Thioredoxin reductase from Bacillus cereus exhibits distinct reduction and NADPH-binding properties.
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ABSTRACT:
SUBMITTER: Shoor M
PROVIDER: S-EPMC8564101 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Plant chloroplasts have versatile thioredoxin systems including two thioredoxin reductases and multiple types of thioredoxins. Plastid-localized NADPH-dependent thioredoxin reductase (NTRC) contains both reductase (NTRd) and thioredoxin (TRXd) domains in a single polypeptide and forms homodimers. To study the action of NTRC and NTRC domains in vivo, we have complemented the ntrc knockout line of Arabidopsis with the wild type and full-length NTRC genes, in which 2-Cys motifs either in NTRd, or in TRXd were inactivated. The ntrc line was also transformed either with the truncated NTRd or TRXd alone. Overexpression of wild-type NTRC promoted plant growth by increasing leaf size and biomass yield of the rosettes. Complementation of the ntrc line with the full-length NTRC gene containing an active reductase but an inactive TRXd, or vice versa, recovered wild-type chloroplast phenotype and, partly, rosette biomass production, indicating that the NTRC domains are capable of interacting with other chloroplast thioredoxin systems. Overexpression of truncated NTRd or TRXd in ntrc background did not restore wild-type phenotype. Modeling of the three-dimensional structure of the NTRC dimer indicates extensive interactions between the NTR domains and the TRX domains further stabilize the dimeric structure. The long linker region between the NTRd and TRXd, however, allows flexibility for the position of the TRXd in the dimer. Supplementation of the TRXd in the NTRC homodimer model by free chloroplast thioredoxins indicated that TRXf is the most likely partner to interact with NTRC. We propose that overexpression of NTRC promotes plant biomass yield both directly by stimulation of chloroplast biosynthetic and protective pathways controlled by NTRC and indirectly via free chloroplast thioredoxins. Our data indicate that overexpression of chloroplast thiol redox-regulator has a potential to increase biofuel yield in plant and algal species suitable for sustainable bioenergy production.
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Project description:Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR, HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 A resolution and refined to an R(cryst) of 19.0% and an R(free) of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25 degrees and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described previously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR-Trx interactions mediate the FO to FR transformation.
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Project description:Thioredoxin, thioredoxin reductase and NADPH form the thioredoxin system and are the major cellular protein disulphide reductase. We report here that Escherichia coli thioredoxin and thioredoxin reductase interact with unfolded and denatured proteins, in a manner similar to that of molecular chaperones that are involved in protein folding and protein renaturation after stress. Thioredoxin and/or thioredoxin reductase promote the functional folding of citrate synthase and alpha-glucosidase after urea denaturation. They also promote the functional folding of the bacterial galactose receptor, a protein without any cysteines. Furthermore, redox cycling of thioredoxin/thioredoxin reductase in the presence of NADPH and cystine stimulates the renaturation of the galactose receptor, suggesting that the thioredoxin system functions like a redox-powered chaperone machine. Thioredoxin reductase prevents the aggregation of citrate synthase under heat-shock conditions. It forms complexes that are more stable than those formed by thioredoxin with several unfolded proteins such as reduced carboxymethyl alpha-lactalbumin and unfolded bovine pancreatic trypsin inhibitor. These results suggest that the thioredoxin system, in addition to its protein disulphide isomerase activity possesses chaperone-like properties, and that its thioredoxin reductase component plays a major role in this function.
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Project description:2-Cys peroxiredoxins (Prxs) play important roles in the protection of chloroplast proteins from oxidative damage. Arabidopsis NADPH-dependent thioredoxin reductase isotype C (AtNTRC) was identified as efficient electron donor for chloroplastic 2-Cys Prx-A. There are three isotypes (A, B, and C) of thioredoxin reductase (TrxR) in Arabidopsis. AtNTRA contains only TrxR domain, but AtNTRC consists of N-terminal TrxR and C-terminal thioredoxin (Trx) domains. AtNTRC has various oligomer structures, and Trx domain is important for chaperone activity. Our previous experimental study has reported that the hybrid protein (AtNTRA-(Trx-D)), which was a fusion of AtNTRA and Trx domain from AtNTRC, has formed variety of structures and shown strong chaperone activity. But, electron transfer mechanism was not detected at all. To find out the reason of this problem with structural basis, we performed two different molecular dynamics (MD) simulations on AtNTRC and AtNTRA-(Trx-D) proteins with same cofactors such as NADPH and flavin adenine dinucleotide (FAD) for 50 ns. Structural difference has found from superimposition of two structures that were taken relatively close to average structure. The main reason that AtNTRA-(Trx-D) cannot transfer the electron from TrxR domain to Trx domain is due to the difference of key catalytic residues in active site. The long distance between TrxR C153 and disulfide bond of Trx C387-C390 has been observed in AtNTRA-(Trx-D) because of following reasons: i) unstable and unfavorable interaction of the linker region, ii) shifted Trx domain, and iii) different or weak interface interaction of Trx domains. This study is one of the good examples for understanding the relationship between structure formation and reaction activity in hybrid protein. In addition, this study would be helpful for further study on the mechanism of electron transfer reaction in NADPH-dependent thioredoxin reductase proteins.
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Project description:The thioredoxin system plays a central role in the intracellular redox maintenance in the majority of cells. The canonical system consists of an NADPH-dependent thioredoxin reductase (TrxR) and thioredoxin (Trx), a disulfide reductase. Although Trx is encoded in almost all sequenced genomes of methanogens, its incorporation into their unique physiology is not well understood. Methanosarcina acetivorans contains a single TrxR (MaTrxR) and seven Trx (MaTrx1-MaTrx7) homologues. We previously showed that MaTrxR and at least MaTrx7 compose a functional NADPH-dependent thioredoxin system. Here, we report the characterization of all seven recombinant MaTrxs. MaTrx1, MaTrx3, MaTrx4 and MaTrx5 lack appreciable disulfide reductase activity, unlike previously characterized MaTrx2, MaTrx6 and MaTrx7. Enzyme assays demonstrated that, of the MaTrxs, only the reduction of disulfide-containing MaTrx7 is linked to the oxidation of reduced coenzymes. NADPH is shown to be supplied to the MaTrxR-MaTrx7 system through the oxidation of the primary methanogen electron carriers F420H2 and ferredoxin, indicating that it serves as a primary intracellular reducing system in M. acetivorans. Bioinformatic analyses also indicate that the majority of methanogens likely utilize an NADPH-dependent thioredoxin system. The remaining MaTrxs may have specialized functions. MaTrx1 and MaTrx3 exhibited thiol oxidase activity. MaTrx3 and MaTrx6 are targeted to the membrane of M. acetivorans and likely function in the formation and the reduction of disulfides in membrane and/or extracellular proteins, respectively. This work provides insight into the incorporation of Trx into the metabolism of methanogens, and this reveals that methanogens contain Trx homologues with alternative properties and activities.
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Project description:Tight control of cellular redox homeostasis is essential for protection against oxidative damage and for maintenance of normal metabolism as well as redox signaling events. Under oxidative stress conditions, the tripeptide glutathione can switch from its reduced form (GSH) to oxidized glutathione disulfide (GSSG), and thus, forms an important cellular redox buffer. GSSG is normally reduced to GSH by 2 glutathione reductase (GR) isoforms encoded in the Arabidopsis genome, cytosolic GR1 and GR2 dual-targeted to chloroplasts and mitochondria. Measurements of total GR activity in leaf extracts of wild-type and 2 gr1 deletion mutants revealed that approximately 65% of the total GR activity is attributed to GR1, whereas approximately 35% is contributed by GR2. Despite the lack of a large share in total GR activity, gr1 mutants do not show any informative phenotype, even under stress conditions, and thus, the physiological impact of GR1 remains obscure. To elucidate its role in plants, glutathione-specific redox-sensitive GFP was used to dynamically measure the glutathione redox potential (E(GSH)) in the cytosol. Using this tool, it is shown that E(GSH) in gr1 mutants is significantly shifted toward more oxidizing conditions. Surprisingly, dynamic reduction of GSSG formed during induced oxidative stress in gr1 mutants is still possible, although significantly delayed compared with wild-type plants. We infer that there is functional redundancy in this critical pathway. Integrated biochemical and genetic assays identify the NADPH-dependent thioredoxin system as a backup system for GR1. Deletion of both, NADPH-dependent thioredoxin reductase A and GR1, prevents survival due to a pollen lethal phenotype.
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Project description:In natural growth habitats, plants face constant, unpredictable changes in light conditions. To avoid damage to the photosynthetic apparatus on thylakoid membranes in chloroplasts, and to avoid wasteful reactions, it is crucial to maintain a redox balance both within the components of photosynthetic electron transfer chain and between the light reactions and stromal carbon metabolism under fluctuating light conditions. This requires coordinated function of the photoprotective and regulatory mechanisms, such as non-photochemical quenching (NPQ) and reversible redistribution of excitation energy between photosystem II (PSII) and photosystem I (PSI). In this paper, we show that the NADPH-dependent chloroplast thioredoxin system (NTRC) is involved in the control of the activation of these mechanisms. In plants with altered NTRC content, the strict correlation between lumenal pH and NPQ is partially lost. We propose that NTRC contributes to downregulation of a slow-relaxing constituent of NPQ, whose induction is independent of lumenal acidification. Additionally, overexpression of NTRC enhances the ability to adjust the excitation balance between PSII and PSI, and improves the ability to oxidize the electron transfer chain during changes in light conditions. Thiol regulation allows coupling of the electron transfer chain to the stromal redox state during these changes.
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Project description:Biological remediation of toxic oxygen-containing anions such as nitrate that are common in the environment is of great significance. Therefore, it is necessary to understand the specific role of nitrate and nitrite reductase in the bioremediation process. Bacillus cereus LJ01, which was isolated from traditional Chinese soybean paste, effectively degraded nitrite (such as NaNO2) at 0-15 mmol L-1 in LB medium. Moreover, the nitrite-degrading active substance (ASDN) was isolated and purified from B. cereus LJ01. The nitrite-degrading activity of nitrite reductase (named LJ01-NiR) was 4004.89 U mg-1. The gene encoding the assimilation of nitrite reductase in B. cereus LJ01 was cloned and overexpressed in E. coli. The purified recombinant LJ01-NiR has a wide range of activities under temperature (20-60 °C), pH (6.5-8.0) and metal ions (Fe3+, Fe2+, Cu2+, Mn2+, and Al3+). Kinetic parameters of LJ01-NiR, including the values of K m and V max were 1.38 mM and 2.00 μmol g-1 min-1, respectively. The results showed that LJ01-NiR could degrade nitrite with or without an electron donor. In addition, sequence analysis revealed that LJ01-NiR was a ferredoxin-dependent nitrite reductase given the presence of conserved [Fe4-S4] cluster and heme-binding domain. The nitrite ion binds to the LJ01-NiR active site by forming three hydrogen bonds with the residues ASN72, ALA133 and ASN140. Due to its high nitrite-degrading activity, LJ01-NiR could potentially be used for environmental pollution treatment.
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Project description:The gene encoding hemolysin II (HlyII) was amplified from Bacillus cereus genomic DNA and a truncated mutant, HlyII(DeltaCT), was constructed lacking the 94 amino acid extension at the C terminus. The proteins were produced in an E. coli cell-free in vitro transcription and translation system, and were shown to assemble into SDS-stable oligomers on rabbit erythrocyte membranes and liposomes. The hemolytic activity of HlyII was measured with rabbit erythrocytes yielding an HC(50) value of 1.64 ng mL(-1), which is over 15 times more potent than staphylococcal alpha-hemolysin. HlyII(DeltaCT) was about eight times less potent than HlyII in this assay. Limited proteolysis of the oligomers formed by HlyII and HlyII(DeltaCT) on red cell membranes showed that the C-terminal extension is sensitive to digestion, while HlyII(DeltaCT) is protease resistant and migrates with an electrophoretic mobility similar to that of digested HlyII. HlyII forms moderately anion selective, rectifying pores (I(+80)/I(-80) = 0.57, 1 M KCl, pH 7.4) in planar lipid bilayers of diphytanoylphosphatidylcholine with a unitary conductance of 637 pS (1 M KCl, 5 mM HEPES, pH 7.4) and exhibits no gating over a wide range of applied potentials (-160 to +160 mV). In addition, it was demonstrated that HlyII forms a homoheptameric pore by using gel shift electrophoresis aided by a genetically encoded oligoaspartate tag. Although they share limited primary sequence identity (30%), these data confirm that HlyII is a structural and functional homolog of staphylococcal alpha-hemolysin.
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