Adenanthin Is an Efficient Inhibitor of Peroxiredoxins from Pathogens, Inhibits Bacterial Growth, and Potentiates Antibiotic Activities.

The emergence and re-emergence of bacterial strains resistant to multiple drugs represent a global health threat, and the search for novel biological targets is a worldwide concern. AhpC are enzymes involved in bacterial redox homeostasis by metabolizing diverse kinds of hydroperoxides. In pathogenic bacteria, AhpC are related to several functions, as some isoforms are characterized as virulence factors. However, no inhibitor has been systematically evaluated to date. Here we show that the natural ent-kaurane Adenanthin (Adn) efficiently inhibits AhpC and molecular interactions were explored by computer assisted simulations. Additionally, Adn interferes with growth and potentializes the effect of antibiotics (kanamycin and PMBN), positioning Adn as a promising compound to treat infections caused by multiresistant bacterial strains.

Structural and functional diversity of asparaginases: Overview and recommendations for a revised nomenclature.

Asparaginases (ASNases) are a large and structurally diverse group of enzymes ubiquitous amongst archaea, bacteria and eukaryotes, that catalyze hydrolysis of asparagine to aspartate and ammonia. Bacterial ASNases are important biopharmaceuticals for the treatment of acute lymphoblastic leukemia, although some patients experience adverse allergic side effects during treatment with these protein therapeutics. ASNases are currently divided into three families: plant-type ASNases, Rhizobium etli-type ASNases and bacterial-type ASNases. This system is outdated as both bacterial-type and plant-type families also include archaeal, bacterial and eukaryotic enzymes, each with their own distinct characteristics. Herein, phylogenetic studies allied to tertiary structural analyses are described with the aim of proposing a revised and more robust classification system that considers the biochemical diversity of ASNases. Accordingly, based on distinct peptide domains, phylogenetic data, structural analysis and functional characteristics, we recommend that ASNases now be divided into three new distinct classes containing subgroups according to structural and functional aspects. Using this new classification scheme, 25 ASNases were identified as candidates for future new lead discovery.

Use of logistic models to evaluate the response of superovulation treatment and embryo production in Santa Inês ewes.

The study aimed to verify the influence of the FecGE mutation in superovulated ewes and to evaluate the probability of logistic models to determine the response capacity of these ewes to superovulatory treatment. Santa Inês ewes (n = 29) were genotyped for the FecGE mutation and separated for their genotype group in carriers of the mutant E allele (FecGE/E, FecG+/E) and non-carrier (FecG+/+) alleles. The ewes underwent hormonal treatment for superovulation. Aside from the genotypes, variables included in the statistical model were reproductive status (empty, early lactation, or late lactation), age (> or < 6 years), and number of births (nulliparous, primiparous, multiparous). The carriers of the mutation could be discriminated from the non-carriers based on the number of corpora lutea, rate of frozen embryos, and fecundity. Recovery rate was significantly higher (P < 0.05) in FecGE/E (94.31%) compared to FecG+/E (63.15%) and FecG+/+ (61.90%) (P < 0.05), whereas fecundity rate of FecG+/+ ewes (50.76%) was significantly higher than FecG+/E (18.96%) and FecGE/E (32.53%) (P < 0.05). We determined in this study that the response to superovulation and embryo production can be discriminated between FecGE/E and FecG+/E ewes in relation to the FecG+/+ genotype. Logistic models that included reproductive status and mutation, or reproductive status and age, or reproductive status and number of births were effective in predicting the response to superovulatory treatment.

Relevance of peroxiredoxins in pathogenic microorganisms.

The oxidative and nitrosative responses generated by animals and plants are important defenses against infection and establishment of pathogenic microorganisms such as bacteria, fungi, and protozoa. Among distinct oxidant species, hydroperoxides are a group of chemically diverse compounds that comprise small hydrophilic molecules, such as hydrogen peroxide and peroxynitrite, and bulky hydrophobic species, such as organic hydroperoxides. Peroxiredoxins (Prx) are ubiquitous enzymes that use a highly reactive cysteine residue to decompose hydroperoxides and can also perform other functions, like molecular chaperone and phospholipase activities, contributing to microbial protection against the host defenses. Prx are present in distinct cell compartments and, in some cases, they can be secreted to the extracellular environment. Despite their high abundance, Prx expression can be further increased in response to oxidative stress promoted by host defense systems, by treatment with hydroperoxides or by antibiotics. In consequence, some isoforms have been described as virulence factors, highlighting their importance in pathogenesis. Prx are very diverse and are classified into six different classes (Prx1-AhpC, BCP-PrxQ, Tpx, Prx5, Prx6, and AhpE) based on structural and biochemical features. Some groups are absent in hosts, while others present structural peculiarities that differentiate them from the host's isoforms. In this context, the intrinsic characteristics of these enzymes may aid the development of new drugs to combat pathogenic microorganisms. Additionally, since some isoforms are also found in the extracellular environment, Prx emerge as attractive targets for the production of diagnostic tests and vaccines. KEY POINTS: • Peroxiredoxins are front-line defenses against host oxidative and nitrosative stress. • Functional and structural peculiarities differ pathogen and host enzymes. • Peroxiredoxins are potential targets to microbicidal drugs.

Glutaredoxin-like protein (GLP)-a novel bacteria sulfurtransferase that protects cells against cyanide and oxidative stresses.

The pathogen Xylella fastidiosa belongs to the Xanthomonadaceae family, a large group of Gram-negative bacteria that cause diseases in many economically important crops. A predicted gene, annotated as glutaredoxin-like protein (glp), was found to be highly conserved among the genomes of different genera within this family and highly expressed in X. fastidiosa. Analysis of the GLP protein sequences revealed three protein domains: one similar to monothiol glutaredoxins (Grx), an Fe-S cluster and a thiosulfate sulfurtransferase/rhodanese domain (Tst/Rho), which is generally involved in sulfur metabolism and cyanide detoxification. To characterize the biochemical properties of GLP, we expressed and purified the X. fastidiosa recombinant GLP enzyme. Grx activity and Fe-S cluster formation were not observed, while an evaluation of Tst/Rho enzymatic activity revealed that GLP can detoxify cyanide and transfer inorganic sulfur to acceptor molecules in vitro. The biological activity of GLP relies on the cysteine residues in the Grx and Tst/Rho domains (Cys33 and Cys266, respectively), and structural analysis showed that GLP and GLPC266S were able to form high molecular weight oligomers (> 600 kDa), while replacement of Cys33 with Ser destabilized the quaternary structure. In vivo heterologous enzyme expression experiments in Escherichia coli revealed that GLP can protect bacteria against high concentrations of cyanide and hydrogen peroxide. Finally, phylogenetic analysis showed that homologous glp genes are distributed across Gram-negative bacterial families with conservation of the N- to C-domain order. However, no eukaryotic organism contains this enzyme. Altogether, these results suggest that GLP is an important enzyme with cyanide-decomposing and sulfurtransferase functions in bacteria, whose presence in eukaryotes we could not observe, representing a promising biological target for new pharmaceuticals.

Functional and structural evaluation of the antileukaemic enzyme L-asparaginase II expressed at low temperature by different Escherichia coli strains.

Acute lymphoblastic leukaemia (ALL) affects lymphoblastic cells and is the most common neoplasm during childhood. Among the pharmaceuticals used in the treatment protocols for ALL, Asparaginase (ASNase) from Escherichia coli (EcAII) is an essential biodrug. Meanwhile, the use of EcAII in neoplastic treatments causes several side effects, such as immunological reactions, hepatotoxicity, neurotoxicity, depression, and coagulation abnormalities. Commercial EcAII is expressed as a recombinant protein, similar to novel enzymes from different organisms; in fact, EcAII is a tetrameric enzyme with high molecular weight (140 kDa), and its overexpression in recombinant systems often results in bacterial cell death or the production of aggregated or inactive EcAII protein, which is related to the formation of inclusion bodies. On the other hand, several commercial expression strains have been developed to overcome these expression issues, but no studies on a systematic evaluation of the E. coli strains aiming to express recombinant asparaginases have been performed to date. In this study, we evaluated eleven expression strains at a low temperature (16 °C) with different characteristics to determine which is the most appropriate for asparaginase expression; recombinant wild-type EcAII (rEcAII) was used as a prototype enzyme and the secondary structure content, oligomeric state, aggregation and specific activity of the enzymes were assessed. Structural analysis suggested that a correctly folded tetrameric rEcAII was obtained using ArcticExpress (DE3), a strain that co-express chaperonins, while all other strains produced poorly folded proteins. Additionally, the enzymatic assays showed high specific activity of proteins expressed by ArcticExpress (DE3) when compared to the other strains used in this work.

Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite.

Organic hydroperoxide resistance (Ohr) enzymes are unique Cys-based, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr from Xylella fastidiosa and were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase-coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 107-108 M-1⋅s-1 range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 107 M-1⋅s-1 at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a ∆ohr mutant of Pseudomonas aeruginosa toward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the ∆ohr strain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the ∆ohr mutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H2O2 insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wild-type strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host-pathogen interactions.

Bioaffinity Fishing Procedure Using Secretory Phospholipase A2 for Screening for Bioactive Components: Modulation of Pharmacological Effect Induced by sPLA2 from Crotalus durissus terrificus by Hispidulin from Moquiniastrum floribundum.

Bioaffinity capturing of molecules allows the discovery of bioactive compounds and decreases the need for various stages in the natural compound isolation process. Despite the high selectivity of this technique, the screening and identification methodology depends on the presence of a protein to capture potential ligands. However, some proteins, such as snake secretory phospholipase A2 (sPLA2), have never been investigated using this approach. The purpose of this study was to evaluate the use of a new method for screening natural compounds using a bioaffinity-guided ultrafiltration method on Crotalus durissus terrificus sPLA2 followed by HPLC-MS to identify the compounds, and this method could be used to discover new anti-inflammatory compounds from the various organisms originating from biodiversity. Different extracts were selected to evaluate their ability to inhibit sPLA2 activity. The extracts were incubated with sPLA2 and the resulting mixture was ultrafiltrated to elute unbound components. The resulting compounds were identified by HPLC-MS. We identified hispidulin as one of the components present in the Moquiniastrum floribundum leaf and evaluated the ability of this isolated compound to neutralize the inflammatory activity of sPLA2 from Crotalus durissus terrificus.

Heavy metal concentrations in Brazilian port areas and their relationships with microorganisms: can pollution in these areas change the microbial community?

The objectives of this study were to analyze the difference in ways in which metals polluting Brazilian port areas influence bacterial communities and the selection of resistant strains. The hypothesis tested was that port areas would have microbial communities significantly different from a pristine area, mainly due to a greater load of metals found in these areas. Sediment samples were collected in two port areas (Santos and São Sebastião) and one pristine area (Ubatuba). Total DNA was extracted and MiSeq sequencing was performed. A hundred strains were isolated from the same samples and were tested for metal resistance. The community composition was similar in the two port regions, but differed from the pristine area. Microbial diversity was significantly lower in the port areas. The phyla Proteobacteria, Cyanobacteria, and Thermodesulfobacteria exhibited positive correlations with copper and zinc concentrations. Chloroflex, Nitrospirae, Planctomycetes, and Chlorobi exhibited negative correlations with copper, chromium, and zinc. Cr and Zn had higher concentrations at port areas and were responsible to select more metal-resistant strains. Some genera were found to be able to easily develop metal resistance. The most isolated genera were Bacillus, Vibrio, and Pseudomonas. This type of study can illustrate, even in very complex natural environments, the influence of pollution on the community as a whole and the consequences of these changes.

Evaluation of the Inhibitory Potential of Casuarictin, an Ellagitannin Isolated from White Mangrove (Laguncularia racemosa) Leaves, on Snake Venom Secretory Phospholipase A2.

Ellagitannins constitute the largest group of hydrolyzable tannins of plants, and, from this group, casuarictin (Casu) was identified in some plant species. However, to our knowledge, no investigation of secretory phospholipase A2 (sPLA2) inhibition by Casu has been performed yet. Casuarictin was isolated by chromatography n-butanol (n-BuOH) partition of Laguncularia racemosa leaves. The pharmacological and biological effects of Casu were evaluated on isolated sPLA2 from the rattlesnake (Crotalus durissus terrificus) and using a plant bacterial strain. The compound was able to form a protein complex consisting of a stable sPLA2 + Casu complex. Analyses carried out with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF) revealed that the molecular mass of sPLA2 increased from 14,425.62 to 15,362.74 Da. The enzymatic activity of the sPLA2 + Casu complex was significantly lower than that of native sPLA2. Besides, molecular interactions of Casu with sPLA2 were able to virtually abolish the native edematogenic effect as well as myonecrosis induced by the protein when injected 10 min after sPLA2. Therefore, Casu may be considered a potential anti-inflammatory that can be used to treat edema and myonecrosis induced by serine-secreting phospholipase A2. In addition, the compound also showed great antimicrobial potential.

Diversity and Distribution of Heavy Metal-Resistant Bacteria in Polluted Sediments of the Araça Bay, São Sebastião (SP), and the Relationship Between Heavy Metals and Organic Matter Concentrations.

Heavy metals influence the population size, diversity, and metabolic activity of bacteria. In turn, bacteria can develop heavy metal resistance mechanisms, and this can be used in bioremediation of contaminated areas. The purpose of the present study was to understand how heavy metals concentration influence on diversity and distribution of heavy metal-resistant bacteria in Araça Bay, São Sebastião, on the São Paulo coast of Brazil. The hypothesis is that activities that contribute for heavy metal disposal and the increase of metals concentrations in environment can influence in density, diversity, and distribution of heavy metal-resistant bacteria. Only 12 % of the isolated bacteria were sensitive to all of the metals tested. We observed that the highest percentage of resistant strains were in areas closest to the São Sebastião channel, where port activity occurs and have bigger heavy metals concentrations. Bacterial isolated were most resistant to Cr, followed by Zn, Cd, and Cu. Few strains resisted to Cd levels greater than 200 mg L(-1). In respect to Cr, 36 % of the strains were able to grow in the presence of as much as 3200 mg L(-1). Few strains were able to grow at concentrations of Zn and Cu as high as 1600 mg L(-1), and none grew at the highest concentration of 3200 mg L(-1). Bacillus sp. was most frequently isolated and may be the dominant genus in heavy metal-polluted areas. Staphylococcus sp., Planococcus maritimus, and Vibrio aginolyticus were also isolated, suggesting their potential in bioremediation of contaminated sites.

Influence of biostimulation and temporary weaning on follicular dynamics and pregnancy rates in Nelore cows (Bos taurus indicus).

The objective of this study was to evaluate the effect of biostimulation and temporary weaning on the follicular dynamics and pregnancy rates in Nelore cows. We used three groups of 75 cows: a control group without biostimulation and suckling calves (WB), a group that was biostimulated and had suckling calves (BE) and a group that was biostimulated and subjected to temporary weaning for 56 h (BETW). Ovarian dynamics were examined using ultrasonography. All groups showed follicular atresia. The interval between beginning of the treatment and wave emergence was 3.25 ± 0.30 days for BE, 3.40 ± 0.27 days for BETW and 3.37 ± 0.50 days for WB. The time between implant removal and ovulation was 64.50 ± 1.88 h for BE, 66.75 ± 1.35 h for BETW and 60.85 ± 3.10 h for WB. Eight cows were submitted to ultrasound analysis, and the percentages of cows that had ovulatory follicles of the new follicular wave with maximum diameters greater than 0.80 cm were 100 % (8/8) in BE (1.28 ± 0.12 cm), 100 % (8/8) in BETW (1.52 ± 0.07 cm) and 87.5 % (7/8) in WB (1.21 ± 0.10 cm). The pregnancy rate was 52 % (39/75) in BE, 69.3 % (52/75) in BETW and 37.3 % (28/75) in WB. The association of biostimulation and temporary weaning increased follicular development, ovulation synchronisation and, consequently, the pregnancy rate in beef cows.

Time-efficient germ cell transplantation from goldfish (Carassius auratus) into adult common carp (Cyprinus carpio).

Germ cell transplantation in fish is a promising technique for surrogate broodstock parents with broader application in aquaculture and conserving endangered and valuable genetic resources. Herein, we describe the establishment of an intrapapillary xenogeneic transplant of germ cells from sexually mature goldfish (C. auratus) males into common carp (C. carpio) males cytoablated with a thermochemical treatment (two doses of busulfan at 40 mg/kg at 35°C). To analyze the presence and development of donor germ cells in recipient testes, donor germ cells were labeled with PKH26, a fluorescent cell membrane dye, before transplantation. Our results demonstrated that thermochemical treatment caused effective spermatogenesis suppression and pronounced germ cell loss. Moreover, transplanted spermatogonial cells were able to colonize the recipients' testes, resume spermatogenesis, and generate spermatozoa within eight weeks after germ cell transplantation. These findings suggested that recipient testes provided suitable conditions for the survival, colonization, proliferation, and differentiation of donor spermatogonia from a related species. This study indicated that recipients' testes exhibited a high degree of plasticity to accept and support xenogeneic donor germ cells, which were able to form sperm in a short time frame. This approach has significant implications for assisted animal reproduction, biotechnology, conservation, and the production of valuable genetic resources and endangered fish species.

Exploring functional and structural features of chemically related natural prenylated hydroquinone and benzoic acid from Piper crassinervium (Piperaceae) on bacterial peroxiredoxin inhibition.

Multiple drug resistance (MDR) bacterial strains are responsible by 1.2 million of human deaths all over the world. The pathogens possess efficient enzymes which are able to mitigate the toxicity of reactive oxygen species (ROS) produced by some antibiotics and the host immune cells. Among them, the bacterial peroxiredoxin alkyl hydroperoxide reductase C (AhpC) is able to decompose efficiently several kinds of hydroperoxides. To decompose their substrates AhpC use a reactive cysteine residue (peroxidatic cysteine-CysP) that together with two other polar residues (Thr/Ser and Arg) comprise the catalytic triad of these enzymes and are involved in the substrate targeting/stabilization to allow a bimolecular nucleophilic substitution (SN2) reaction. Additionally to the high efficiency the AhpC is very abundant in the cells and present virulent properties in some bacterial species. Despite the importance of AhpC in bacteria, few studies aimed at using natural compounds as inhibitors of this class of enzymes. Some natural products were identified as human isoforms, presenting as common characteristics a bulk hydrophobic moiety and an α, ß-unsaturated carbonylic system able to perform a thiol-Michael reaction. In this work, we evaluated two chemically related natural products: 1,4-dihydroxy-2-(3',7'-dimethyl-1'-oxo-2'E,6'-octadienyl) benzene (C1) and 4-hydroxy-2-(3',7'-dimethyl-1'-oxo-2'E,6'-octadienyl) benzoic acid (C2), both were isolated from branches Piper crassinervium (Piperaceae), over the peroxidase activity of AhpC from Pseudomonas aeruginosa (PaAhpC) and Staphylococcus epidermidis (SeAhpC). By biochemical assays we show that although both compounds can perform the Michael addition reaction, only compound C2 was able to inhibit the PaAhpC peroxidase activity but not SeAhpC, presenting IC50 = 20.3 µM. SDS-PAGE analysis revealed that the compound was not able to perform a thiol-Michael addition, suggesting another inhibition behavior. Using computer-assisted simulations, we also show that an acidic group present in the structure of compound C2 may be involved in the stabilization by polar interactions with the Thr and Arg residues from the catalytic triad and several apolar interactions with hydrophobic residues. Finally, C2 was not able to interfere in the peroxidase activity of the isoform Prx2 from humans or even the thiol proteins of the Trx reducing system from Escherichia coli (EcTrx and EcTrxR), indicating specificity for P. aeruginosa AhpC.

In Silico Evaluation of Quercetin Methylated Derivatives on the Interaction with Secretory Phospholipases A2 from Crotalus durissus terrificus and Bothrops jararacussu.

Quercetin derivatives have already shown their anti-inflammatory potential, inhibiting essential enzymes involved in this process. Among diverse pro-inflammatory toxins from snake venoms, phospholipase A2 is one of the most abundant in some species, such as Crotalus durissus terrificus and Bothrops jararacussu from the Viperidae family. These enzymes can induce the inflammatory process through hydrolysis at the sn-2 position of glycerophospholipids. Hence, elucidating the main residues involved in the biological effects of these macromolecules can help to identify potential compounds with inhibitory activity. In silico tools were used in this study to evaluate the potential of quercetin methylated derivatives in the inhibition of bothropstoxin I (BthTX-I) and II (BthTX-II) from Bothrops jararacussu and phospholipase A2 from Crotalus durissus terrificus. The use of a transitional analogous and two classical inhibitors of phospholipase A2 guided this work to find the role of residues involved in the phospholipid anchoring and the subsequent development of the inflammatory process. First, main cavities were studied, revealing the best regions to be inhibited by a compound. Focusing on these regions, molecular docking assays were made to show main interactions between each compound. Results reveal that analogue and inhibitors, Varespladib (Var) and p-bromophenacyl bromide (BPB), guided quercetins derivatives analysis, revealing that Leu2, Phe5, Tyr28, glycine in the calcium-binding loop, His48, Asp49 of BthTX-II and Cdtspla2 were the main residues to be inhibited. 3MQ exhibited great interaction with the active site, similar to Var results, while Q anchored better in the BthTX-II active site. However, strong interactions in the C-terminal region, highlighting His120, seem to be crucial to decreasing contacts with phospholipid and BthTX-II. Hence, quercetin derivatives anchor differently with each toxin and further in vitro and in vivo studies are essential to elucidate these data.

Gallic Acid as a Non-Selective Inhibitor of α/ß-Hydrolase Fold Enzymes Involved in the Inflammatory Process: The Two Sides of the Same Coin.

(1) Background: Gallic acid (GA) has been characterized as an effective anti-inflammatory, antivenom, and promising drug for therapeutic use. (2/3) Methods and Results: GA was identified from ethanolic extract of fresh pitanga (Eugenia uniflora) leaves, which was identified using commercial GA. Commercial GA neutralized the enzymatic activity of secretory PLA2 (sPLA2) by inhibiting the active site and inducing changes in the secondary structure of the enzyme. Pharmacological edema assays showed that GA strongly decreased edema when the compound was previously incubated with sPLA2. However, prior treatment of GA (30 min before) significantly increased the edema and myotoxicity induced by sPLA2. The molecular docking results of GA with platelet-acetylhydrolase (PAF-AH) and acetylcholinesterase reveal that this compound was able to interact with the active site of both molecules, inhibiting the hydrolysis of platelet-activating factor (PAF) and acetylcholine (ACh). (4) Conclusion: GA has a great potential application; however, our results show that this compound can also induce adverse effects in previously treated animals. Additionally, the increased edema and myotoxicity observed experimentally in GA-treated animals may be due to the inhibition of PAF-AH and Acetylcholinesterase.

Structural and biochemical characterization of peroxiredoxin Qbeta from Xylella fastidiosa: catalytic mechanism and high reactivity.

The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 10(7) and 10(6) M(-1) S(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie approximately 12.3 A apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account.

Effects of Serine or Threonine in the Active Site of Typical 2-Cys Prx on Hyperoxidation Susceptibility and on Chaperone Activity.

Typical 2-Cys peroxiredoxins (2-Cys Prx) are ubiquitous Cys-based peroxidases, which are stable as decamers in the reduced state, and may dissociate into dimers upon disulfide bond formation. A peroxidatic Cys (CP) takes part of a catalytic triad, together with a Thr/Ser and an Arg. Previously, we described that the presence of Ser (instead of Thr) in the active site stabilizes yeast 2-Cys Prx as decamers. Here, we compared the hyperoxidation susceptibilities of yeast 2-Cys Prx. Notably, 2-Cys Prx containing Ser (named here Ser-Prx) were more resistant to hyperoxidation than enzymes containing Thr (Thr-Prx). In silico analysis revealed that Thr-Prx are more frequent in all domains of life, while Ser-Prx are more abundant in bacteria. As yeast 2-Cys Prx, bacterial Ser-Prx are more stable as decamers than Thr-Prx. However, bacterial Ser-Prx were only slightly more resistant to hyperoxidation than Thr-Prx. Furthermore, in all cases, organic hydroperoxide inhibited more the peroxidase activities of 2-Cys Prx than hydrogen peroxide. Moreover, bacterial Ser-Prx displayed increased thermal resistance and chaperone activity, which may be related with its enhanced stability as decamers compared to Thr-Prx. Therefore, the single substitution of Thr by Ser in the catalytic triad results in profound biochemical and structural differences in 2-Cys Prx.

Thiol Peroxidases as Major Regulators of Intracellular Levels of Peroxynitrite in Live Saccharomyces cerevisiae Cells.

Thiol peroxidases (TP) are ubiquitous and abundant antioxidant proteins of the peroxiredoxin and glutathione peroxidase families that can catalytically and rapidly reduce biologically relevant peroxides, such as hydrogen peroxide and peroxynitrite. However, the TP catalytic cycle is complex, depending on multiple redox reactions and partners, and is subjected to branching and competition points that may limit their peroxide reductase activity in vivo. The goals of the present study were to demonstrate peroxynitrite reductase activity of TP members in live cells in real time and to evaluate its catalytic characteristics. To these ends, we developed a simple fluorescence assay using coumarin boronic acid (CBA), exploiting that fact that TP and CBA compete for peroxynitrite, with the expectation that higher TP peroxynitrite reductase activity will lower the CBA oxidation. TP peroxynitrite reductase activity was evaluated by comparing CBA oxidation in live wild type and genetically modified Δ8 (TP-deficient strain) and Δ8+TSA1 (Δ8 strain that expresses only one TP member, the TSA1 gene) Saccharomyces cerevisiae strains. The results showed that CBA oxidation decreased with cell density and increased with increasing peroxynitrite availability. Additionally, the rate of CBA oxidation decreased in the order Δ8 > Δ8+TSA1 > WT strains both in control and glycerol-adapted (expressing higher TP levels) cells, showing that the CBA competition assay could reliably detect peroxynitrite in real time in live cells, comparing CBA oxidation in strains with reduced and increased TP expression. Finally, there were no signs of compromised TP peroxynitrite reductase activity during experimental runs, even at the highest peroxynitrite levels tested. Altogether, the results show that TP is a major component in the defense of yeast against peroxynitrite insults under basal and increasing stressful conditions.

Reduction of sulfenic acids by ascorbate in proteins, connecting thiol-dependent to alternative redox pathways.

Sulfenic acids are the primary product of thiol oxidation by hydrogen peroxide and other oxidants. Several aspects of sulfenic acid formation through thiol oxidation were established recently. In contrast, the reduction of sulfenic acids is still scarcely investigated. Here, we characterized the kinetics of the reduction of sulfenic acids by ascorbate in several proteins. Initially, we described the crystal structure of our model protein (Tsa2-C170S). There are other Tsa2 structures in distinct redox states in public databases and all of them are decamers, with the peroxidatic cysteine very accessible to reductants, convenient features to investigate kinetics. We determined that the reaction between Tsa2-C170S-Cys-SOH and ascorbate proceeded with a rate constant of 1.40 ± 0.08 × 103 M-1 s-1 through a competition assay developed here, employing 2,6-dichlorophenol-indophenol (DCPIP). A series of peroxiredoxin enzymes (Prx6 sub family) were also analyzed by this competition assay and we observed that the reduction of sulfenic acids by ascorbate was in the 0.4-2.2 × 103 M-1 s-1 range. We also evaluated the same reaction on glyceraldehyde 3-phosphate dehydrogenase and papain, as the reduction of their sulfenic acids by ascorbate were reported previously. Once again, the rate constants are in the 0.4-2.2 × 103 M-1 s-1 range. We also analyzed the reduction of Tsa2-C170S-SOH by ascorbate by a second, independent method, following hydrogen peroxide reduction through a specific electrode (ISO-HPO-2, World Precision Instruments) and employing a bi-substrate, steady state approach. The kcat/KMAsc was 7.4 ± 0.07 × 103 M-1 s-1, which was in the same order of magnitude as the value obtained by the DCPIP competition assay. In conclusion, our data indicates that reduction of sulfenic acid in various proteins proceed at moderate rate and probably this reaction is more relevant in biological systems where ascorbate concentrations are high.