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Heat stress due to global warming adversely affects plant physiology and metabolism, significantly reducing agricultural productivity. Plants have evolved various adaptive mechanisms to cope with such stresses, involving a range of heat stress-responsive proteins. This study investigates the molecular functions and structural changes of OsTDX (Oryza sativa TPR repeat-containing thioredoxin) in rice under heat stress, focusing on its roles as a disulfide reductase and molecular chaperone. OsTDX, sharing a 52 % overall amino acid identity with AtTDX, predominantly forms high molecular weight (HMW) complexes under heat stress conditions. Functional assays revealed that OsTDX exhibited increased disulfide reductase activity in a dose-dependent manner and significantly enhanced holdase chaperone activity, particularly under specific heat stress conditions (60 °C). The structural shift from low molecular weight (LMW) to HMW forms was accompanied by increased hydrophobicity, as indicated by bis-ANS fluorescence intensity measurements. In conclusion, OsTDX exhibits dual functions as a disulfide reductase and a holdase chaperone, with its chaperone activity significantly enhanced under heat stress through structural changes to HMW complexes. These findings contribute to understand the molecular mechanisms of heat tolerance in rice and highlight the potential role of OsTDX in the development of heat-tolerant crops to address crop yield declines due to global warming. • OsTDX, which contains Trx and TPR domains, represents a disulfide reductase. • OsTDX undergoes structural changes by heat and acts as a molecular chaperone. • It can play an important role in solving crop yield declines due to global warming. [ABSTRACT FROM AUTHOR]

Many thioredoxin-h (Trx-h) proteins, cytosolic isotypes of Trxs, have been functionally characterized in plants; however, the physiological function of Arabidopsis Trx-h2, which harbors two active site cysteine (Cys) residues and an N-terminal extension peptide containing a fatty acid acylation site, remains unclear. In this study, we investigated the physiological function of Trx-h2 by performing several abiotic stress treatments using trx-h1–3 knockout mutant lines, and found that the reductase function of Trx-h2 is critical for cold resistance in Arabidopsis. Plants overexpressing Trx-h2 in the trx-h2 mutant background (Trx-h2OE/ trx-h2) showed strong cold tolerant phenotypes compared with Col-0 (wild type) and trx-h2 mutant plants. By contrast, Trx-h2(C/S)OE/ trx-h2 plants expressing a variant Trx-h2 protein, in which both active site Cys residues were substituted by serine (Ser) residues, showed high cold sensitivity, similar to trx-h2 plants. Moreover, cold-responsive (COR) genes were highly up-regulated in Trx-h2OE/ trx-h2 plants but not in trx-h2 and Trx-h2(C/S)OE/ trx-h2 plants under cold conditions. These results explicitly suggest that the cytosolic Trx-h2 protein relays the external cold stress signal to downstream cold defense signaling cascades through its protein disulfide reductase function. • Redox-dependent regulation of cold stress tolerance in plants. • Trx-h2 increases the expression of cold-responsive (COR) genes at low temperature. • Disulfide reductase function of Trx-h2 is critical for its role in cold tolerance. [ABSTRACT FROM AUTHOR]

Effective hydrolysis of feather waste by keratinase is an important way to develop high-quality alternative proteins for feed. It has been shown that the reduction of disulfide bonds is the key rate-limiting step in the hydrolysis of feathers by keratinases. However, the reductive activity of keratinase itself is low. In order to increase the reductive activity of the catalytic system, we screened endogenous disulfide reductase from Bacillus subtilis and constructed a synergistic catalytic system, KerZ1/yjbI-S2-P1, consisting of keratinase and disulfide reductase by co-expression optimization. The reductive activity and feather hydrolytic activity of the synergistic system were 5.67-fold and 5-fold higher than the original system. Feathers were hydrolyzed to a higher extent compared with the original system. The total amino acid and soluble protein concentrations in the products increased by 99% and 59%, respectively. This study provides a more environmentally friendly and efficient reduction capability for keratinase to hydrolysis waste feathers, and also demonstrates a new way of alternative protein feedstock mining. [ABSTRACT FROM AUTHOR]

The di‐copper center CuA is an essential metal cofactor in cytochrome oxidase (Cox) of mitochondria and many prokaryotes, mediating one‐electron transfer from cytochrome c to the site for oxygen reduction. CuA is located in subunit II (CoxB) of Cox and protrudes into the periplasm of Gram‐negative bacteria or the mitochondrial intermembrane space. How the two copper ions are brought together to build CoxB·CuA is the subject of this review. It had been known that the reductase TlpA and the metallochaperones ScoI and PcuC are required for CuA formation in bacteria, but the mechanism of copper transfer has emerged only recently for the Bradyrhizobium diazoefficiens system. It consists of the following steps: (a) TlpA keeps the active site cysteine pair of CoxB in its dithiol state as a prerequisite for metal insertion; (b) ScoI·Cu2+ rapidly forms a transient complex with apo‐CoxB; (c) PcuC, loaded with Cu1+ and Cu2+, dissociates this complex to CoxB·Cu2+, and a second PcuC·Cu1+·Cu2+ transfers Cu1+ to CoxB·Cu2+, yielding mature CoxB·CuA. Variants of this pathway might exist in other bacteria or mitochondria. [ABSTRACT FROM AUTHOR]

Pig hair keratin accumulates an abundance of potential protein and amino acids, which could be used for the source of amino acid feed. Due to the highly cross-linking by disulfide, hydrogen bonding and hydrophobic interations, Keratin is insoluble and not degraded by normal proteases such as pepsin, trypsin and papainl. Keratin degrading microbial strains can directly utilize keratin as carbon and nitorgen source, which can secret a variety of enzymes, such as keratinase, disulfide reductase. Currently, the kerain-biodegradation by microorganisms represents an alternative attractive method to improve the nutritional value of keratin wastes. In order to obtain the microorganisms with high pig-hair keratin degrading ability, the strains were isolated from environment soil by taking keratin as sole culture carbon and nitrogen source. The identification of Strain X-3 was studied and the keratin-degradation characteristics were further investigated from the aspects of thiolysis and enzymolysis. It was expected to provide theoretical guidance for the resue of feather waste resource. In this study, six strains with pig hair degrading ability were obtained, and strain X-3 could degrade pig hair keratin more effectively than other five strains. The degrading rate of pig hair keratin was up to 77.3% after 7 days fermentation, which was higher than that of the reported strains. The colonies of strain X-3 were irregular in shape, moist and rough on peptone beef medium. The cell of the strain was in the shape of rod, Gram positive and sporulating under the microscope. An aceession number MK182787 was obtained after the information of 16S rDNA sequence of strain X-3 was submitted to NCBI. The phylogenetic tree was constructed based on the sequence of 16S rDNA by the software Mega 5.0.The strain X-3 was identified as Bacillus coagulans on the basis of morphology, physiological characteristics and 16S rDNA sequence phylogenetic analysis. The wool, chicken feather, goose feather,duck feather keratin-degrading rate was 62.3%, 86.7%, 88.4%, 72.4%, respectively after 7 days fermentation. Bacillus coagulans X-3 was a broad spectrum keratin degrading strain because it showed efficient degrading properties to different keratins. The maximum of keratinase activity (84.1 U/mL) and reductase activity (23.8 U/mL) were detected during keratin degradation. High keratinase activity and disulfide reductase activity were detected during the fermentatin process and the keratin degrading rate changed relating to enzyme activity, which indicates enzyme plays important role in the process of keratin degradation. Sulfur in the keratins was transferred into sulfate, sulfite and thiol compounds in the degradation process. The sulfur in the keratin was mainly removed into the medium in the form of sulfate. The soluable protein, sulfate, sulfite and thiol content began to increase quickly accompanied the keratin degradation. However, further rearch will be required to determine the relationship of thiolysis and pig hair keratin degradation. Up to date, Bacillus coagulans has not been reported on the pig hair keratin degrading area, thus, we provide a new efficient keratin-degarding strain to utilize pig hair. Bacillus coagulans X-3 has a great potential roles in amino acid feedstock and environmental protection for its excellent keratin-degrading property and biological safety. [ABSTRACT FROM AUTHOR]

Aims: Living cells employ thioredoxin and glutaredoxin disulfide oxido-reductases to protect thiol groups in intracellular proteins. FrnE protein of Deinococcus radiodurans (drFrnE) is a disulfide oxido-reductase that is induced in response to Cd2+ exposure and is involved in cadmium and radiation tolerance. The aim of this study is to probe structure, function, and cellular localization of FrnE class of proteins. Results: Here, we show drFrnE as a novel cytoplasmic oxido-reductase that could be functional in eubacteria under conditions where thioredoxin/glutaredoxin systems are inhibited or absent. Crystal structure analysis of drFrnE reveals thioredoxin fold with an alpha helical insertion domain and a unique, flexible, and functionally important C-terminal tail. The C-tail harbors a novel 239-CX4C-244 motif that interacts with the active site 22-CXXC-25 motif. Crystal structures with different active site redox states, including mixed disulfide (Cys22-Cys244), are reported here. The biochemical data show that 239-CX4C-244 motif channels electrons to the active site cysteines. drFrnE is more stable in the oxidized form, compared with the reduced form, supporting its role as a disulfide reductase. Using bioinformatics analysis and fluorescence microscopy, we show cytoplasmic localization of drFrnE. We have found 'true' orthologs of drFrnE in several eubacterial phyla and, interestingly, all these groups apparently lack a functional glutaredoxin system. Innovation and Conclusion: We show that drFrnE represents a new class of hitherto unknown intracellular oxido-reductases that are abundantly present in eubacteria. Unlike other well-known oxido-reductases, FrnE harbors an additional dithiol motif that acts as a conduit to channel electrons to the active site during catalytic turnover. Antioxid. Redox Signal. 28, 296-310. [ABSTRACT FROM AUTHOR]

In most cells, the thioredoxin (Trx) and glutathione systems are essential in maintaining redox homeostasis. The selenoprotein thioredoxin glutathione reductase (TGR) is a hybrid enzyme in which a glutaredoxin (Grx) domain is linked to a thioredoxin reductase (TrxR). Notably, the protein is also capable of reducing glutathione disulfide (GSSG), thus representing an important link between the two redox systems. In this study, we recombinantly produced human TGR (hTGR wild‐type) by fusing its open reading frame with a bacterial selenocysteine insertion sequence element and co‐expressing the construct in Escherichia coli together with the selA, selB, and selC genes. Additionally, the Sec→Cys mutant (hTGRU642C) of the full‐length protein, the isolated TrxR domain (hTGR151–643) and the Grx domain containing a monothiol active site (hTGR1–150) were produced and purified. All four proteins were kinetically characterized in direct comparison using Trx, DTNB, HED, or GSSG as the oxidizing substrate. Interestingly, the HED reduction activity was Sec independent and comparable in the full‐length protein and the isolated Grx domain, whereas the TrxR and glutathione reductase reactions were clearly selenocysteine dependent, with the GR reaction requiring the Grx domain. Site‐directed mutagenesis studies revealed novel insights into the mechanism of GSSG reduction. Furthermore, we identified several glutathionylation sites in hTGR, including Cys93, Cys133, and Cys619, and an inhibitory effect of these modifications on enzyme activity. In contrast to other TGRs, for example, from platyhelminth parasites, hTGR did not exhibit hysteretic behavior. These findings provide new insights into the reaction mechanism and regulation of monothiol Grx‐containing TGRs. Database: EC numbers: 1.8.1.9; 1.8.1.B1 [ABSTRACT FROM AUTHOR]

Keratinases (E.C.3.4.99.11) constitute a group of enzymes capable of disrupting the highly stable keratin structure consisting of disulphide, hydrogen, and hydrophobic bonds in the form of α-helices and β-sheets B. licheniformis MB-2 and Bacillus sp. MTS are two feather-degrading bacteria isolated from Tompaso crater at North Sulawesi and sulfuric land around Tangkuban Perahu in West Java. They were both capable of breaking down whole chicken feathers. In addition both isolates were capable of degrading other proteinous substrates rich in beta structure such as coccon, silk, human hair and fish scales. Result of fermentation experiment implied that addition of nitrogen sources (0.02% yeast extract and 0.02% tryptone) to the basal medium increased keratinase production. Our experiments showed that keratinase production of Bacillus sp. MTS was higher and faster than that from B. licheniformis MB-2. Maximum extracellular keratinase activity of the enzyme derived from B. licheniformis was obtained during stationary phase at 72 h, while Bacillus sp. MTS was reached at 48 h. Disulfide reductase activity also detected in the extracellular fluid of Bacillus sp. MTS. The maximum condition for extracellular keratinase activity was 55oC and the enzyme showed two maximum pHs : pH 8.0 and pH 10. The zymogram analysis indicated sixth protein bands of 17, 25, 32, 53, 96 and 122 kD which were able to hydrolyze gelatin substrate in-situ. (Animal Production 12(1): 60-68 (2010)Key Words : Bacillus, feather, keratinase, disulfide reductase

The use of oxygen as the final electron acceptor in aerobic organisms results in an improvement in the energymetabolism. However, as a byproduct of the aerobic metabolism, reactive oxygen species are produced, leaving to the potential risk of an oxidative stress. To contend with such harmful compounds, living organisms have evolved antioxidant strategies. In this sense, the thiol-dependent antioxidant defense systems play a central role. In all cases, cysteine constitutes the major building block on which such systems are constructed, being present in redox substrates such as glutathione, thioredoxin, and trypanothione, as well as at the catalytic site of a variety of reductases and peroxidases. In some cases, the related selenocysteine was incorporated at selected proteins. In invertebrate parasites, antioxidant systems have evolved in a diversity of both substrates and enzymes, representing a potential area in the design of anti-parasite strategies. The present review focus on the organization of the thiol-based antioxidant systems in invertebrate parasites. Differences between these taxa and its final mammal host is stressed. An understanding of the antioxidant defense mechanisms in this kind of parasites, as well as their interactions with the specific host is crucial in the design of drugs targeting these organisms. [ABSTRACT FROM AUTHOR]

In bacteria, many Dsb-family proteins play diverse roles in the conversion between the oxidized and reduced states of cysteine residues of substrate proteins. Most Dsb enzymes catalyze disulfide formation in periplasmic or secreted substrate proteins. Recently, a DsbM protein has been found in a Gram-negative bacterium, and was characterized as a cytosolic Dsb member with the conserved C XXC motif on the basis of sequence homology to the Dsb-family proteins. The protein was implicated in the reduction of the cytoplasmic redox-sensor protein OxyR in Pseudomonas aeruginosa. Here, crystal structures of DsbM from P. aeruginosa are presented, revealing that it consists of a modified thioredoxin domain containing the C XXC motif and a lid domain surrounding the C XXC motif. In a glutathione-linked structure, a glutathione molecule is linked to the C XXC motif of DsbM and is bound in an elongated cavity region in the thioredoxin domain, which is also suited for substrate peptide binding. A striking structural similarity to a human glutathione S-transferase was found in the glutathione-binding pocket. Further, biochemical evidence is presented suggesting that DsbM is directly involved in the reduction of the disulfide of Cys199 and Cys208 in OxyR, resulting in the acceleration of OxyR reduction in the absence of reactive oxygen species stress. These findings may help to expand the understanding of the diverse roles of redox-related proteins that contain the C XXC motif. [ABSTRACT FROM AUTHOR]

Thioredoxins are small ubiquitous redox proteins that are involved in many biological processes. Proteins with thiol-disulfide bonds are essential regulators of cellular redox homeostasis and diagnostic markers for redox-dependent diseases. Here, we identified and characterized the thioredoxin domain-containing protein 12 (EaTXNDC12) gene in red spotted grouper (Epinephelus akaara), evaluated transcriptional responses, and investigated the activity of the recombinant protein using functional assays. EaTXNDC12 is a 19.22-kDa endoplasmic reticulum (ER)-resident protein with a 522-bp open reading frame and 173 amino acids, including a signal peptide. We identified a conserved active motif (66WCGAC70) and ER retention motif (170GDEL173) in the EaTXNDC12 amino acid sequence. Relative EaTXNDC1 2 mRNA expression was analyzed using 12 different tissues, with the highest expression seen in brain tissue, while skin tissue showed the lowest expression level. Furthermore, mRNA expression in response to immune challenges was analyzed in the head kidney, blood, and gill tissues. EaTXNDC12 was significantly modulated in response to bacterial endotoxin lipopolysaccharide (LPS), nervous necrosis virus (NNV), and polyinosinic:polycytidylic acid (poly(I:C)) challenges in all of the tested tissues. Recombinant EaTXNDC12 (rEaTXNDC12) displayed antioxidant ability in an insulin reductase assay, and a capacity for free radical inhibition in a 2,2-diphenyl-1-picryl-hydrazyl-hydrate assay. In addition, a DNA nicking assay revealed that purified rEaTXNDC12 exhibited concentration-dependent DNA protection activity, while results from 2-hydroxyethyl disulfide and L-dehydroascorbic assays indicated that rEaTXNDC12a possesses reducing ability. Furthermore, fathead minnow (FHM) cells transfected with EaTXNDC12-pcDNA demonstrated significantly upregulated cell survival against H 2 O 2 -induced apoptosis. Collectively, the results of this study strengthen our knowledge of EaTXNDC12 with respect to cellular redox hemostasis and immune regulation in Epinephelus akaara. • Thioredoxin domain-containing protein 12 (TXNDC12) in red spotted grouper was identified and characterized. • The EaTXNDC12 was ubiquitously expressed in all the tissues. • EaTXNDC1 2 mRNA expression level significantly increased in blood, gill, and head kidney after the immune challenge. • Recombinant EaTXNDC12 showed thiol-disulfide reduction activity. • EaTXNDC12 increased the cell viability and reduced cell apoptosis upon H 2 O 2 infection of FHM cells. [ABSTRACT FROM AUTHOR]

Thioredoxin (Trx) is a major thiol-disulfide reductase that plays a role in many biological processes, including DNA replication and redox signaling. Although selenocysteine (Sec)-containing Trxs have been identified in certain bacteria, their enzymatic properties have not been characterized. In this study, we expressed a selenoprotein Trx from Treponema denticola , an oral spirochete, in Escherichia coli and characterized this selenoenzyme and its natural cysteine (Cys) homologue using E. coli Trx1 as a positive control. 75 Se metabolic labeling and mutation analyses showed that the SECIS (Sec insertion sequence) of T. denticola selenoprotein Trx is functional in the E. coli Sec insertion system with specific selenium incorporation into the Sec residue. The selenoprotein Trx exhibited approximately 10-fold higher catalytic activity than the Sec-to-Cys version and natural Cys homologue and E. coli Trx1, suggesting that Sec confers higher catalytic activity on this thiol-disulfide reductase. Kinetic analysis also showed that the selenoprotein Trx had a 30-fold higher K m than Cys-containing homologues, suggesting that this selenoenzyme is adapted to work efficiently with high concentrations of substrate. Collectively, the results of this study support the hypothesis that selenium utilization in oxidoreductase systems is primarily due to the catalytic advantage provided by the rare amino acid, Sec. [ABSTRACT FROM AUTHOR]

Glutaredoxin2 (Grx2) is a mitochondrial isozyme of the cytosolic glutaredoxin1 (thioltransferase or TTase). Both belong to the large oxidoreductase family and play an important role in maintaining thiol/disulfide redox homeostasis in the cells. Grx2 is recently found in the lens where its activities of disulfide reductase and peroxidase, similar to TTase, can protect the lens against oxidative stress. Since other eye tissues are also highly sensitive to oxidative stress, and TTase's distribution in the eye is known, we focused on this study by investigating the Grx2 distribution in the ocular tissues in comparison to the lens. Fresh porcine eyes were dissected into cornea, iris, ciliary body, the lens, vitreous humor, retina, and optic nerve. Each tissue (pooled from three eyes) was homogenized and processed for mitochondrial isolation. The mitochondrial fraction was analyzed for Grx2 protein using Western blotting with anti-Grx2 antibody, and Grx2 activity using the published procedure. The eye tissues were also measured for Grx2 mRNA expression by RT-PCR with GAPDH as the control. Grx2-rich mouse liver and purified recombinant mouse Grx2 were used as positive controls for the above analyses. It was found that Grx2 was present in all the tested ocular tissues, except vitreous humor. In comparison with the mouse liver, the protein levels of Grx2 in porcine ciliary body and the lens were 27-fold and 0.75-fold, respectively. Comparing to the lens, Grx2 protein was highest in the ciliary body (13.5-fold), followed by retina (9.2-fold), iris and optic nerve (2-fold), and cornea (1.2-fold). Enzyme activity assays showed that the retina had the highest Grx2 specific activity (3.9 mU/mg protein), followed by ciliary body (3.1 mU/mg), the lens (0.58 mU/mg), and optic nerve (0.32 mU/mg). Grx2 gene expression in these ocular tissues was further confirmed by RT-PCR analysis. Grx2 mRNA expression showed the highest in ciliary body, followed by retina, optic nerve, cornea, iris, and the lens. No Grx2 mRNA, protein or enzyme activity could be found in the vitreous humor. The results indicate that Grx2 level was higher in eye tissues rich in vasculature and mitochondria (i.e. ciliary body and retina), corroborating with the levels of mRNA expression and Grx2 activity. The rich presence of Grx2 in these tissues is also consistent with their known sensitivity to oxidative stress. [ABSTRACT FROM AUTHOR]

Multiple isoforms of Arabidopsis thaliana h-type thioredoxins (AtTrx-hs) have distinct structural and functional specificities. AtTrx-h3 acts as both a disulfide reductase and as a molecular chaperone. We prepared five representative AtTrx-hs and compared their protein structures and disulfide reductase and molecular chaperone activities. AtTrx-h2 with an N-terminal extension exhibited distinct functional properties with respect to other AtTrx-hs. AtTrx-h2 formed low-molecular-mass structures and exhibited only disulfide reductase activity, whereas the other AtTrx-h isoforms formed high-molecular-mass complexes and displayed both disulfide reductase and molecular chaperone activities. The domains that determine the unique structural and functional properties of each AtTrx-hs protein were determined by constructing a domain-swap between the N- and C-terminal regions of AtTrx-h2 and AtTrx-h3 (designated AtTrx-h-2N3C and AtTrx-h-3N2C respectively), an N-terminal deletion mutant of AtTrx-h2 [AtTrx-h2-N(Δ19)] and site-directed mutagenesis of AtTrx-h3. AtTrx-h2-N(Δ19) and AtTrx-h-3N2C exhibited similar properties to those of AtTrx-h2, but AtTrx-h-2N3C behaved more like AtTrx-h3, suggesting that the structural and functional specificities of AtTrx-hs are determined by their C-terminal regions. Hydrophobicity profiling and molecular modelling revealed that Ala100 and Ala106 in AtTrx-h3 play critical roles in its structural and functional regulation. When these two residues in AtTrx-h3 were replaced with lysine, AtTrx-h3 functioned like AtTrx-h2. The chaperone function of AtTrx-hs conferred enhanced heat-shock-resistance on a thermosensitive trx1/2-null yeast mutant. [ABSTRACT FROM AUTHOR]

Abstract: Over the last decades, malaria parasites have been rapidly developing resistance against antimalarial drugs, which underlines the need for novel drug targets. Thioredoxin reductase (TrxR) is crucially involved in redox homeostasis and essential for Plasmodium falciparum. Here, we report the first crystal structure of P. falciparum TrxR bound to its substrate thioredoxin 1. Upon complex formation, the flexible C-terminal arm and an insertion loop of PfTrxR are rearranged, suggesting that the C-terminal arm changes its conformation during catalysis similar to human TrxR. Striking differences between P. falciparum and human TrxR are a Plasmodium-specific insertion and the conformation of the C-terminal arm, which lead to considerable differences in thioredoxin binding and disulfide reduction. Moreover, we functionally analyzed amino acid residues involved in substrate binding and in the architecture of the intersubunit cavity, which is a known binding site for disulfide reductase inhibitors. Cell biological experiments indicate that P. falciparum TrxR is indeed targeted in the parasite by specific inhibitors with antimalarial activity. Differences between P. falciparum and human TrxR and details on substrate reduction and inhibitor binding provide the first solid basis for structure-based drug development and lead optimization. [Copyright &y& Elsevier]

Keratinolytic bacteria Bacillus polymyxa B20 and B. cereus B5esz during cultures in medium with chicken feathers as a sole nutrient source, accumulated various amounts of sulfur compounds at different oxidation level, including thiols, thiosulfate, sulfite and sulfate. The main difference observed between the two tested strains was higher release of sulfate by the former and elevated concentration of thiols by the latter. Additionally, the activity of glutathione reductase, that could potentially play a role in keratinolysis was confirmed, mainly in the cell homogenate fraction, rather than extracellular. Keratinases in crude culture fluids exhibited activity towards soluble keratin preparation, as well as native feather keratin. Application of 2-mercaptoethanol and sulfite, agents that potentially could take part in keratin sulfitolysis, led to a conclusion that they could play a role in keratin degradation, other than activation of extracellular enzymes. [ABSTRACT FROM AUTHOR]

Genevestigator analysis has indicated heat shock induction of transcripts for NADPH-thioredoxin reductase, type C (NTRC) in the light. Here we show overexpression of NTRC in Arabidopsis (NTRC°E) resulting in enhanced tolerance to heat shock, whereas NTRC knockout mutant plants (ntrc1) exhibit a temperature sensitive phenotype. To investigate the underlying mechanism of this phenotype, we analyzed the protein’s biochemical properties and protein structure. NTRC assembles into homopolymeric structures of varying complexity with functions as a disulfide reductase, a foldase chaperone, and as a holdase chaperone. The multiple functions of NTRC are closely correlated with protein structure. Complexes of higher molecular weight (HMW) showed stronger activity as a holdase chaperone, while low molecular weight (LMW) species exhibited weaker holdase chaperone activity but stronger disulfide reductase and foldase chaperone activities. Heat shock converted LMW proteins into HMW complexes. Mutations of the two active site Cys residues of NTRC into Ser (C217/454S-NTRC) led to a complete inactivation of its disulfide reductase and foldase chaperone functions, but conferred only a slight decrease in its holdase chaperone function. The overexpression of the mutated C217/454S-NTRC provided Arabidopsis with a similar degree of thermotolerance compared with that of NTRC°E plants. However, after prolonged incubation under heat shock, NTRC°E plants tolerated the stress to a higher degree than C217/454S-NTRC°E plants. The results suggest that the heat shock-mediated holdase chaperone function of NTRC is responsible for the increased thermotolerance of Arabidopsis and the activity is significantly supported by NADPH. [ABSTRACT FROM PUBLISHER]

Abstract: Bacillus sp. MTS isolated from Tangkuban Perahu crater Indonesia was found capable of degrading whole chicken feather effectively. The bacteria produced extracellular alkaline keratinase and disulfide reductase. When grown in feather media, Bacillus sp. MTS produced multi-fractions of both enzymes. The purified enzymes worked optimally at alkaline pHs, for keratinase at pH 8–12, and for disulfide reductase at pH 8–10. Optimum temperature for the extracellular keratinase was within 40–70°C, while that for disulfide reductase was 35°C. When the purified keratinase was mixed with purified disulfide reductase, enzyme activities on the natural keratin substrates (feather and wool) were greatly increased compared to activity of each enzyme alone, activity of proteinase K or activity of purified keratinase in the presence of reducing agents. The mutual action of the two enzymes on feather was examined by Scanning Electron Microscope. [Copyright &y& Elsevier]

Abstract: Thioredoxin-related protein 14 (TRP14) is a novel 14-kDa disulfide reductase with two active site Cys residues in its WCPDC motif, which is comparable to the WCGPC motif of thioredoxin (Trx). Although the active site cysteine of TRP14 is sufficiently nucleophilic, its redox potential is similar to that of Trx1, and it receives the electrons from Trx reductase 1 (TrxR1) as does Trx1. TRP14 does not target the same substrate as Trx1, suggesting that TRP14 and Trx1 might act on distinct substrate proteins. Comparison of the crystal structures of TRP14 and Trx1 reveals distinct surface structures in the vicinity of their active sites. Both TRP14 and Trx1 inhibit the pathways of nuclear factor-κB (NF-κB), mitogen-activated protein kinases, and apoptosis in cells stimulated with tumor necrosis factor-α (TNF-α), but they appear to do so by acting on target proteins, some of which do not overlap. TRP14 inhibits the TNF-α-induced NF-κB activation to a greater extent than Trx1. The dynein light chain LC8 was identified as a new target of disulfide reductase activity of TRP14, and LC8 was shown to bind IκBα in a redox-dependent manner, thereby preventing its phosphorylation by IκB kinase. These findings elucidate the molecular mechanism by which NF-κB activation is regulated through TRP14. [Copyright &y& Elsevier]

We found that Arabidopsis AtTDX, a heat-stable and plant-specific thioredoxin (Trx)-like protein, exhibits multiple functions, acting as a disulfide reductase, foldase chaperone, and holdase chaperone. The activity of AtTDX, which contains 3 tetratricopeptide repeat (TPR) domains and a Trx motif, depends on its oligomeric status. The disulfide reductase and foldase chaperone functions predominate when AtTDX occurs in the low molecular weight (LMW) form, whereas the holdase chaperone function predominates in the high molecular weight (HMW) complexes. Because deletion of the TPR domains results in a significant enhancement of AtTDX disulfide reductase activity and complete loss of the holdase chaperone function, our data suggest that the TPR domains of AtTDX block the active site of Trx and play a critical role in promoting the holdase chaperone function. The oligomerization status of AtTDX is reversibly regulated by heat shock, which causes a transition from LMW to HMW complexes with concomitant functional switching from a disulfide reductase and foldase chaperone to a holdase chaperone. Overexpression of AtTDX in Arabidopsis conferred enhanced heat shock resistance to plants, primarily via its holdase chaperone activity. [ABSTRACT FROM AUTHOR]

Abstract: The naphthoquinone 7-methyljuglone (5-hydroxy-7-methyl-1,4-naphthoquinone) has previously been isolated and identified as an active component of root extracts of Euclea natalensis which displays antitubercular activity. Herein, a series of synthetic and plant-derived naphthoquinone derivates of the 7-methyljuglone scaffold have been prepared and evaluated for antibacterial activity against Mycobacterium tuberculosis. Several of these compounds have been shown to operate as subversive substrates with mycothiol disulfide reductase. The absence of a direct correlation between antitubercular activity and subversive substrate efficiency with mycothiol disulfide reductase, might be a consequence of their non-specific reactivity with multiple biological targets (e.g. other disulfide reductases). [Copyright &y& Elsevier]

The membrane-embedded domain of the unusual electron transporter DsbD (DsbDβ) uses two redox-active cysteines to catalyze electron transfer between thioredoxin-fold polypeptides on opposite sides of the bacterial cytoplasmic membrane. How the electrons are transferred across the membrane is unknown. Here, we show that DsbDβ displays an inherent functional and structural symmetry: first, the two cysteines of DsbDβ can be alkylated from both the cytoplasm and the periplasm. Second, when the two cysteines are disulfide-bonded, cysteine scanning shows that the C-terminal halves of the cysteine-containing transmembrane segments 1 and 4 are exposed to the aqueous environment while the N-terminal halves are not. Third, proline residues located pseudo-symmetrically around the two cysteines are required for redox activity and accessibility of the cysteines. Fourth, mixed disulfide complexes, apparent intermediates in the electron transfer process, are detected between DsbDβ and thioredoxin molecules on each side of the membrane. We propose a model where the two redox-active cysteines are located at the center of the membrane, accessible on both sides of the membrane to the thioredoxin proteins. [ABSTRACT FROM AUTHOR]

An Escherichia coli cell-free protein synthesis cell extract has been created that lacks all known cytoplasmic disulfide reduction pathways but still retains significant reductase activity. Oxidized glutathione was partially stabilized by deleting the gene for glutathione reductase. To avoid previously reported AhpC mutations, thioredoxin reductase was only removed after extract preparation. The trxB gene was extended to encode a hemagglutinin tag so that TrxB could be removed by affinity adsorption. However, significant glutathione reductase activity remained. The unknown glutathione reductase pathway is disabled by iodoacetamide, is inhibited by NADH, and appears to use NADPH as an electron source. [Copyright &y& Elsevier]

Abstract: The macrocyclic spermidine alkaloid lunarine 1 from Lunaria biennis is a competitive, time-dependent inhibitor of the protozoan oxidoreductase trypanothione reductase (TryR), a promising target in drug design against tropical parasitic diseases. Various molecules related to 1 and the alkaloid itself have been synthesized in racemic form and evaluated against TryR in order to determine the key features of 1 that are associated with time-dependent inhibition. Kinetic data are consistent with an inactivation mechanism involving a conjugate addition of an active site cysteine residue onto the C-24–C-25 double bond of the tricyclic nucleus of 1. Comparison of data for synthetic (±)-1, the natural product, and other derivatives 7–10 from L. biennis confirms the importance of the unique structure of the tricyclic core as a motif for inhibitor design and reveals that the non-natural enantiomer may be a more suitable scaffold upon which thiophilic groups may be presented. [Copyright &y& Elsevier]

Abstract: Physiologically significant levels of intracellular coenzyme A were identified in Pyrococcus furiosus, Thermococcus litoralis, and Sulfolobus solfataricus, suggesting a role for CoA as an important low molecular mass thiol in the thermophilic Archaea. In P. furiosus, cells grown in the presence of sulfur showed significantly higher levels of oxidized CoA compared with those grown in the absence of S0. T. litoralis showed strikingly similar CoA levels, although with low disulfide levels in both the presence and absence of S0. S. solfataricus showed similarly high levels of CoA thiol, with correspondingly low levels of the CoA disulfide. These results are consistent with the identification of a coenzyme A disulfide reductase (CoADR) in P. furiosus and horikoshii as well as the presence of CoADR homologues in the genomes of S. solfataricus and T. kodakaraensis. [Copyright &y& Elsevier]

The maturation of c-type cytochromes requires the covalent ligation of the heme cofactor to reduced cysteines of the CXXCH motif of apocytochromes. In contrast to mitochondria of other eukaryotes, plant mitochondria follow a pathway close to that found in α- and γ-proteobacteria. We identified a nuclear-encoded protein, AtCCMH, the Arabidopsis thaliana ortholog of bacterial CcmH/CycL proteins. In bacteria, CcmH and the thioredoxin CcmG are components of a periplasmic thio-reduction pathway proposed to maintain the apocytochrome c cysteines in a reduced state. AtCCMH is located exclusively in mitochondria. AtCCMH is an integral protein of the inner membrane with the conserved RCXXC motif facing the intermembrane space. Reduction assays show that the cysteine thiols in the RCXXC motif of AtCCMH can form a disulfide bond that can be reduced by enzymatic thiol reductants. A reduced form of AtCCMH can reduce the intra-disulfide bridge of a model peptide of apocytochrome c. When expressed in Escherichia coil, AtCCMH coimmunoprecipitates with the bacterial CcmF, a proposed component of the heme lyase. Blue-native PAGE of mitochondrial membrane complexes reveals the colocalization of AtCCMH and AtCcmFN2 in a 500-kDa complex. Yeast two-hybrid assays show an interaction between the AtCCMH intermembrane space domain and A. thaliana apocytochrome c. A. thaliana ccmh/ ccmh knockout plants show lethality at the torpedo stage of embryogenesis. Our results show that AtCCMH is an essential mitochondrial protein with characteristics consistent with its proposed apocytochrome c-reducing and heme Iyase function. [ABSTRACT FROM AUTHOR]

Heterodisulfide reductase (HDR) from methanogenic archaea is an iron-sulfur protein that catalyzes reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol-coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). Via the characterization of a paramagnetic reaction intermediate generated upon oxidation of the enzyme in the presence of coenzyme M, the enzyme was shown to contain a [4Fe-4S] cluster in its active site that catalyzes reduction of the disulfide substrate in two one-electron reduction steps. The formal thiyl radical generated by the initial one-electron reduction of the disulfide is stabilized via reduction and coordination of the resultant thiol to the [4Fe-4S] cluster. [ABSTRACT FROM AUTHOR]

Keratinolytic Bacillus licheniformis RG1 was used to study the mechanism of keratinolysis. Scanning election microscopy studies revealed that bacterial cells grew closely adhered to the barbules of feathers, completely degrading them within 24 h. Biochemical studies indicated that the Bacillus strain produced an extracellular protease, which had keratinolytic potential. The extracellular keratinolytic activity (425 U) was synergistically enhanced by the addition of intracellular disulfide reductases (1712 U). However, these enzymes alone (keratinase and disulfide reductase), without live bacterial cells, failed to degrade the feather. Complete feather degradation was obtained only when living bacterial cells were present, emphasizing that bacterial adhesion plays a key role during the degradation process. The bacterial cells probably provide a continuous supply of reductant to break disulfide bridges. In addition, sulfite detected in the extracellular broth during feather degradation indicated that sulfitolysis may also play a role in feather degradation by the bacterium. [ABSTRACT FROM AUTHOR]

Abstract: Antioxidant defenses include a group of ubiquitous, non-heme peroxidases, designated the peroxiredoxins, which rely on an activated cysteine residue at their active site to catalyze the reduction of hydrogen peroxide, organic hydroperoxides, and peroxynitrite. In the typical 2-Cys peroxiredoxins, a second cysteinyl residue, termed the resolving cysteine, is also involved in intersubunit disulfide bond formation during the course of catalysis by these enzymes. Many bacteria also express a flavoprotein, AhpF, which acts as a dedicated disulfide reductase to recycle the bacterial peroxiredoxin, AhpC, during catalysis. Mechanistic and structural studies of these bacterial proteins have shed light on the linkage between redox state, oligomeric state, and peroxidase activity for the peroxiredoxins, and on the conformational changes accompanying catalysis by both proteins. In addition, these studies have highlighted the dual roles that the oxidized cysteinyl species, cysteine sulfenic acid, can play in eukaryotic peroxiredoxins, acting as a catalytic intermediate in the peroxidase activity, and as a redox sensor in regulating hydrogen peroxide-mediated cell signaling. [Copyright &y& Elsevier]

The bacterial membrane protein DsbD transfers electrons across the cytoplasmic membrane to reduce protein disulfide bonds in extracytoplasmic proteins. Its substrates include protein disulfide isomerases and a protein involved in cytochrome c assembly. Two membrane-embedded cysteines in DsbD alternate between the disulfide-bonded (oxidized) and reduced states in this process. [Copyright &y& Elsevier]

The oxygen sensor fumarate nitrate reductase regulator (FNR) of Escherichia coli contains in the active (anaerobic) state a [4Fe–4S]2+ cluster which is lost after exposure to O2. In aerobically prepared apoFNR, or in FNR obtained by treatment of [4Fe–4S] · FNR with O2 in vitro, intramolecular cysteine disulfides are found, including the cysteine residues which serve as ligands for the Fe–S cluster. It is shown here that the reconstitution of [4Fe–4S] · FNR from this form of aerobic apoFNR was preceded by a long lag phase when glutathione was used as the reducing agent. Addition of E. coli glutaredoxins (Grx) 1, 2 or 3 decreased the lag phase greatly and stimulated the reconstitution rate slightly (about twofold). Reconstitution of anaerobically prepared apoFNR, which has a lower cysteine disulfide content, showed only a short lag phase, which further decreased in the presence of Grx. It is concluded that in the lag phase the cysteine disulfides of apoFNR become reduced for the incorporation of the [4Fe–4S] cluster and that this reaction is stimulated by Grx. Thioredoxin (Trx) 1 showed no stimulation of FNR reconstitution in vitro. It is suggested that the function of Grx might be of significance for the insertion of FeS cluster in proteins containing disulfides. [Copyright &y& Elsevier]

A novel keratin-degrading bacterium Stenotrophomonas sp. strain D-1, isolated from deer fur, produced two types of extracellular proteins: proteolytic and disulfide bond-reducing. The results on the biochemical properties suggest that this protease belongs to the serine protease, and the disulfide bond-reducing protein could be the disulfide reductase type. None of these enzymes showed keratinolytic activity independently. However, after mixing of the two enzymes, the keratinolytic activity was increased tremendously (more than 50-fold) over that of the protease only. This keratinolytic activity was more than 2-fold higher than that of the combination with proteinase K (also known for its high keratinolytic activity). Since the two enzymes discovered in this study acted cooperatively and resulted in higher keratinolytic activity, a new mechanism of keratin degradation has been revealed. To our knowledge, this is the first report on the cooperative action of two enzymes resulting in the effective degradation of keratin. [Copyright &y& Elsevier]

Copper is essential for the stability and activity of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Copper is bound to COX1 and COX2, two core subunits of CcO, forming the CuB and CuA sites, respectively. Biogenesis of these two copper sites of CcO occurs separately and requires a number of evolutionarily conserved proteins that form the mitochondrial copper delivery pathway. Pathogenic mutations in some of the proteins of the copper delivery pathway, such as SCO1, SCO2, and COA6, have been shown to cause fatal infantile human disorders, highlighting the biomedical significance of understanding copper delivery mechanisms to CcO. While two decades of studies have provided a clearer picture regarding the biochemical roles of SCO1 and SCO2 proteins, some discrepancy exists regarding the function of COA6, the new member of this pathway. Initial genetic and biochemical studies have linked COA6 with copper delivery to COX2 and follow-up structural and functional studies have shown that it is specifically required for the biogenesis of the CuA site by acting as a disulfide reductase of SCO and COX2 proteins. Its role as a copper metallochaperone has also been proposed. Here, we critically review the recent literature regarding the molecular function of COA6 in CuA biogenesis. [ABSTRACT FROM AUTHOR]

Recently a newly discovered pyridine nucleotide-disulfide oxidoreductase was reported to be essential for the degradation of epoxyalkanes by the Xanthobacter Py2 [Swaving, J., De Bont, J. A. M., Westphal, A. & De Kok, A. (1996) J. Bacteriol. 178, 6644-6646]. The disulfide oxidoreductase has now been purified from propene-grown Xanthobacter Py2. This enzyme (component II) is a NADPH-dependent FAD-containing homodimeric protein. The physiological substrate for this enzyme is unknown. The enzyme was active with the following dithiol substrates in decreasing order : 1,3-propanedithiol, reduced lipoamide and dithiothreitol, and inactive with glutathione and monothiols. In the reversed direction, only activity with 5,5′-dithiobis(2-nitrobenzoate) could be measured. Compared with other disulfide reductases it has a high activity with 5,5′-dithiobis(2-nitrobenzoate) and a low diaphorase and oxidase activity. Steady-state kinetic studies at pH 8.5 with 1,3-propanedithiol show that the enzyme operates by a ternary complex mechanism in the direction of NADP+ reduction. Anaerobic incubation of the enzyme with 1,3-propanedithiol resulted in slow reduction of the enzyme to yield the thiolate-FAD charge-transfer complex, the rate depending on the pH. At pH 7, where reduction was not detectable within 2 h, rapid mixing of NADP+ with the enzyme-propanedithiol mixture resulted in the formation of a complex between the reduced enzyme and NADP+ within the dead time of the instrument (5.6 ms). This is followed by slow formation of NADPH, concomitant with the appearance of the flavin C(4a)-thiol adduct, as judged from the spectral changes. This suggests that the rate-limiting step is the transfer of a hydride ion from the half-reduced enzyme to NADP+. Stopped-flow experiments involving reduction by NADPH show a biphasic behavior. The rapid formation (kobs = 40 s-1) of a transient intermediate with... [ABSTRACT FROM AUTHOR]

The activities of formylmethanofuran dehydrogenase, methylenetetrahydromethanopterin dehydrogenase, methylenetetrahydromethanopterin reductase, and heterodisulfide reductase were tested in cell extracts of 10 different methanogenic bacteria grown on H/CO or on other methanogenic substrates. The four activities were found in all the organisms investigated: Methanobacterium thermoautotrophicum,M. wolfei, Methanobrevibacter arboriphilus, Methanosphaera stadtmanae, Methanosarcina barkeri (strains Fusaro and MS), Methanothrix soehngenii, Methanospirillum hungatei, Methanogenium organophilum, and Methanococcus voltae. Cell extracts of H/CO grown M. barkeri and of methanol grown M. barkeri showed the same specific activities suggesting that the four enzymes are of equal importance in CO reduction to methane and in methanol disproportionation to CO and CH. In contrast, cell extracts of acetate grown M. barkeri exhibited much lower activities of formylmethanofuran dehydrogenase and methylenetetrahydromethanopterin dehydrogenase suggesting that these two enzymes are not involved in methanogenesis from acetate. In M. stadtmanae, which grows on H and methanol, only heterodisulfide reductase was detected in activities sufficient to account for the in vivo methane formation rate. This finding is consistent with the view that the three other oxidoreductases are not required for methanol reduction to methane with H. [ABSTRACT FROM AUTHOR]

The disulfide reducing activities of GSSG-and CoASSG-reductases were measured on partially purified extracts from a variety of prokaryotes and eukaryotes. Glutathione-reductase was found in varying amounts in all eukaryotes and prokaryotes, used in this study, with the exception of the two strict anaerobes Clostridium tartarivorum and Desulfovibrio vulgaris, and the two primitive Archaebacteria Methanosarcina barkeri and Halobacterium halobium. CoASSG-reductase was found in some eukaryotes and prokaryotes, but showed no clear pattern of distribution other than its absence whenever GSSG-reductase was not present. The absence of GSSG-reductase activity in organisms lacking GSH, confirms that glutathione metabolism is not universal and suggests that this enzyme might be useful as a marker in classifying organisms. The data suggest that glutathione-reductase occurs as a result of the change from a reducing to a oxidizing atmosphere in the primitive Earth. [ABSTRACT FROM AUTHOR]

Sulfide oxidation is catalyzed by ancient membrane-bound sulfide:quinone oxidoreductases (SQR) which are classified into six different types. For catalysis of sulfide oxidation, all SQRs require FAD cofactor and a redox-active centre in the active site, usually formed between conserved essential cysteines. SQRs of different types have variation in the number and position of cysteines, highlighting the potential for diverse catalytic mechanisms. The photosynthetic purple sulfur bacterium, Thiocapsa roseopersicina contains a type VI SQR enzyme (TrSqrF) having unusual catalytic parameters and four cysteines likely involved in the catalysis. Site-directed mutagenesis was applied to identify the role of cysteines in the catalytic process of TrSqrF. Based on biochemical and kinetic characterization of these TrSqrF variants, Cys121 is identified as crucial for enzyme activity. The cofactor is covalently bound via a heterodisulfide bridge between Cys121 and the C8M group of FAD. Mutation of another cysteine present in all SQRs (Cys332) causes remarkably decreased enzyme activity (14.6% of wild type enzyme) proving important, but non-essential role of this residue in enzyme catalysis. The sulfhydril-blocking agent, iodoacetamide can irreversibly inactivate TrSqrF but only if substrates are present and the enzyme is actively catalyzing its reaction. When the enzyme is inhibited by iodoacetamide, the FAD cofactor is released. The inhibition studies support a mechanism that entails opening and reforming of the heterodisulfide bridge during the catalytic cycle of TrSqrF. Our study thus reports the first detailed structure-function analysis of a type VI SQR enzyme which enables the proposal of a distinct mechanism of sulfide oxidation for this class. Unlabelled Image • Cysteine profile of type VI sulfide:quinone oxidoreductases (SQRs) differs from that of other type of SQRs. • The first homology model of a type VI SQR suggests a novel catalytic centre. • Cys121 binds FAD cofactor covalently, which bond is temporarily broken during the catalytic cycle. • Cys121 essential, Cys332 is very important for SqrF enzyme catalysis. • The first model for the catalytic mechanism of a type VI sulfide:quinone oxidoreductase is presented. [ABSTRACT FROM AUTHOR]

Peroxiredoxins are cysteine-dependent peroxide reductases that group into 6 different, structurally discernable classes. In 2011, our research team reported the application of a bioinformatic approach called active site profiling to extract active site-proximal sequence segments from the 29 distinct, structurally-characterized peroxiredoxins available at the time. These extracted sequences were then used to create unique profiles for the six groups which were subsequently used to search GenBank(nr), allowing identification of ∼3500 peroxiredoxin sequences and their respective subgroups. Summarized in this minireview are the features and phylogenetic distributions of each of these peroxiredoxin subgroups; an example is also provided illustrating the use of the web accessible, searchable database known as PREX to identify subfamily-specific peroxiredoxin sequences for the organism Vitis vinifera (grape).