartate, or the simultaneous substitution of both arginines with lysine residues, completely abolished export. In each case, the now cytoplasmically localised TorA retained full enzymatic activity with the artificial electron donor benzyl viologen. However, the mutant strains were incapable of anaerobic growth with trimethylamine N-oxide and the non-fermentable carbon-source glycerol. The growth phenotype of the mutant strains was exploited in a genetic screen with the aim of identifying second-site suppressor mutations that allowed export of the modified TorA precursors.