Exhibited Peroxidase Activity Research Articles

Characterisation of conformational and functional features of alkyl hydroperoxide reductase E-like protein

Alkyl hydroperoxide reductase E (AhpE) is a member of the peroxidase family of enzymes that catalyse the reduction of peroxides, however its structural and functional roles are still unclear in details. In this study, we used the Thermococcus kodakarensis AhpE-like protein as a model to investigate structure–function relationships including the molecular properties of DNA binding activity. Multiple sequence alignment, structural comparison and biochemical analyses revealed that TkAhpE includes conserved peroxidase residues in the active site, and exhibits peroxidase activity with structure-dependent holdase chaperone function. Following electrophoretic mobility shift assays and electron microscopy analysis demonstrated distinctive binding features of TkAhpE to the DNA showing that their dimeric conformer can bind to the double-stranded DNA, but not to the single-stranded DNA, indicating its striking molecular features to double-stranded DNA-specific interactions. Based on our results, we provided that TkAhpE is a multifunctional peroxidase displaying structure-dependent molecular chaperone and DNA binding activities.

Peroxidase to Cytochrome b Type Transition in the Active Site of Heme-Bound Amyloid β Peptides Relevant to Alzheimer's Disease.

Recent evidence has established the colocalization of amyloid-rich plaques and heme-rich deposits in the human cerebral cortex as a common postmortem feature in Alzheimer's disease (AD). The amyloid β (Aβ) peptides have been shown to bind heme, and the resultant heme-Aβ complexes can generate toxic partially reduced oxygen species (PROS) and exhibit peroxidase activity. The heme-Aβ active site exhibits a concentration-dependent equilibrium between a high-spin mono-His-bound species similar to a peroxidase-type active site and a bis-His-bound six-coordinate low-spin species similar to that of a cytochrome b type active site. The ν(Fe-His) (241 cm(-1)) vibration has been identified in the high-spin heme-Aβ active site by resonance Raman spectroscopy. The formation of the low-spin heme-Aβ species is promoted by the His14 and noncoordinating second-sphere Arg5 residues. The high-spin state produces more PROS than the low-spin species. Nonbiological constructs modeling different forms of Aβ (oligomers, fibrils, etc.) suggest that the detrimental high-spin state is likely to dominate under most physiological conditions.

Redox-dependent chaperone/peroxidase function of 2-Cys-Prx from the cyanobacterium Anabaena PCC7120: role in oxidative stress tolerance.

BackgroundCyanobacteria, progenitors of plant chloroplasts, provide a suitable model system for plants to study adaptation towards different abiotic stresses. Genome of the filamentous, heterocystous, nitrogen-fixing cyanobacterium Anabaena PCC7120 harbours a single gene (alr4641) encoding a typical 2-Cys-Peroxiredoxins (2-Cys-Prxs). 2-Cys-Prxs are thiol-based peroxidases that also function as molecular chaperones in plants and other systems. The Alr4641 protein from Anabaena PCC7120 shows high level biochemical similarities with the plant 2-Cys-Prx. The physiological role played by the Alr4641 protein in Anabaena was addressed in this study.ResultsIn Anabaena PCC7120, alr4641 transcript /Alr4641 protein was induced in response to abiotic stresses and its promoter was active in the vegetative cells as well as heterocysts. The wild-type Alr4641 protein or Alr4641 lacking the peroxidatic cysteine (Alr4641C56S) or the resolving cysteine (Alr4641C178S) existed as higher oligomers in their native form. The wild-type or the mutant Alr4641 proteins showed similar chaperone activity, but only the wild-type protein exhibited peroxidase activity indicating that unlike peroxidase activity, chaperone activity was not dependent on cysteines. In contrast to other 2-Cys-Prxs, chaperone/peroxidase activity of Alr4641 was dependent on its redox state and not oligomerization status. Alr4641 could protect plasmid DNA from oxidative damage and physically associate with NADPH-dependent thioredoxin reductase (NTRC). Like 2-Cys-Prxs from plants (e.g. rice), Alr4641 could detoxify various peroxides using NTRC as reductant. On exposure to H2O2, recombinant Anabaena PCC7120 strain over-expressing Alr4641 (An4641+) showed reduced content of reactive oxygen species (ROS), intact photosynthetic functions and consequently better survival than the wild-type Anabaena PCC7120, indicating that Alr4641 can protect Anabaena from oxidative stress.ConclusionsThe peroxidase/chaperone function of Alr4641, its inherent transcriptional/translational induction under different abiotic stresses and localization in both vegetative cells and heterocysts could be an adaptive strategy to battle various oxidative stresses that Anabaena encounters during its growth. Moreover, the recombinant Anabaena strain over expressing Alr4641 showed higher resistance to oxidative stress, suggesting its potential to serve as stress-tolerant biofertilizers in rice fields.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0444-2) contains supplementary material, which is available to authorized users.

Physiological and pathological views of peroxiredoxin 4

Peroxiredoxins (PRDXs) form an enzyme family that exhibits peroxidase activity using electrons from thioredoxin and other donor molecules. As the signaling roles of hydrogen peroxide in response to extracellular stimuli have emerged, the involvement of PRDX in the hydrogen peroxide-mediated signaling has become evident. Among six PRDX members in mammalian cells, PRDX4 uniquely possesses a hydrophobic signal peptide at the amino terminus, and, hence, it undergoes either secretion or retention by the endoplasmic reticulum (ER) lumen. The role of PRDX4 as a sulfoxidase in ER is now attracting much attention regarding the oxidative protein folding of nascent proteins. Contrary to this role in the ER, the functional significance of PRDX4 in the extracellular milieu is virtually unknown despite its implications as a biomarker under pathological conditions in some diseases. Other than its systemically expressed form, a variant form of PRDX4 is transcribed from the upstream promoter/exon 1 of the systemic promoter/exon 1 and is uniquely expressed in sexually matured testes. Circumstantial evidence, together with deduced functions from the systemic form, suggests that there are potential roles for testicular PRDX4 in the reproductive processes such as the regulation of hormonal signals and the oxidative packaging of sperm chromatin. Elucidation of these PRDX4 functions under in vivo situations is expected to show the whole picture of how PRDX4 has evolved in multicellular organisms.

Coexistence of native-like and non-native partially unfolded ferricytochrome c on the surface of cardiolipin-containing liposomes.

Cytochrome c, in spite of adopting a rather rigid structure around its prosthetic heme group, is rather diverse with regard to its function and structural variability. On the surface of the inner membrane of mitochondria it serves as an electron transfer carrier. However, at conditions which have not yet been unambiguously identified, cytochrome c can adopt a variety of non-native conformations, some of which exhibit peroxidase activity. Cardiolipin-containing liposomes have served as ideal model system to investigate the various modes of interaction between cytochrome c and the inner mitochrondrial membrane. We probed the binding of horse heart ferricytochrome c to liposomes formed with 20% tetraoleoyl cardiolipin (TOCL) and 80% dioleoyl-sn-glycero-3-phosphocholine (DOPC) as a function of lipid/protein ratio by fluorescence and visible circular dichroism spectroscopy. The obtained binding isotherms suggest that they reflect reversible binding processes, which excludes the possibility of significant protein insertion into the membrane. A global analysis of our data revealed the existence of two binding sites on the protein which causes rather different degrees of protein unfolding. We found that these two modes of interaction between protein and liposome led to conformational changes. While site 1 is relatively unaffected by NaCl, site 2 shows a more native-like state or a higher population of the native state in the presence of NaCl. At the highest utilized concentration of NaCl, there is only a 40% inhibition of the binding to site 2. We interpret our finding for this binding site as reflecting an equilibrium between electrostatically bound proteins with a high degree of unfolding and less unfolded proteins which bind either via H-bonding between lysine side chains and PO2(-) or hydrophobic interactions. With regard to site 2 binding, our results are reminiscent of the two-state equilibrium between a compact C and an extended E-state proposed by Pletneva and co-workers (Hanske et al. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 125-230). We conjecture that the nonelectrostatically bound proteins should have higher abilities to maintain the redox potential that is required for the function as an electron transfer protein.

Coexistence of Native-Like and Non-Native Misfolded Ferricytochrome C on the Surfac of Cardiolipin Containing Liposomes

Cytochrome c, in spite of adopting a rather rigid structure around its prosthetic heme group, is rather diverse with regard to its function and structural variability. On the surface of the inner membrane of mitochondria it serves as an electron transfer carrier. However, at conditions which have not yet been unambiguously identified, cytochrome c can adopt a variety of non-native conformations, some of which exhibit peroxidase activity. Cardiolipin-containing liposomes have served as ideal model system to investigate the various modes of interaction between cytochrome c and the inner mitochrondrial membrane. We probed the binding of horse heart ferricytochrome c to liposomes formed with 20% tetraoleoyl cardiolipin (TOCL) and 80% dioleoyl-sn-glycero-3-phosphocholine (DOPC) as a function of lipid/protein ratio by fluorescence and visible circular dichroism spectroscopy. The obtained binding isotherms suggest that they reflect reversible binding processes, which excludes the possibility of significant prote...

Enzymatic Activity of the Human BK Channel: A Function Beyond Electrical Signaling

BK (Slo1) channels open probability is acutely regulated by heme, which associates with their intracellular multi-ligand sensor, the gating ring. We found that the gating ring region encompassing the heme-binding site shares structural homology with Cytochrome C (CytC), the well-known hemoprotein. In addition to its role in electron shuttling, CytC exhibits various catalytic properties such as peroxidase activity, i.e. the oxidation of suitable substrates using peroxides. To probe for peroxidase activity of the CytC-like domain in a purified BK channel gating ring, we used the chromophore 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the oxidizable substrate. We found that the BK gating ring complexed with heme in the presence of H2O2 catalyzes the formation of ABTS+• cation radical, monitored by absorption at 415nm. The initial rate of ABTS+• formation was linearly correlated with [gating ring] between 0.05-0.3 μM. Disruption of the heme regulatory motif (C615S/H616R) significantly decreased the initial rate of ABTS+• formation. The kinetic parameters of the enzymatic reaction were determined by performing two-substrate Michaelis-Menten analysis, which yielded for gating ring: kcat/KmH2O2 ≈12 s−1mM−1 and kcat/KmABTS ≈0.5 s−1mM−1. For CytC, we estimated kcat/KmH2O2 ≈1.4 s−1mM−1 and kcat/KmABTS ≈0.035 s−1mM−1. These results suggest that, under our experimental conditions, the gating ring catalytic efficiency is ∼10 times higher than CytC. Finally, we found that HEK cells expressing BK channels (blocked with 100 nM Iberiotoxin) are significantly more resistant to oxidative insult (200 μM H2O2) than cells expressing BK channels with impared heme binding (C615S/H616R) (p<0.05) as revealed by the increased cell viability (MTT assay). Thus, the BK channel exhibits peroxidase activity and confers a protective effect against oxidative cell damage. These results redefine the role of BK channels, assigning a catalytic property, in addition to their established K+ conducting properties.

Characterization of a 2-Cys peroxiredoxin IV in Marsupenaeus japonicus (kuruma shrimp) and its role in the anti-viral immunity

Accumulating evidence suggests that peroxiredoxins (Prx) are key molecules in the pathogenesis of various infectious diseases and are potential therapeutic targets for major diseases such as cancers. In this study, we report a peroxiredoxin IV (Prx IV) in Marsupenaeus japonicus, designated as MjPrx IV, which exhibited peroxidase activity and participated in the anti-white spot syndrome virus (WSSV) immune response. MjPrx IV is a 245-amino acid polypeptide with a predicted 19-amino acid signal peptide, an Ahpc-TSA domain, and a 1-Cys PrxC domain. Phylogenetic analysis revealed that the protein belongs to the Prx IV subfamily. MjPrx IV transcripts were detected in the gills, hepatopancreas, heart, stomach, ovaries, spermary, and intestine tissues, and are upregulated in the gonads, gills and hemocytes of shrimp after WSSV challenge. The mature MjPrx IV peptide was recombinantly expressed in an Escherichia coli system. The protein exhibited peroxidase activity. Furthermore, dsRNA suppression of MjPrx IV increased WSSV replication in shrimp, whereas rMjPrx IV injection into shrimp decreased WSSV replication. These data suggest that MjPrx IV has an important role in shrimp antiviral immunity. To our knowledge, this study is the first to report a shrimp Prx IV that has anti-WSSV activity.

High-Throughput Quantitative Screening of Peroxidase-Mimicking DNAzymes on a Microarray by Using Electrochemical Detection

Some guanine-rich DNA sequences, which are called DNAzymes, can adopt G-quadruplex structures and exhibit peroxidase activity by binding with hemin. Although known DNAzymes show less activity than horseradish peroxidase, they have the potential to be widely used for the detection of target molecules in enzyme-linked immunosorbent assays if sequences that exhibit higher activity can be identified. However, techniques for achieving this have not yet been described. Therefore, we compared the DNAzyme activities of more than 1000 novelistically designed sequences with that of the original DNAzyme by using an electrochemical detection system on a 12K DNA microarray platform. To the best of our knowledge, this is the first description of an array-based assessment of peroxidase activity of G-quadruplex-hemin complexes. By using this novel assay system, more than 200 different mutants were found that had significantly higher activities than the original DNAzyme sequence. This microarray-based DNAzyme evaluation system is useful for identifying highly active new DNAzymes that might have potential as tools for developing DNA-based biosensors with aptamers.

Identification of a Novel Calcium Binding Motif Based on the Detection of Sequence Insertions in the Animal Peroxidase Domain of Bacterial Proteins

Proteins of the animal heme peroxidase (ANP) superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20), where it was found to be involved in Ca2+ coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33–79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca2+ binding with a KD of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821) is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of life.

Gold Nanoparticles Supported on Nanoparticulate Ceria as a Powerful Agent against Intracellular Oxidative Stress

Ceria-supported gold nanoparticles are prepared exhibiting peroxidase activity and acting as radical traps. Au/CeO(2) shows a remarkable biocompatibility as demonstrated by measuring cellular viability, proliferation, and lack of apoptosis for two human cell lines (Hep3B and HeLa). The antioxidant activity of Au/CeO(2) against reactive oxygen species (ROS) is demonstrated by studying the cellular behavior of Hep3B and HeLa in a model of cellular oxidative stress. It is determined that Au/CeO(2) exhibits higher antioxidant activity than glutathione, the main cytosolic antioxidant compound, and its CeO(2) carrier. Overall the result presented here shows the potential of implementing well-established nanoparticulated gold catalysts with remarkable biocompatibility in cellular biology.

Cloning and characterization of two glutathione S‐transferases from pyrethroid‐resistant Culex pipiens

The Marin strain of Culex pipiens Say is a pyrethroid-resistant population that was collected in Marin County, California, in 2001 and subsequently maintained in the laboratory under regular permethrin exposure. In this study, two cDNAs, CpGSTd1 and CpGSTd2, encoding glutathione S-transferase (GST) were cloned from Cx. pipiens Marin. Phylogenetic analysis of the deduced amino acid sequences, CpGSTD1 and CpGSTD2, of these genes indicated that they belong to the Delta class of insect GSTs. The nucleotide and deduced amino acid sequences of CpGSTd1 and CpGSTd2 were 59 and 48% identical respectively. CpGSTD1 and CpGSTD2 were expressed in Escherichia coli and purified by affinity chromatography. The recombinant GSTs exhibited unique selectivity towards the general GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB), and also differed in their sensitivity to known inhibitors of GSTs. CpGSTD1 exhibited peroxidase activity with cumene hydroperoxide, while CpGSTD2 appeared to lack this activity. CpGSTD1 was able to metabolize 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), while DDT metabolism by CpGSTD2 was not detectable. CpGSTD1 and CpGSTD2 showed no detectable metabolism of permethrin. Gene expression of CpGSTd1 and CpGSTd2 in Marin mosquitoes was elevated about twofold in comparison with that found in a pyrethroid-sensitive mosquito strain. The results indicate that CpGSTD1 and CpGSTD2 have unique biochemical characteristics, but they do not appear to play major roles in permethrin resistance in Marin mosquitoes.

A label-free, G-quadruplex DNAzyme-based fluorescent probe for signal-amplified DNA detection and turn-on assay of endonuclease

A novel G-quadruplex DNAzyme molecular beacon (G-DNAzymeMB) strategy is developed for assays of target DNA and restriction endonuclease. The detection system consists of G-DNAzymeMB strand and a blocker DNA by using the fluorescence of 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) catalyzed by G-DNAzymeMB as a signal reporter. G-DNAzymeMB exhibits peroxidase activity in its free hairpin structure, and forms a catalytically inactive hybrid when hybridized with blocker DNA. Upon displacement of blocker DNA by target DNA or cleavage by restriction endonuclease, G-DNAzymeMB is released and two lateral portions of G-DNAzymeMB form a G-quadruplex structure, resulting in the recovery of catalytic activity which acts as a cofactor to catalyze H2O2-mediated oxidation of H2DCFDA. For DNA detection system, exonuclease III (Exo III)-catalyzed amplification strategy is introduced to improve the sensitivity and target DNA could be detected as low as 0.1pM. With respect to restriction endonuclease detection system, 0.1U/mL EcoRI endonuclease could be detected and this method could be easily transported to other restriction endonuclease analysis by simply changing the recognition sequence. These results demonstrate that the proposed G-DNAzymeMB strategy could be used as a label-free, simple, sensitive and cost-effective approach in analysis of target DNA and restriction endonuclease.

Characterization of G‐Quadruplex/Hemin Peroxidase: Substrate Specificity and Inactivation Kinetics

Recently, G-quadruplex/hemin (G4/hemin) complexes have been found to exhibit peroxidase activity, and this feature has been extensively exploited for colorimetric detection of various targets. To further understand and characterize this important DNAzyme, its substrate specificity, inactivation mechanism, and kinetics have been examined by comparison with horseradish peroxidase (HRP). G4/hemin DNAzyme exhibits broader substrate specificity and much higher inactivation rate than HRP because of the exposure of the catalytic hemin center. The inactivation of G4/hemin DNAzyme is mainly attributed to the degradation of hemin by H(2)O(2) rather than the destruction of G4. Both the inactivation rate and catalytic oxidation rate of G4/hemin DNAzyme depend on the concentration of H(2)O(2), which suggests that active intermediates formed by G4/hemin and H(2)O(2) are the branch point of catalysis and inactivation. Reducing substrates greatly inhibit the inactivation of G4/hemin DNAzyme by rapidly reacting with the active intermediates. A possible catalytic and inactivation process of G4/hemin has been proposed. These results imply a potential cause for the hemin-mediated cellular injury and provide insightful information for the future application of G4/hemin DNAzyme.

Reactive metabolites of desipramine and clomipramine: The kinetics of formation and reactivity with DNA

Tricyclic antidepressants (TCAs), along with phenothyazines and some industrial chemicals, are shown to react with enzymes that exhibit peroxidase activity. These reactions result in the formation of reactive intermediates having unpaired electrons. The peroxidase oxidation and reactivity of two TCAs, desipramine and clomipramine, were investigated. As a model of peroxidase, horseradish peroxidase (HRP) was employed. The products of the peroxidase catalyzed oxidation of desipramine and clomipramine were identified as N-dealkylated compounds iminodibenzyl and 3-chloroiminodibenzyl using the GC/MS technique. Both drugs formed broad UV/vis absorption spectra in the presence of HRP and H2O2, indicating the formation of a radical cations—reactive intermediate of the oxidation reaction. The dynamics of the formation of the desipramine intermediate was studied using UV/vis spectroscopy. The extinction coefficient was measured for the reactive intermediate, 7.80×103M−1cm−1, as well as the apparent Michaelis–Menten and catalytic constants, 4.4mM and 2.3s−1, respectively. Both desipramine and clomipramine degraded DNA in the presence of HRP/H2O2, as was revealed by agarose gel electrophoresis and PCI extraction. Manipulating the kinetic parameters of drug’s radical formation and determining the extent of degradation to biomolecules could be potentially used for designing effective agents exhibiting specific reactivity.

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.

Heme–Cu Bound Aβ Peptides: Spectroscopic Characterization, Reactivity, and Relevance to Alzheimer’s Disease

Recently, it has been shown that heme binds to Aβ peptides which may play a major role in Alzheimer's disease (AD). This study illustrates that Aβ peptides can bind both Cu and heme cofactors at the same time. Both cofactors have unique spectroscopic and electrochemical features which are unaffected in the presence of the other, implying that they are electronically, chemically, and electrochemically uncoupled. These data clearly indicate that Cu cannot bind to three histidine residues simultaneously in Cu-Aβ complexes as previously proposed, since one of the histidines is involved in binding heme. The heme-Aβ and the heme-Cu-Aβ peptide complexes function as peroxidases. Interestingly, the Cu-Aβ complex also exhibits peroxidase activity, which may have significant implications in AD. Both Cu(+)-Aβ and heme (Fe(2+))-Aβ complexes reduce O(2) to H(2)O(2) quantitatively. Only one of the two electrons that are required for the reduction of O(2) to H(2)O(2) is derived from the reduced metal site, while the Tyr(10) residue of the native Aβ peptide donates the second electron. This Tyr(10) residue, the source of electron for the generation of partially reduced oxygen species (PROS, e.g., H(2)O(2)) is absent in rodents, which do not get affected by AD. When both heme and Cu are bound to the Aβ peptides, which is likely to happen physiologically, the amount of toxic PROS generated is maximum, implying that heme-Cu-Aβ complexes could potentially be most toxic for AD.

Vascular peroxidase-1 is rapidly secreted, circulates in plasma, and supports dityrosine cross-linking reactions

Members of the peroxidase–cyclooxygenase superfamily catalyze biochemical reactions essential to a broad spectrum of biological processes, including host defense, thyroid hormone biosynthesis, and modification of extracellular matrix, as well as contributing to the pathogenesis of chronic inflammatory diseases. We recently identified a novel member of this family, vascular peroxidase-1 (VPO1), that is highly expressed in the human cardiovascular system. Its biosynthesis and enzymatic properties are largely unknown. Here, we report that VPO1 was rapidly and efficiently secreted into the extracellular space when the gene was stably expressed in human embryonic kidney (HEK) cells. Secreted VPO1 is a monomer with complex N-linked oligosaccharides and exhibits peroxidase activity. Biosynthesis of endogenous VPO1 by cultured human umbilical vein endothelial cells (HUVECs) shares features exhibited by heterologous expression of recombinant VPO1 (rVPO1) in HEK cells. The proinflammatory agents lipopolysaccharide and tumor necrosis factor-α induce expression of VPO1 mRNA and protein in HUVECs. Furthermore, murine and bovine sera and human plasma contain enzymatically active VPO1. rVPO1 exhibits spectral and enzymatic properties characteristic of the peroxidase–cyclooxygenase family, except with regard to its heat stability. rVPO1 catalyzes tyrosyl radical formation and promotes dityrosine cross-linking. Taken together, these data demonstrate that VPO1 is a glycosylated heme peroxidase that is actively secreted into circulating plasma by vascular endothelial cells and shares several features with other members of the peroxidase–cyclooxygenase family, including the catalysis of dityrosine formation.

In silico prediction and characterization of 3D structure and binding properties of catalase from the commercially important crab, Scylla serrata

The enzyme catalase breaks down H(2)O(2), a potentially harmful oxidant, to H(2)O and O(2). Besides oxidase activity, the enzyme also exhibits peroxidase activity. Therefore, it plays an important role in maintaining health and regulating pathophysiology of the organisms. However, 3D structure of this important enzyme in invertebrates particularly in crabs is not yet available. Therefore, an attempt has been made to predict the structure of the crab catalase and to envisage its catalytic interaction with H(2)O(2). A three dimensional model of crab catalase was constructed using the NADPH binding site on Beef Liver catalase from Bos taurus (PDBID: 7CAT) as template by comparative modeling approach. Backbone conformation of the modeled structure by PROCHECK revealed that more than 98% of the residues fell in the allowed regions, ERRAT results confirmed good quality of modeled structure and VERIFY3D profile was satisfying. Molecular docking has been used to know the binding modes of hydrogen peroxide with the crab catalase protein. The receptor structures used for docking were derived from molecular dynamics (MD) simulations of homology modeled structure. The docking results showed that the three important determinant residues Arg68, Val70 and Arg108 in catalase were binding with H(2)O(2) as they had strong hydrogen bonding contacts with the substrate. Our analysis provides insight into the structural properties of crab catalase and defines its active sites for binding with substrate. These data are important for further studies of catalase of invertebrates in general and that of crabs in particular.

Characteristics of soybean 1-Cys peroxiredoxin and its behavior in seedlings under flooding stress

By previous proteomic analysis, the amount of proteins exhibiting similarity to 1-Cys peroxiredoxin (Prx), a thiol-dependent peroxidase, was shown to be higher in seedlings of soybean (Glycine max (L.) Merr.) suffering from flooding stress than in normally grown seedlings. In this study, we characterized soybean 1-Cys Prx to elucidate the relationship of the protein to flooding stress. In the soybean genome, two genes corresponding to 1-Cys Prx (designated as GmPer1a, GmPer1b) exist. GmPer1a encodes a polypeptide containing the putative catalytic site, and a recombinant GmPer1a protein exhibited peroxidase activity. On the other hand, the GmPer1b contains a stop codon inside the deduced polypeptide-coding region, indicating that GmPer1b might be a pseudogene. The GmPer1a was expressed in developing seeds and transiently in germinating seeds of soybean. It was no longer expressed in 2-day-old seedlings, and was not induced by flooding treatment. The GmPer1 protein was synthesized in developing seeds, and was degraded during germination and growth. In addition, two forms of GmPer1 protein existed in both submerged and normally grown seedlings, and the amount of both forms was higher in the submerged seedlings. These suggest that both normal and post- translationally modified forms of GmPer1 might remain in seedlings suffering from flooding stress as a result of growth retardation.