Molecular cloning and characterization of a 2-Cys peroxiredoxin from Taenia solium

A Taenia solium 2-Cys peroxiredoxin (Ts2-CysPrx) clone was isolated from a T. solium adult cDNA library. The clone encodes a polypeptide comprising 197 amino acids with a predictive Mr = 21,836. It has the 2 classical cysteine domains from the typical 2-Cys peroxiredoxins, and its primary amino acid sequence shows higher identity with 2 Echinococcus 2-Cys peroxiredoxins. Northern and Southern blot hybridizations exhibit an mRNA with a size of -1.0 kb, encoded by 1 gene. Ts2-CysPrx was expressed in Escherichia coli and purified by anion-exchange chromatography. Biochemical analysis showed Ts2-CysPrx is a dimer composed by monomers of -22 kDa that presented activity with hydrogen peroxide (H2O2) and cumene hydroperoxide. It presented the catalytic mechanism for a typical 2-CysPrx because the homodimeric oxidized form is reduced to a monomeric form by thioredoxin (Trx) and by dithiothreitol (DTT) and was converted to a homodimeric oxidized form by H2O2. Western blot studies using antibodies against Ts2-CysPrx revealed that the protein is expressed during the entire T. solium life cycle, as in other Taenia species. Immunohistochemical studies indicated that Ts2-CysPrx is localized on the tegument and in tegumentary and muscle cells of cysticerci. We also show that T. crassiceps cysticerci can tolerate H2O2 levels of 2.5 mM for 2.5 hr.     

Biochemical and molecular characterization of the mitochondrial peroxiredoxin PsPrxII F from Pisum sativum.

The pea peroxiredoxin homologue PsPrxII F of the Arabidopsis thaliana mitochondrial AtPrxII F was isolated as cDNA and genomic DNA, and characterized in respect to its biochemical and molecular properties. The deduced amino acid sequence contains an N-terminal targeting address for mitochondrial import. Mitochondrial location of PsPrxII F was confirmed by immunocytochemistry. The mature enzyme, without the transit peptide, has a molecular mass of 18.75 kDa, and, at positions 59 and 84, carries the two catalytic cysteinyl residues which are characteristic for this particular Prx subgroup. Activity of site-directed mutagenized C84S-variant lacking the so-called resolving Cys dropped to about 12% of WT Prx while C59S lost its peroxidatic activity completely. Likewise, WT PsPrxII F and C84S-variant but not C59S protected plasmid DNA against strand breakage in a mixed function oxidation assay. WT PrxII F and the variant proteins aggregated to high mass oligomers not yet described for type II Prx. Upon oxidation with hydrogen peroxide PsPrxII F focussed in a series of spots of distinct pI but similar molecular masses in two-dimensional gels indicating different oxidation states of the protein. Using this technique, partial oxidation was also detected in leaf extracts and isolated mitochondria. PsPrxII F mRNA and protein accumulated in cold and heavy metals treated pea plants suggesting a particular function under stress.     

Molecular characterization of a 2-Cys peroxiredoxin induced by abiotic stress in mungbean

A mungbean low temperature-inducible VrPrx1 encoding 2-Cys peroxiredoxin (2-Cys Prx) was cloned by subtractive suppression hybridization. The deduced VrPrx1 amino acid sequence showed highest sequence homology to 2-Cys Prxs of Phaseolus vulgaris (95%), Pisum sativum (89%), and Arabidopsis thaliana (87%). VrPrx1 RNA and protein levels were increased by low temperature, hydrogen peroxide (H₂O₂), and wounding but decreased by high salinity, drought, and exogenous abscisic acid. Recombinant His-tagged VrPrx1 recombinant protein protected DNA and glutamine synthetase activity from degradation via the thiol/Fe(III) oxygen mixed-function oxidation system, and exhibited peroxidase activity to H₂O₂ in the presence of the reducing agent dithiothreitol (DTT) in vitro. The oxidized dimers and oligomers of the VrPrx1 recombinant protein were reduced to monomers by DTT or thioredoxin. Subcellular localization studies confirmed that VrPrx1-GFP was targeted to the plastid. To evaluate the function of VrPrx1 in planta, the antioxidant activities and photosynthetic efficiency were investigated in VrPrx1-overexpressing Arabidopsis plants. VrPrx1 ectopic expression conferred improved photosynthetic efficiency under oxidative stress conditions. Hence, mungbean VrPrx1 may play an important role in protecting the photosynthetic apparatus against oxidative and abiotic stress conditions.     

Cloning and characterization of the P subunit of glycine decarboxylase from pea (Pisum sativum).

A pea leaf cDNA library constructed in lambda gt11 was screened with an antibody raised to the P subunit of glycine decarboxylase. One of the positive clones isolated was sequenced and shown to contain an open reading frame, which encoded the entire P subunit polypeptide. Aligning the deduced amino acid sequence with the amino acid sequence determined directly from the NH2 terminus of the mature P subunit shows the presence of a putative 86 amino acid leader sequence, presumably required for import into the mitochondria, and gives a Mr of the mature protein of 105,000. Comparison of this deduced amino acid sequence with the sequence of a pyridoxal phosphate-containing peptide isolated from the P subunit of chicken liver glycine decarboxylase shows remarkable conservation. The P subunit, however, shows little sequence homology with other published amino acid decarboxylases. Expression of the P subunit mRNA shows a pattern very similar to that of the corresponding polypeptide: it is strongly light induced and is expressed at a much higher level in leaves than in other tissues. Southern blot analysis suggests that the P subunit is encoded by a small multigene family.     

Engineering of 2-Cys peroxiredoxin for enhanced stress-tolerance

A typical 2-cysteine peroxiredoxin (2-Cys Prx)-like protein (PpPrx) that alternatively acts as a peroxidase or a molecular chaperone in Pseudomonas putida KT2440 was previously characterized. The dual functions of PpPrx are regulated by the existence of an additional Cys(112) between the active Cys(51) and Cys(171) residues. In the present study, additional Cys residues (Cys(31), Cys(112), and Cys(192)) were added to PpPrx variants to improve their enzymatic function. The optimal position of the additional Cys residues for the dual functionality was assessed. The peroxidase activities of the S31C and Y192C mutants were increased 3- to 4-fold compared to the wild-type, while the chaperone activity was maintained at > 66% of PpPrx. To investigate whether optimization of the dual functions could enhance stress-tolerance in vivo, a complementation study was performed. The S31C and Y192C mutants showed a much greater tolerance than other variants under a complex condition of heat and oxidative stresses. The optimized dual functions of PpPrx could be adapted for use in bioengineering systems and industries, such as to develop organisms that are more resistant to extreme environments.     

ATP-dependent modulation and autophosphorylation of rapeseed 2-Cys peroxiredoxin

2-Cys peroxiredoxins (2-Cys Prx) are ubiquitous thiol-containing peroxidases that have been implicated in antioxidant defense and signal transduction. Although their biochemical features have been extensively studied, little is known about the mechanisms that link the redox activity and non-redox processes. Here we report that the concerted action of a nucleoside triphosphate and Mg(2+) on rapeseed 2-Cys Prx reversibly impairs the peroxidase activity and promotes the formation of high molecular mass species. Using protein intrinsic fluorescence in the analysis of site-directed mutants, we demonstrate that ATP quenches the emission intensity of Trp179, a residue close to the conserved Cys175. More importantly, we found that ATP facilitates the autophosphorylation of 2-Cys Prx when the protein is successively reduced with thiol-bearing compounds and oxidized with hydroperoxides or quinones. MS analyses reveal that 2-Cys Prx incorporates the phosphoryl group into the Cys175 residue yielding the sulfinic-phosphoryl [Prx-(Cys175)-SO(2)PO(3)(2-)] and the sulfonic-phosphoryl [Prx-(Cys175)-SO(3)PO(3)(2-)] anhydrides. Hence, the functional coupling between ATP and 2-Cys Prx gives novel insights into not only the removal of reactive oxygen species, but also mechanisms that link the energy status of the cell and the oxidation of cysteine residues.     

Cloning, overexpression, and characterization of peroxiredoxin and NADH peroxiredoxin reductase from Thermus aquaticus

The genes for peroxiredoxin (Prx) and NADH:peroxiredoxin oxidoreductase (PrxR) have been cloned from the thermophilic bacterium Thermus aquaticus. prx is located upstream from prxR, the two genes being separated by 13 bases. The amino acid sequences show that Prx is related to two-cysteine peroxiredoxins from a range of organisms and that PrxR resembles NADH-dependent flavoenzymes that catalyze the reduction of peroxiredoxins in mesophilic bacteria. The sequence of PrxR also resembles those of thioredoxin reductases (TrxR) from thermophiles but with an N-terminal extension of about 200 residues. PrxR has motifs for two redox-active disulfides, one in the FAD-binding site, as occurs in TrxR, and the other in the N-terminal extension. The molecular masses of the monomers of Prx and PrxR are 21.0 and 54.9 kDa, respectively; both enzymes exist as multimers. The recombinant flavoenzyme requires 3 mol equivalents of dithionite for full reduction, as is consistent with 1 FAD and 2 disulfides per monomer. PrxR and Prx together catalyze the anaerobic reduction of hydrogen peroxide. The activity of Prx is much less than has been observed with homologous proteins. Prx appears to be inactivated by cumene hydroperoxide. PrxR itself has low peroxidase activity.     

Plasmodium falciparum 2-Cys peroxiredoxin reacts with plasmoredoxin and peroxynitrite

Thioredoxin peroxidase 1 (TPx1) of the malarial parasite Plasmodium falciparum is a 2-Cys peroxiredoxin involved in the detoxification of reactive oxygen species and - as shown here - of reactive nitrogen species. As novel electron acceptor of reduced TPx1, we characterised peroxynitrite; the rate constant for ONOO- reduction by the enzyme (1 x 10(6) M(-1) s(-1) at pH 7.4 and 37 degrees C) was determined by stopped-flow measurements. As reducing substrate of TPx1, we identified - aside from thioredoxin - plasmoredoxin; this 22-kDa protein occurs only in malarial parasites. When studying the potential roles of Cys74 and Cys170 of Tpx1 in catalysis, as well as in oligomerisation behaviour, we found that replacement of Cys74 by Ala influenced neither the dimerisation nor enzymatic activity of TPx1. In the C170A mutant, however, the kcat/Km for reduced Trx as a substrate was shown to be approximately 50-fold lower and, in contrast to the wild-type enzyme, covalently linked dimers were not formed. For the catalytic cycle of TPx1, we conclude that oxidation of the peroxidatic Cys50 by the oxidising substrate is followed by the formation of an intermolecular disulfide bond between Cys50 and Cys170' of the second subunit, which is then attacked by an external electron donor such as thioredoxin or plasmoredoxin.     

Crystal Structure of AhpE from Mycobacterium tuberculosis, a 1-Cys peroxiredoxin

All living systems require protection against the damaging effects of reactive oxygen species. The genome of Mycobacterium tuberculosis, the cause of TB, encodes a number of peroxidases that are thought to be active against organic and inorganic peroxides, and are likely to play a key role in the ability of this organism to survive within the phagosomes of macrophages. The open reading frame Rv2238c in M.tuberculosis encodes a 153-residue protein AhpE, which is a peroxidase of the 1-Cys peroxiredoxin (Prx) family. The crystal structure of AhpE, determined at 1.87 A resolution (R(cryst)=0.179, R(free)=0.210), reveals a compact single-domain protein with a thioredoxin fold. AhpE forms both dimers and octamers; a tightly-associated dimer and a ring-like octamer, generated by crystallographic 4-fold symmetry. In this native structure, the active site Cys45 is in its oxidized, sulfenic acid (S-O-H) state. A second crystal form of AhpE, obtained after soaking in sodium bromide and refined at 1.90 A resolution (R(cryst)=0.242, R(free)=0.286), reveals the reduced structure. In this structure, a conformational change in an external loop, in two of the four molecules in the asymmetric unit, allows Arg116 to stabilise the Cys45 thiolate ion, and concomitantly closes a surface channel. This channel is identified as the likely binding site for a physiological reductant, and the conformational change is inferred to be important for the reaction cycle of AhpE.     

Cloning and expression of cytosolic phosphoglycerate kinase from pea (Pisum sativum L.)

In common with several other respiratory and photosynthetic enzymes, a sub-population of cytosolic phosphoglycerate kinase (PGK) occurs in the nucleus in pea leaves and shoots. The full-length cDNA encoding pea cytosolic PGK has been cloned and sequenced, revealing not only the PGK 'signature' but also a nuclear localization signal (NLS). A translational fusion of PGK and GFP was used to transform tobacco BY-2 cells resulting in GFP locating to the cell nuclei.     

Crystal structure of decameric 2-Cys peroxiredoxin from human erythrocytes at 1.7 A resolution

BACKGROUND: The peroxiredoxins (Prxs) are an emerging family of multifunctional enzymes that exhibit peroxidase activity in vitro, and in vivo participate in a range of cellular processes known to be sensitive to reactive oxygen species. Thioredoxin peroxidase B (TPx-B), a 2-Cys type II Prx from erythrocytes, promotes potassium efflux and down-regulates apoptosis and the recruitment of monocytes by endothelial tissue. RESULTS: The crystal structure of human decameric TPx-B purified from erythrocytes has been determined to 1.7 [corrected)] A resolution. The structure is a toroid comprising five dimers linked end-on through predominantly hydrophobic interactions, and is proposed to represent an intermediate in the in vivo reaction cycle. In the crystal structure, Cys51, the site of peroxide reduction, is oxidised to cysteine sulphinic acid. The residue Cys172, lies approximately 10 A away from Cys51 [corrected]. CONCLUSIONS: The oxidation of Cys51 appears to have trapped the structure into a stable decamer, as confirmed by sedimentation analysis. A comparison with two previously reported dimeric Prx structures reveals that the catalytic cycle of 2-Cys Prx requires significant conformational changes that include the unwinding of the active-site helix and the movement of four loops. It is proposed that the stable decamer forms in vivo under conditions of oxidative stress. Similar decameric structures of TPx-B have been observed by electron microscopy, which show the protein associated with the erythrocyte membrane.     

A 2-Cys peroxiredoxin regulates peroxide-induced oxidation and activation of a stress-activated MAP kinase

Oxidative stress-induced cell damage is an important component of many diseases and ageing. In eukaryotes, activation of JNK/p38 stress-activated protein kinase (SAPK) signaling pathways is critical for the cellular response to stress. 2-Cys peroxiredoxins (2-Cys Prx) are highly conserved, extremely abundant antioxidant enzymes that catalyze the breakdown of peroxides to protect cells from oxidative stress. Here we reveal that Tpx1, the single 2-Cys Prx in Schizosaccharomyces pombe, is required for the peroxide-induced activation of the p38/JNK homolog, Sty1. Tpx1 activates Sty1, downstream of previously identified redox sensors, by a mechanism that involves formation of a peroxide-induced disulphide complex between Tpx1 and Sty1. We have identified conserved cysteines in Tpx1 and Sty1 that are essential for normal peroxide-induced Tpx1-Sty1 disulphide formation and Tpx1-dependent regulation of peroxide-induced Sty1 activation. Thus we provide new insight into the response of SAPKs to diverse stimuli by revealing a mechanism for SAPK activation specifically by oxidative stress.     

Structural determinants of multimerization and dissociation in 2-Cys peroxiredoxin chaperone function

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Cloning of a gene for an acyl-CoA dehydrogenase from Pisum sativum L. and purification and characterization of its product as an isovaleryl-CoA dehydrogenase

Isovaleryl-CoA dehydrogenase (IVD, EC ) catalyzes the third step in the catabolism of leucine in mammals. Deficiency of this enzyme leads to the clinical disorder isovaleric acidemia. IVD has been purified and characterized from human and rat liver, and the x-ray crystallographic structure of purified recombinant human IVD has been reported. Nothing is known about IVD activity in plants, although cDNA clones from Arabidopsis thaliana and partial sequences from Gossypium hirsutum and Oryza sativa have been identified as putative IVDs based on sequence homology and immuno cross-reactivity. In this report we describe the identification and characterization of an IVD from pea, purification of the enzyme using a novel and rapid auxin affinity chromatography matrix, and cloning of the corresponding gene. At the amino acid level, pea IVD is 60% similar to human and rat IVD. The specific activity and abundance of plant IVD was found to be significantly lower than for its human counterpart and exhibits developmental regulation. Substrate specificity of the plant enzyme is similar to the human IVD, and it cross-reacts to anti-human IVD antibodies. Molecular modeling of the pea enzyme based on the structure of human IVD indicates a high degree of structural similarity among these enzymes. Glu-244, shown to function as the catalytic base in human IVD along with most of the amino acids that make up the acyl CoA binding pocket, is conserved in pea IVD. The genomic structure of the plant IVD gene consists of 13 exons and 12 introns, spanning approximately 4 kilobases, and the predicted RNA splicing sites exhibit the extended consensus sequence described for other plant genes.     

Biochemical characterization of a novel 2-Cys peroxiredoxin from <Emphasis Type="Italic">Antrodia camphorata</Emphasis>

Peroxiredoxins (Prxs) play important roles in antioxidation and cell signaling. A gene encoding a novel 2-Cys Prx was identified based on sequence homology in an expressed sequence tag database of the Antrodia camphorata, a medicinal mushroom found only in Taiwan. The 2-Cys Prx cDNA (940 bp) encodes a protein of 188 amino acid residues with calculated molecular mass of 20,965 Da and a pI of 5.89. The coding region was subcloned into pAVD10, transformed into Escherichia coli, and expressed as a His-tagged fusion protein. The purified enzyme was characterized under various conditions. The Prx retained 68% activity after being heated at 60°C for 2 min. It was stable under a broad pH range from 5 to 11. The enzyme activity was slightly decreased in the presence of 1% sodium dodecyl sulfate. The enzyme was somewhat susceptible to chymotrypsin treatment but resistant to digestion by trypsin. Jenq-Kuen Huang, Chuian-Fu Ken, and Hui-Ming Huang contributed equally to this paper.     

Crystal structure of a novel Plasmodium falciparum 1-Cys peroxiredoxin

Plasmodium falciparum, the causative agent of malaria, is sensitive to oxidative stress and therefore the family of antioxidant enzymes, peroxiredoxins (Prxs) represent a target for antimalarial drug design. We present here the 1.8 A resolution crystal structure of P.falciparum antioxidant protein, PfAOP, a Prx that in terms of sequence groups with mammalian PrxV. The structure is compared to all 11 known Prx structures to gain maximal insight into its properties. We describe the common Prx fold and show that the dimeric PfAOP can be mechanistically categorized as a 1-Cys Prx. In the active site the peroxidatic Cys is over-oxidized to cysteine sulfonic acid, making this the first Prx structure seen in that state. Now with structures of Prxs in Cys-sulfenic, -sulfinic and -sulfonic acid oxidation states known, the structural steps involved in peroxide binding and over-oxidation are suggested. We also describe that PfAOP has an alpha-aneurism (a one residue insertion), a feature that appears characteristic of the PrxV-like group. In terms of crystallographic methodology, we enhance the information content of the model by identifying bound water sites based on peak electron densities, and we use that information to infer that the oxidized active site has suboptimal interactions that may influence catalysis. The dimerization interface of PfAOP is representative of an interface that is widespread among Prxs, and has sequence-dependent variation in geometry. The interface differences and the structural features (like the alpha-aneurism) may be used as markers to better classify Prxs and study their evolution.     

2-Cys peroxiredoxin function in intracellular signal transduction: therapeutic implications

H(2)O(2) is a reactive oxygen species that has drawn much interest because of its role as a second messenger in receptor-mediated signaling. Mammalian 2-Cys peroxiredoxins have been shown to eliminate efficiently the H(2)O(2) generated in response to receptor stimulation. 2-Cys peroxiredoxins are members of a novel peroxidase family that catalyze the H(2)O(2) reduction reaction in the presence of thioredoxin, thioredoxin reductase and NADPH. Several lines of evidence suggest that 2-Cys peroxiredoxins have dual roles as regulators of the H(2)O(2) signal and as defenders of oxidative stress. In particular, 2-Cys peroxiredoxin appears to provide selective, specific and localized control of receptor-mediated signal transduction. Thus, the therapeutic potential of 2-Cys peroxiredoxins is clear for diseases, such as cancer and cardiovascular diseases, that involve reactive oxygen species.