Redox-active Disulfide Bridge Research Articles

Expression of OsTPX Gene Improves Cellular Redox Homeostasis and Photosynthesis Efficiency in Synechococcus elongatus PCC 7942.

Cyanobacterial 2-Cys peroxiredoxin (thioredoxin peroxidase, TPX) comprises a family of thiol antioxidant enzymes critically involved in cell survival under oxidative stress. In our previous study, a putative TPX was identified using a proteomics analysis of rice (Oryza sativa L. japonica, OsTPX) seedlings exposed to oxidative stress. This OsTPX gene is structurally similar to the Synechococcus elongatus TPX gene in the highly conserved redox-active disulfide bridge (Cys114, Cys236) and other highly conserved regions. In the present study, the OsTPX gene was cloned into rice plants and S. elongatus PCC 7942 strain to study hydrogen peroxide (H2O2) stress responses. The OsTPX gene expression was confirmed using semi-quantitative RT-PCR and western blot analysis. The OsTPX gene expression increased growth under oxidative stress by decreasing reactive oxygen species and malondialdehyde level. Additionally, the OsTPX gene expression in S. elongatus PCC 7942 (OT) strain exhibited a reduced loss of chlorophyll and enhanced photosynthesis efficiency under H2O2 stress, thereby increasing biomass yields twofold compared with that of the control wild type (WT) strain. Furthermore, redox balance, ion homeostasis, molecular chaperone, and photosynthetic systems showed upregulation of some genes in the OT strain than in the WT strain by RNA-Seq analysis. Thus, OsTPX gene expression enhances oxidative stress tolerance by increasing cell defense regulatory networks through the cellular redox homeostasis in the rice plants and S. elongatus PCC 7942.

Unprecedented pathway of reducing equivalents in a diflavin-linked disulfide oxidoreductase

Flavoproteins participate in a wide variety of physiologically relevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FAD molecules per monomer in redox communication with an active disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein "DDOR" (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-based transfer of reducing equivalents in bacterial membranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications.

Substitution of Thr55 by Gly and Lys48 by Asp in OsTrx20 using site-directed mutagenesis

Thioredoxins are small (12–13kDa) ubiquitous proteins containing a redox active disulfide bridge. The primary structure of one of the rice Trx isoforms, OsTrx20, in which Thr is substituted for the largely conserved Gly in position 55 in the active site and Lys is substituted for the conserved Asp/Asn in position 48 is considerably different with other h-type Trx isoforms. In order to probe the functional roles of Thr-55 and Lys-48 in OsTrx20, Thr was replaced with Gly and Lys with Asp using site-directed mutagenesis. The wild type OsTrx20 as well as single mutants T55GOsTrx20, K48DOsTrx20 and the double mutant T55G-K48DOstrx20 were heterologously expressed in Escherichia coli and purified. The changes in the ability to reduce insulin for OsTrx20 and mutants as well as OsTrx23 which has a Trx typical active site were monitored in the pH range 6.5-8. The results showed that whereas the activity of wild type OsTrx20 is dependent on pH and decreases remarkably at high pH values, the activities of mutants T55GOsTrx20, K48DOsTrx20, T55G-K48DOsTrx20 and wild type OsTrx23 slightly change under different pH conditions. These results support the significant involvement of residues Thr-55 and Lys-48 in instability of OsTrx20 activity under pH variations.

Molecular characterization of thioredoxin-1 and thioredoxin reductase activity in mud crab Scylla paramamosain.

The thioredoxin (Trx) system consists of thioredoxin reductase (TrxR), Trx, and nicotinamide adenine dinucleotide phosphate (NADPH). TrxR is an NADPH-dependent oxidoreductase. Trx is a ubiquitous small protein with a redox-active disulfide bridge that plays important regulatory roles in some vital metabolic reactions. In this study, a cDNA sequence (SpTrx1) showing high identity to the first Trx gene was isolated from a hepatopancreas cDNA library of the mud crab Scylla paramamosain. The full-length cDNA of SpTrx1 consisted of 672 bp and contained a complete open reading frame of 318 bp encoding a polypeptide of 105 amino acids. Quantitative real-time polymerase chain reaction analysis revealed that SpTrx1 expression was ubiquitous in various organs of S. paramamosain, including the gill, muscle, heart, hemolymph, testis, and hepatopancreas. SpTrx1 expression was upregulated significantly after Vibrio parahaemolyticus challenge: it obviously rose at 48 h and reached the highest level at 72 h. Furthermore, TrxR activity was detected in the gill, heart, muscle, hemolymph, and hepatopancreas. The relative TrxR activity in different tissues after V. parahaemolyticus injection had the same tendency in each tissue (P < 0.01) as SpTrx1 expression. The TrxR activity increased 2 h after injection, peaked at 8 h, slowly decreased from 12 to 24 h, and returned to normal levels at 48 h. The consistency of the expression between the Trx transcript and TrxR activity demonstrated that Trx was closely related to TrxR in the Trx system in S. paramamosain, suggesting that it may participate in the immune system of mud crabs.

Second thioredoxin from the mud crab Scylla paramamosain: cDNA cloning and mRNA expression analysis

Thioredoxin, is a kind of ubiquitous small proteins with a redox active disulfide bridge, and has been found to play important regulatory roles in some vital biochemical reactions of metabolism. In the present study, a hepatopancreas cDNA library from the mud crab Scylla paramamosain Estampador, 1949 was constructed using the SMART technique. A complete cDNA sequence that showing high identity with the conserved sequence of the second thioredoxin gene (Trx2) was isolated from the cDNA library and subjected to further investigation. The full-length Trx2 of Scylla paramamosain consisted of 1336 bp and contained a complete open reading frame of 396 bp. The gene encoded a polypeptide of 131 amino acids and constituted a predicted molecular mass of 14.45 kDa and a theoretical isoelectric point of 8.08. Quantitative real-time PCR (qRT-PCR) analyses revealed that Trx2 expression was ubiquitous in various organs of Scylla paramamosain, including the gill, muscle, heart, haemocyte, testis, and hepatopancreas. The Trx2 expression level was the highest in the hepatopancreas and gill, and the lowest in the haemocyte. The result of the challenge experiment showed that Trx2 expression was up-regulated significantly after Vibrio parahaemolyticus challenge and reached the highest level at 6 h. This study provides useful information regarding the structure and functions of Trx2, suggesting that it may be a potential biomarker gene for evaluation of environmental stress in marine species.

Three thioredoxin targets in the inner envelope membrane of chloroplasts function in protein import and chlorophyll metabolism

Thioredoxins (Trxs) are ubiquitous small proteins with a redox-active disulfide bridge. In their reduced form, they constitute very efficient protein disulfide oxidoreductases. In chloroplasts, two types of Trxs (f and m) coexist and play central roles in the regulation of the Calvin cycle and other processes. Here, we identified a class of Trx targets in the inner plastid envelope membrane of chloroplasts that share a CxxC motif approximately 73 aa from their carboxyl-terminal end. Members of this group belong to a superfamily of Rieske iron-sulfur proteins involved in protein translocation and chlorophyll metabolism. These proteins include the protein translocon protein TIC55, the precursor NADPH:protochlorophyllide oxidoreductase translocon protein PTC52, which operates as protochlorophyllide a-oxygenase, and the lethal leaf spot protein LLS1, which is identical with pheophorbide a oxygenase. The role of these proteins in dark/light regulation and oxidative control by the Trx system is discussed.

GliP, a Multimodular Nonribosomal Peptide Synthetase in Aspergillus fumigatus, Makes the Diketopiperazine Scaffold of Gliotoxin

The fungal metabolite gliotoxin has a redox-active disulfide bridge spanning carbons 3 and 6 of a diketopiperazine (DKP) scaffold. The proposed DKP synthetase, GliP, from Aspergillus fumigatus Af293, is a three module (A1-T1-C1-A2-T2-C2-T3) 236 kDa protein that can be overproduced in soluble form in Escherichia coli. Once primed on its three thiolation domains with phosphopantetheine prosthetic groups, GliP activates and tethers l-Phe on T1 and l-Ser on T2, before generating the l-Phe-l-Ser-S-T2 dipeptidyl enzyme intermediate. Release of the dipeptide as the cyclic DKP happens slowly both in wild-type GliP and in enzyme forms where C2 and T3 have been mutationally inactivated. The lack of a thioesterase domain in GliP may account both for the slow release and for the directed fate of intramolecular cyclization to create the DKP scaffold for subsequent elaboration to gliotoxin.

Isolation of a thioredoxin h cDNA from grapefruit peel tissue that is induced upon infection by Penicillium digitatum and elicitation of pathogen resistance

Thioredoxins are ubiquitous small proteins with a redox-active disulfide bridge; they participate in redox regulation of selected target proteins, such as transcription factors, receptors and various metabolic and regulatory enzymes. In the present study, we isolated by PCR cDNA subtraction analysis a grapefruit 135-bp polymerase chain reaction (PCR) fragment whose expression was enhanced in the fruit peel tissue following the application of Candida oleophila yeast cells, an elicitation treatment that induces resistance towards the green mold pathogen Penicillium digitatum (Pers.:Fr.) Sacc., and that showed high sequence homology with various thioredoxin h ( TRXh) genes. The full-length cDNA sequence of cTRXh (for citrus TRXh) was further isolated by screening a grapefruit flavedo cDNA library; it had a total length of 785 bp with an open reading frame of 369 bp, and encoded a predicted polypeptide of 123 amino acids with a molecular mass of 13.4 kDa. Expression analysis studies revealed that cTRXh mRNA levels increased in grapefruit peel tissue upon infection by P. digitatum, and following the elicitation of fruit disease resistance by application of C. oleophila yeast cells, wounding, exposure to ethylene, and a hot water rinsing and brushing treatment. These results indicate that cTRXh may be part of the molecular mechanisms involved in the induction of pathogen defense responses in grapefruit, and may be involved in the signaling cascades leading to enhanced resistance.

Characterization of Arabidopsis Mutants for the Variable Subunit of Ferredoxin:thioredoxin Reductase

The ferredoxin/thioredoxin reductase (FTR) is the key enzyme of a light dependent redox regulatory system controlling enzyme activities in oxygenic photosynthetic cells. It is composed of two dissimilar subunits. The catalytic subunit contains a [4Fe-4S] cluster and a redox-active disulfide bridge as the active site. The function of the second subunit, named the variable subunit because it has less conserved primary sequence and length, is not yet known. In order to get insights into the physiological role and importance of FTR, we studied two Arabidopsis mutant lines in which one of two genes encoding FTRA subunit was disrupted by T-DNA insertion. In FTRA1 mutants, the absence of the corresponding transcript was not compensated by the increase in the level of FTRA2 mRNA. Mutant plants exhibited phenotypic perturbations when compared with wild-type plants. Disruptants were found significantly more sensitive to oxidative stress as imposed under high light or in the presence of paraquat. Mutants were further characterized at the biochemical level. Despite the fact that no difference was found by immunodetection of FTR polypeptides, evidence for an impaired FTR system occurring in the mutants was obtained by measuring the endogenous activation rate of one of its targets. In the leaves of mutants placed under normal culture conditions, NADP-dependent malate dehydrogenase (NADP-MDH) activation rate was abnormally low. A partially compensating increase of the enzyme activity was found as well as a higher amount of 2-cys-peroxiredoxin. Our results provide in planta confirmation of the antioxidant role previously proposed for some of the plastidial thioredoxins from Arabidopsis thaliana. The variable subunit of the FTR proved to be important, but its precise role remains to be established.

Heterodimer formation between thioredoxin f and fructose 1,6-bisphosphatase from spinach chloroplasts

Chloroplast fructose 1,6-bisphosphatase (FBPase) is activated by reduction of a regulatory disulfide through thioredoxin f (Trx f). In the course of this reduction a transient mixed disulfide is formed linking covalently Trx f with FBPase, which possesses three Cys on a loop structure, two of them forming the redox-active disulfide bridge. The goal of this study was to identify the Cys involved in the transient mixed disulfide. To stabilize this reaction intermediate, mutant proteins with modified active sites were used. We identified Cys-155 of the FBPase as the one engaged in the formation of the mixed disulfide intermediate with Cys-46 of Trx f.

PLANT THIOREDOXIN SYSTEMS REVISITED.

Thioredoxins, the ubiquitous small proteins with a redox active disulfide bridge, are important regulatory elements in plant metabolism. Initially recognized as regulatory proteins in the reversible light activation of key photosynthetic enzymes, they have subsequently been found in the cytoplasm and in mitochondria. The various plant thioredoxins are different in structure and function. Depending on their intracellular location they are reduced enzymatically by an NADP-dependent or by a ferredoxin (light)-dependent reductase and transmit the regulatory signal to selected target enzymes through disulfide/dithiol interchange reactions. In this review we summarize recent developments that have provided new insights into the structures of several components and into the mechanism of action of the thioredoxin systems in plants.

A Sulfhydryl Oxidase from Chicken Egg White

A dimeric glycoprotein containing one FAD per approximately 80,000 Mr subunit has been isolated from chicken egg white and found to have sulfhydryl oxidase activity with a range of small molecular weight thiols. Dithiothreitol was the best substrate of those tested, with a turnover number of 1030/min, a Km of 150 microM, and a pH optimum of about 7.5. Oxidation of thiol substrates generates hydrogen peroxide in aerobic solution. Anaerobically, the ferricenium ion is a facile alternative electron acceptor. Reduction of the oxidase with dithionite or dithiothreitol under anaerobic conditions yields a two-electron intermediate (EH2) showing a charge transfer band (lambdamax 560 nm; epsilonobs 2.5 mM-1 cm-1). Complete bleaching of the flavin and discharge of the charge transfer complex require a total of four electrons. Borohydride and catalytic photoreduction give the same spectral changes. EH2, but not the oxidized enzyme, is inactivated by iodoacetamide with alkylation of 2.7 cysteine residues/subunit. These data indicate that the oxidase contains a redox-active disulfide bridge generating a thiolate to oxidized flavin charge transfer complex at the EH2 level. Sulfite treatment does not form the expected flavin adduct with the native enzyme but cleaves the active site disulfide, yielding an air-stable EH2-like species. The close functional resemblance of the oxidase to the pyridine nucleotide-dependent disulfide oxidoreductase family is discussed.

Amino acid sequence of the maize ferredoxin:thioredoxin reductase variable subunit.

The ferredoxin:thioredoxin reductase (FTR) is the essential enzyme of the light dependent regulatory system controlling enzyme activities in oxygenic, photosynthetic cells. This protein is composed of two dissimilar subunits, a catalytic subunit containing a [4Fe-4S] cluster and a redox-active disulfide bridge as the active site, and a variable subunit, whose function is not known yet. Whereas size and primary structure of the catalytic subunit from different organisms seem to be well conserved, they are quite variable for the variable subunit. Here we report the complete amino acid sequence of the variable subunit of maize (Zea mays) FTR established by protein sequencing. The subunit contains 97 residues and has a calculated molecular mass of 10939 Da. A sequence comparison shows 40% identity with the variable subunit from spinach and 38% with the one from Anacystis. The identical residues are grouped in three consensus domains, one near the N-terminus, one in the middle of the subunit and one near the C-terminus. We have obtained some evidence indicating that the N-terminal consensus domain is possibly involved in the interaction with the catalytic subunit.

Reduction of ferredoxin:thioredoxin reductase by artificial electron donors

The ferredoxin:thioredoxin reductase is an essential enzyme of the light dependent regulatory system in oxygenic photosynthesis. It is composed of two dissimilar subunits and contains a 4Fe-4S cluster and a redox-active disulfide bridge. Artificial electron donors of redox potentials below -300 mV are capable of reducing the disulfide bridge. Based on our results we speculate that a group of more negative potential than the disulfide bridge is the first acceptor of the electrons in FTR. The chemical reduction of FTR has been used successfully for the detection of the enzyme during its purification.

Amino Acid Sequence of Spinach Ferredoxin:Thioredoxin Reductase Catalytic Subunit and Identification of Thiol Groups Constituting a Redox-Active Disulfide and a [4Fe-4S] Cluster

Ferredoxin:thioredoxin reductase is a [4Fe-4S] protein involved in the light regulation of carbon metabolism in oxygenic photosynthesis. This enzyme catalyses the reduction of thioredoxins with light-generated electrons. Ferredoxin:thioredoxin reductase is composed of two dissimilar subunits, a catalytic subunit, and a variable subunit. The catalytic subunit of spinach ferredoxin:thioredoxin reductase, which contains the redox-active disulfide bridge, was sequenced by conventional protein sequencing techniques and the functional roles of all eight cysteine residues were examined by chemical modifications. The polypeptide chain with a calculated molecular mass of 12,959 Da consists of 113 amino acids and has a calculated isoelectric point of 5.30. Six of the eight cysteine residues are clustered as Cys-Pro-Cys and Cys-His-Cys groups. Cys19 and Cys27 are free cysteines with no catalytic function, Cys54 and Cys84 constitute the redox-active disulfide bridge of the active site, and the remaining four, Cys52, Cys71, Cys73, and Cys82 bind the Fe-S cluster.

Amino acid sequence of spinach ferredoxin:thioredoxin reductase catalytic subunit and identification of thiol groups constituting a redox-active disulfide and a [4Fe-4S] cluster.

Ferredoxin:thioredoxin reductase is a [4Fe-4S] protein involved in the light regulation of carbon metabolism in oxygenic photosynthesis. This enzyme catalyses the reduction of thioredoxins with light-generated electrons. Ferredoxin:thioredoxin reductase is composed of two dissimilar subunits, a catalytic subunit, and a variable subunit. The catalytic subunit of spinach ferredoxin:thioredoxin reductase, which contains the redox-active disulfide bridge, was sequenced by conventional protein sequencing techniques and the functional roles of all eight cysteine residues were examined by chemical modifications. The polypeptide chain with a calculated molecular mass of 12,959 Da consists of 113 amino acids and has a calculated isoelectric point of 5.30. Six of the eight cysteine residues are clustered as Cys-Pro-Cys and Cys-His-Cys groups. Cys19 and Cys27 are free cysteines with no catalytic function, Cys54 and Cys84 constitute the redox-active disulfide bridge of the active site, and the remaining four, Cys52, Cys71, Cys73, and Cys82 bind the Fe-S cluster.

28] Ferredoxin: Thioredoxin system

This chapter focuses on the ferredoxin: thioroxin system. The ferredoxin: thioroxin system is a well-established regulatory system in oxygenic photosynthesis. It regulates, by light-driven thiol-disulfide exchange, the activity of key photosynthetic enzymes. Electrons provided by the excitation of chlorophyll are transferred via ferredoxin and ferredoxin : thioredoxin reductase (FTR) to two types of enzyme-specific thioredoxins, f and m. The thioredoxins as well as FFR each contain one redoxactive disulfide bridge involved in catalysis, and FTR has in addition a 4Fe-4S cluster. The ferredoxin : thioroxin system has been demonstrated in several organisms, and its components have been isolated and characterized. The best known system is the one from spinach. Both types of spinach chloroplast thioredoxins have been cloned and expressed in Escherichia coli, which considerably simplifies their purification. The FTR has also been cloned, but not overexpressed yet. Recombinant thioredoxin f, strongly expressed in E. coli, is present in the cells mostly in insoluble form deposited in inclusion bodies. This presents the advantage that the inclusion bodies can be easily purified and the insoluble thioredoxin freed from the bulk of contaminating proteins.

The primary structure of Escherichia coli glutaredoxin. Distant homology with thioredoxins in a superfamily of small proteins with a redox-active cystine disulfide/cysteine dithiol.

An immunosorbent method using antiglutaredoxin-Sepharose was developed for purification of glutaredoxin in high yield from a mutant strain of Escherichia coli K 12 lacking thioredoxin reductase (C 10-17). The primary structure of the protein was determined by analyses of [14C]carboxymethylated glutaredoxin and its proteolytic fragments obtained by digestions with trypsin, clostripain, chymotrypsin and staphylococcal Glu-specific extracellular protease. The single active-center disulfide has the structure-Cys-Pro-Tyr-Cys-, with the half-cystine residues located at positions 11 and 14 in the polypeptide chain. In total the protein was deduced to have 85 residues corresponding to a molecular weight of 9674 for the reduced form of glutaredoxin, making it one of the smallest known enzymes (a glutathione-disulfide transhydrogenase). The half-cystines are identically spaced and similarly positioned in the N-terminal part of the protein when compared with a corresponding functionally active disulfide/dithiol in thioredoxins. Glutaredoxin is also distantly homologous with thioredoxins from phage T4 and E. coli, but extensive differences, even around the redox-active disulfide, distinguish glutaredoxin from the thioredoxins. Allowing for deletions in the glutaredoxin sequence (or insertions in the T4 thioredoxin sequence) at four places, there are identical residues at 25 positions of the 77 compared (= 32% identity). The results establish that glutaredoxin belongs to the same superfamily of small redox proteins as the thioredoxins. The structures are, however, subject to large changes, only four positions have residues identical among all presently analyzed forms. The fluorescence of reduced and oxidized glutaredoxin demonstrates an increase in the quantum yield of the tyrosine emission upon reduction with dithiothreitol. Differences in the spectra support the presence of tyrosine adjacent to the redox-active disulfide bridge. They also confirm that glutaredoxin lacks the disulfide-adjacent tryptophan residues of E. coli thioredoxin. There are known to be great differences between the bacterial E. coli and phage T4 forms of thioredoxin. The glutaredoxin structure is most similar to the phage type, both with respect to size of the polypeptide chain and to actual sequence. From the structural results and the previously known functional similarities it appears possible that the phage thioredoxin may have evolved from an early glutaredoxin gene. The mixed properties are compatible with this conclusion, the superfamily assignment, and the differences in biological activity.

The catalytic mechanism of glutathione reductase as derived from x-ray diffraction analyses of reaction intermediates.

The mode of binding of NADPH and oxidized glutathione to the flavoenzyme glutathione reductase has been determined by x-ray crystallography. Furthermore, two intermediates of the reaction have been produced in the crystal and have been structurally elucidated. All these analyses were done at 0.3 nm resolution. The results allow the stereochemical description of the mechanism of the enzyme. The dinucleotide NADPH is bound in an extended conformation with the nicotinamide ring stacking onto the re-face of the flavin part of FAD, and adenine located at the protein surface. The binding of NADPH results in the 2-electron reduced form of the enzyme, EH2. This form has also been analyzed without any ligand bound. In EH2 the redoxactive disulfide bridge of the protein, which lies at the si-face of the flavin ring, is opened and the sulfur of Cys-58 moves by about 0.1 nm into a position where it can attack one of the sulfurs of the substrate oxidized glutathione. This interchange leads to a mixed glutathione-protein disulfide, which can be stabilized in crystals and has been analyzed. By selectively reacting Cys-58 with iodoacetamide the crystalline enzyme can be blocked in its EH2 state. The imidazole of His-467' is near to all sulfurs taking part in the disulfide bridge exchange and is therefore certainly crucial for catalysis. The crystallographic results establish that electrons flow from NADPH to the substrate GSSG via flavin and the redoxactive protein disulfide bridge. This is consistent with the scheme that has been postulated from biochemical, spectroscopic, and model studies.