Crystal structure of sulfonic peroxiredoxin Ahp1 in complex with thioredoxin Trx2 mimics a conformational intermediate during the catalytic cycle.

Peroxiredoxin (Prx) is a thiol-based peroxidase that eliminates reactive oxygen species to avoid oxidative damage. Alkyl hydroperoxide reductase Ahp1 is a novel and specific typical 2-cysteine Prx. Here, we present the crystal structure of sulfonic Ahp1 complexed with thioredoxin Trx2 at 2.12 Å resolution. This structure implies that the transient Ahp1-Trx2 complex during the catalytic cycle already have an ability to decompose the peroxides. Structural analysis reveals that the segment glutamine23-lysine32 juxtaposed to the resolving cysteine (CR) of Ahp1 moves inward to generate a compact structure upon peroxidatic cysteine (CP) overoxidation, resulting in the breakdown of several conserved hydrogen bonds formed by Ahp1-Trx2 complex interaction. Structural comparisons suggest that the structure of sulfonic Ahp1 represents a novel conformation of Ahp1, which can mimic a conformational intermediate between the reduced and oxidized forms. Therefore, this study may provide a new structural insight into the intermediate state in which the segment glutamine23-lysine32 juxtaposed to the cysteine31 (CR) undergoes a conformational change upon cysteine62 (CP) oxidation to prepare for the formation of an intermolecular CP-CR disulfide bond during Ahp1 catalytic cycle.

Thioredoxin H (TrxH) contributes to adversity adaptation and pathogenicity of Edwardsiella piscicida.

Thioredoxins (Trxs) play an important role in defending against oxidative stress and keeping disulfide bonding correct to maintain protein function. Edwardsiella piscicida, a severe fish pathogen, has been shown to encode several thioredoxins including TrxA, TrxC, and TrxH, but their biological roles remain unknown. In this study, we characterized TrxH of E. piscicida (named TrxHEp) and examined its expression and function. TrxHEp is composed of 125 residues and possesses typical thioredoxin H motifs. Expression of trxHEp was upregulated under conditions of oxidative stress, iron starvation, low pH, and during infection of host cells. trxHEp expression was also regulated by ferric uptake regulator (Fur), an important global regulatory of E. piscicida. Compared to the wild type TX01, a markerless trxHEp in-frame mutant strain TX01∆trxH exhibited markedly compromised tolerance of the pathogen to hydrogen peroxide, acid stress, and iron deficiency. Deletion of trxHEp significantly retarded bacterial biofilm growth and decreased resistance against serum killing. Pathogenicity analysis shows that the inactivation of trxHEp significantly impaired the ability of E. piscicida to invade host cells, reproduce in macrophages, and infect host tissues. Introduction of a trans-expressed trxH gene restored the lost virulence of TX01∆trxH. There is likely to be a complex relationship of functional complementation or expression regulation between TrxH and another two thioredoxins, TrxA and TrxC, of E. piscicida. This is the first functional report of TrxH in fish pathogens, and the findings suggest that TrxHEp is essential for coping with adverse circumstances and contributes to host infection of E. piscicida.

Isolation and functional characterization of two thioredoxin h isoforms from grape.

Thioredoxins (Trxs) are small ubiquitous proteins that participate in dithiol-disulfide exchange reactions. In contrast to animals and prokaryotes, plants possess different types of Trxs that play a vital role in a number of different cellular processes. Two full-length cDNAs encoding different Trx h isoforms, designated VvTrx h2 and VvTrx h3, were isolated and cloned from grape (Vitis vinifera L. cv. Askari) berry tissue by rapid amplification of cDNA ends (RACE) method. VvTrx h2 and VvTrx h3 were heterologously expressed in Escherichia coli and their activities were compared using DTT-dependent insulin reduction and 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) reduction activities. The NADPH-dependent DTNB reduction assay demonstrated that the both VvTrx h isoforms were reduced by NADPH-dependent thioredoxin reductase (NTR) from E. coli. Under heat shock treatment, the recombinant VvTrx h proteins formed the oligomeric structures at above 50 °C with a decrease in their disulfide reductase activities. The redox-dependent structural changes of VvTrx h2 and VvTrx h3 revealed that their oligomeric structures were changed into monomers and significantly increased their disulfide reductase activities. Furthermore, the both recombinant proteins were able to conserve a DTNB reduction activity even after 15 min heating at 99 °C.

Helicoverpa-inducible Thioredoxin h from Cicer arietinum: structural modeling and potential targets.

Thioredoxins are small and universal proteins, which are involved in the cell redox regulation. In plants, they participate in a broad range of biochemical processes like self-incompatibility, seed germination, pathogen & pest defense and oxidative stress tolerance. The h-type of thioredoxin (Trx-h) protein represents the largest Trx family. Herein, we characterized the Helicoverpa - inducible Trx h from an important legume, Cicer arietinum, CaHaTrx-h, 'CGFS' type Trxs, which encodes for a 113 amino acids long protein and possess characteristic motifs "FLKVDVDE" and "VVDFTASWCGPCRFIAPIL" and 73% sequence identity with AtTrx-h. Homology modeling and simulation of the target showed that the extended ß-sheet regions remain stable during the simulation while the helical regions fluctuate between alpha and 3-10 helical forms and highlights the flexibility of helix2-helix3 and terminal regions probably to accommodate an approaching protein target and facilitate their interaction. During the simulation, the structure exists in five energy minima clusters with biggest cluster size belonging to 20-25 ns time frames. PR-5 and Mannitol Dehydrogenase were nominated as potential targets and share close interaction with CaHaTrx-h via disulfide bond reduction. The study is an effort in the direction of understanding stress-related mechanisms in crop plants to overcome losses in agricultural yield.

Recombinant ACHT1 from Arabidopsis thaliana: crystallization and X-ray crystallographic analysis.

Thioredoxins (Trxs) play important roles in chloroplasts by linking photosynthetic light reactions to a series of plastid functions. They execute their function by regulating the oxidation and reduction of disulfide bonds. ACHT1 (atypical cysteine/histidine-rich Trx1) is a thylakoid-associated thioredoxin-type protein found in the Arabidopsis thaliana chloroplast. Recombinant ACHT1 protein was overexpressed in Escherichia coli, purified and crystallized by the vapour-diffusion method. The crystal diffracted to 1.7 Šresolution and a complete X-ray data set was collected. Preliminary crystallographic analysis suggested that the crystals belonged to space group C2221, with unit-cell parameters a = 102.7, b = 100.6, c = 92.8 Å.

Thioredoxin h isoforms from rice are differentially reduced by NADPH/thioredoxin or GSH/glutaredoxin systems.

Rice (Oryza sativa L.) has multiple potential genes encoding thioredoxin (Trx) h and NADP-thioredoxin reductase (NTR). These NTR and Trx h isoforms, known as cytoplasmic NTR/Trx system along with multiple members of glutaredoxin (Grx) family constitute a complex redox control system in rice. In the present study, we investigated the kinetic parameters of two rice NTRs, OsNTRA and OsNTRB, toward three endogenous Trx h isoforms, OsTrx1, OsTrx20, and OsTrx23. The results showed that in contrast with OsTrx1 and OsTrx23, the isoform OsTrx20 was not reduced by OsNTR isoforms. The kcat/Km values of OsNTRB and OsNTRA toward OsTrx1 was six- and 13-fold higher than those values toward OsTrx23, respectively, suggesting that OsNTR isoforms do not reduce different OsTrx h isoforms, equivalently. Furthermore, the possible reduction of OsTrx isoforms by the glutathione (GSH)/Grx system was investigated through the heterologous expression of a gene encoding OsGrx9, a bicysteinic CPYC Grx found in rice. Whereas OsTrx23 was not reduced by GSH, OsTrx20 and with less efficiently OsTrx1 were reduced by GSH or GSH/Grx. Therefore, it seems that OsTrx1 can be reduced either by OsNTR or GSH/Grx. These data for the first time provides an evidence for cross-talking between NTR/Trx and GSH/Grx systems in rice.

Analysis of Arabidopsis thioredoxin-h isotypes identifies discrete domains that confer specific structural and functional properties.

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(∆19)] and site-directed mutagenesis of AtTrx-h3. AtTrx-h2-N(∆19) 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.

Cloning and characterization of thioredoxin h in the three-pistil line of common wheat.

Thioredoxin h (Trxh) is a ubiquitous protein that reduces disulfides in target proteins, and is itself reduced by NADPH-thioredoxin reductase. In the current study, the complementary DNA sequence and the genomic sequence of the three-pistil (TP) line of common wheat (Triticum aestivum L.) were obtained from spikes through reverse transcription-polymerase chain reaction (RT-PCR) and touchdown-PCR. Sequence alignment of amino acids of TPTrxh then allowed for predictions of its physicochemical properties, secondary structures, tertiary structures, and functional domains. Furthermore, the TPTrxh gene was overexpressed in Escherichia coli and its activity was demonstrated using a dithiothreitol-dependent insulin assay. The expression patterns of TPTrxh were analyzed through real-time RT-PCR in different tissues and across different developmental stages of young spikes. The complementary DNA of TPTrxh was found to be 411 bp in length, encoding 118 amino acids. Its genomic sequence was determined to be 2632 bp, possessing 3 exons and 2 introns. Functional domain analysis indicated that TPTrxh contained a WCGPC motif located at the end of the second ß-fold and on the initial side of the second α-helix. The TPTrxh protein reduces intramolecular and intermolecular disulfide bridges in target proteins. Young spikes contain higher levels of TPTrxh transcripts than do stems and leaves. The transcript levels in the young spikes (2-5 mm in length) of the Chinese Spring TP line increased 2.84-fold relative to those of young spikes (2-5 mm in length) of the Chinese Spring line. These data provide a basis for future research into the function of Trxh, and offer further insight into the molecular mechanism of the TP mutation in wheat.

Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed.

Inactivation of barley limit dextrinase inhibitor by thioredoxin-catalysed disulfide reduction.

Barley limit dextrinase (LD) that catalyses hydrolysis of α-1,6 glucosidic linkages in starch-derived dextrins is inhibited by limit dextrinase inhibitor (LDI) found in mature seeds. LDI belongs to the chloroform/methanol soluble protein family (CM-protein family) and has four disulfide bridges and one glutathionylated cysteine. Here, thioredoxin is shown to progressively reduce disulfide bonds in LDI accompanied by loss of activity. A preferential reduction of the glutathionylated cysteine, as indicated by thiol quantification and molecular mass analysis using electrospray ionisation mass spectrometry, was not related to LDI inactivation. LDI reduction is proposed to cause conformational destabilisation leading to loss of function.

A new isoform of thioredoxin h group in potato, SbTRXh1, regulates cold-induced sweetening of potato tubers by adjusting sucrose content.

UNLABELLED: In order to study the molecular mechanism of the cold-induced sweetening (CIS) of potato tubers, a novel isoform of thioredoxin h group, SbTRXh1, which was up-regulated early in the 4 °C storage of CIS-resistant potato (Solanum berthaultii) tubers, was cloned in present research. The genetic transformation of over-expression (OE) and RNA interference (RNAi) of SbTRXh1 into potato cv. E-Potato 3 (E3) was carried out to clarify its function in CIS regulation. The results showed that the transcripts of SbTRXh1 in either OE- or RNAi-tubers were strongly induced in 4 °C storage and quantitatively related to the reducing sugar (RS) accumulation, indicating that SbTRXh1 is involved in the CIS process of potato tubers. Regression analysis between the transcripts and protein contents of SbTRXh1 showed a very significant logarithmic relationship implying that the expression of SbTRXh1 may be mainly regulated at transcriptional level. Further monitoring the variation of the sugar contents in cold-stored tubers demonstrated a linear relationship between RS and sucrose (Suc). Thus, it can be inferred that SbTRXh1 may function in the Suc-RS pathway for CIS regulation of potato tubers. KEY MESSAGE: SbTRXh1 is primarily demonstrated to be involved in the regulation of cold-induced sweetening (CIS) of potato tubers, and it may function in the Suc-RS pathway for CIS regulation.

Isolation, identification and sequence analysis of a thioredoxin h gene, a member of subgroup III of h-type Trxs from grape (Vitis vinifera L. cv. Askari).

Thioredoxins (Trxs) are small ubiquitous proteins which play a regulatory role in a variety of cellular processes. In contrast to other organisms, plants have a great number of Trx types, consisting of six well-defined groups: f, m, x, and y in chloroplasts, o in mitochondria, and h mainly in cytosol. A full-length cDNA, designated VvCxxS2, encoding Trx h polypeptide was isolated and cloned from grape (Vitis vinifera L. cv. Askari) berries organ by reverse transcription polymerase chain reaction (RT-PCR). The cDNA was 381 bp nucleotides in length with a deduced amino acid of 126 residues, possessing a WCIPS active site, which belongs to the subgroup III of h-type Trxs based on phylogenetic analysis. The calculated molecular mass and the predicted isoelectric point of the deduced polypeptide are 14.25 kDa and 4.68, respectively. Nucleotide sequence analysis of genomic DNA fragment of VvCxxS2 gene revealed that this gene possesses two introns at positions identical to the previously sequenced Trx h genes. A modeling analysis indicated that VvCxxS2 shares a common structure with other Trxs, and is preferably reduced by Grx rather than NADPH-dependent thioredoxin reductase (NTR). The deduced protein sequence showed a high similarity to Trx h from other plants, in particular from castor bean (Ricinus communis), Betula pendula and sweet orange (Citrus sinensis). Semiquantitative RT-PCR experiments indicated that the transcripts of VvCxxS2 gene are present in all plant organs and different developmental stages. In addition, the higher expression of the VvCxxS2 gene was observed in berry organ as compared to the other organs.

Identification and characterization of thioredoxin h isoforms differentially expressed in germinating seeds of the model legume Medicago truncatula.

Thioredoxins (Trxs) h, small disulfide reductases, and NADP-thioredoxin reductases (NTRs) have been shown to accumulate in seeds of different plant species and play important roles in seed physiology. However, little is known about the identity, properties, and subcellular location of Trx h isoforms that are abundant in legume seeds. To fill this gap, in this work, we characterized the Trx h family of Medicago truncatula, a model legume, and then explored the activity and localization of Trx h isoforms accumulating in seeds. Twelve Trx h isoforms were identified in M. truncatula. They belong to the groups previously described: h1 to h3 (group I), h4 to h7 (group II), and h8 to h12 (group III). Isoforms of groups I and II were found to be reduced by M. truncatula NTRA, but with different efficiencies, Trxs of group II being more efficiently reduced than Trxs of group I. In contrast, their insulin disulfide-reducing activity varies greatly and independently of the group to which they belong. Furthermore, Trxs h1, h2, and h6 were found to be present in dry and germinating seeds. Trxs h1 and, to a lesser extent, h2 are abundant in both embryonic axes and cotyledons, while Trx h6 is mainly present in cotyledons. Thus, M. truncatula seeds contain distinct isoforms of Trx h that differ in spatial distribution and kinetic properties, suggesting that they play different roles. Because we show that Trx h6 is targeted to the tonoplast, the possible role of this isoform during germination is finally discussed.

Kinetic and thermodynamic properties of two barley thioredoxin h isozymes, HvTrxh1 and HvTrxh2.

Barley thioredoxin h isozymes 1 (HvTrxh1) and barley thioredoxin h isozymes 2 (HvTrxh2) show distinct spatiotemporal distribution in germinating seeds. Using a novel approach involving measurement of bidirectional electron transfer rates between Escherichia coli thioredoxin, which exhibits redox-dependent fluorescence, and the barley isozymes, reaction kinetics and thermodynamic properties were readily determined. The reaction constants were approximately 60% higher for HvTrxh1 than HvTrxh2, while their redox potentials were very similar. The primary nucleophile, CysN, of the active site Trp-CysN-Gly-Pro-CysC motif has an apparent pKa of 7.6 in both isozymes, as found by iodoacetamide titration, but showed approximately 70% higher reactivity in HvTrxh1, suggesting significant functional difference between the isozymes.

Heat-shock and redox-dependent functional switching of an h-type Arabidopsis thioredoxin from a disulfide reductase to a molecular chaperone.

A large number of thioredoxins (Trxs), small redox proteins, have been identified from all living organisms. However, many of the physiological roles played by these proteins remain to be elucidated. We isolated a high M(r) (HMW) form of h-type Trx from the heat-treated cytosolic extracts of Arabidopsis (Arabidopsis thaliana) suspension cells and designated it as AtTrx-h3. Using bacterially expressed recombinant AtTrx-h3, we find that it forms various protein structures ranging from low and oligomeric protein species to HMW complexes. And the AtTrx-h3 performs dual functions, acting as a disulfide reductase and as a molecular chaperone, which are closely associated with its molecular structures. The disulfide reductase function is observed predominantly in the low M(r) forms, whereas the chaperone function predominates in the HMW complexes. The multimeric structures of AtTrx-h3 are regulated not only by heat shock but also by redox status. Two active cysteine residues in AtTrx-h3 are required for disulfide reductase activity, but not for chaperone function. AtTrx-h3 confers enhanced heat-shock tolerance in Arabidopsis, primarily through its chaperone function.

Crystal structures of barley thioredoxin h isoforms HvTrxh1 and HvTrxh2 reveal features involved in protein recognition and possibly in discriminating the isoform specificity.

H-type thioredoxins (Trxs) constitute a particularly large Trx sub-group in higher plants. Here, the crystal structures are determined for the two barley Trx h isoforms, HvTrxh1 and HvTrxh2, in the partially radiation-reduced state to resolutions of 1.7 A, and for HvTrxh2 in the oxidized state to 2.0 A. The two Trxs have a sequence identity of 51% and highly similar fold and active-site architecture. Interestingly, the four independent molecules in the crystals of HvTrxh1 form two relatively large and essentially identical protein-protein interfaces. In each interface, a loop segment of one HvTrxh1 molecule is positioned along a shallow hydrophobic groove at the primary nucleophile Cys40 of another HvTrxh1 molecule. The association mode can serve as a model for the target protein recognition by Trx, as it brings the Met82 Cgamma atom (gamma position as a disulfide sulfur) of the bound loop segment in the proximity of the Cys40 thiol. The interaction involves three characteristic backbone-backbone hydrogen bonds in an antiparallel beta-sheet-like arrangement, similar to the arrangement observed in the structure of an engineered, covalently bound complex between Trx and a substrate protein, as reported by Maeda et al. in an earlier paper. The occurrence of an intermolecular salt bridge between Glu80 of the bound loop segment and Arg101 near the hydrophobic groove suggests that charge complementarity plays a role in the specificity of Trx. In HvTrxh2, isoleucine corresponds to this arginine, which emphasizes the potential for specificity differences between the coexisting barley Trx isoforms.