Role of Myeloperoxidase of northern snakehead (Channa argus) in Aeromonas veronii infection.

Myeloperoxidase (MPO) is a ferrous lysosomal protein with many immune functions that belongs to the heme peroxidase enzyme. In this study, the functions of MPO in the northern snakehead (Channa argus) were investigated by cloning an MPO cDNA sequence with a full length of 3181 bp. Homology analysis showed that northern snakehead MPO gene had the highest (81%) homology with mandarin fish (Siniperca chuatsi). In healthy northern snakehead, the MPO gene was expressed in the head-kidney, kidney, heart, gill, spleen, liver, and muscles but not midgut. After the northern snakehead was infected with Aeromonas veronii, the MPO gene expression varied in different tissues with low level in spleen, liver, gill and muscle, fluctuated in kidney and head-kidney and showed high level in heart. The result indicated that MPO might play an important role in the antimicrobial immune response of the northern snakehead.

Characterization of a novel DyP-type peroxidase from Streptomyces avermitilis.

DyP-type peroxidases are a heme peroxidase family with unique properties whose members are widely distributed from prokaryotes to eukaryotes. DyP-type peroxidases are subdivided into class P, I and V based on structure-based sequence alignment. Class V enzymes possess degradation activities for anthraquinone dyes, and include extra sequences compared with class P and I. Class V enzymes are mainly found in fungi, with only two such proteins, AnaPX and DyP2, reported in bacteria. Here, we heterologously expressed, purified and biochemically characterized SaDyP2 protein, predicted to belong to class V. SaDyP2 was purified as a ∼50 kDa enzyme containing a heme cofactor and was found to oxidize the typical peroxidase substrates, ABTS and DMP. SaDyP2 was generally thermostable and exhibited a lower optimal pH, a feature typical of DyP-type peroxidases. It also degraded anthraquinone dyes, a specific substrate of DyP-type peroxidases, although the kcat for SaDyP2 was lower than that for other class V enzymes. The Km value of SaDyP2 for anthraquinone dye was similar to that of other enzymes of this class. Homology modeling revealed that the structure of SaDyP2 best fit that of class V enzymes.

Characterization and application of a novel class II thermophilic peroxidase from Myceliophthora thermophila in biosynthesis of polycatechol.

A peroxidase from the thermophilic fungus Myceliophthora thermophila that belongs to ascomycete Class II based on PeroxiBase classification was functionally expressed in methylotrophic yeast Pichia pastoris. The putative peroxidase from the genomic DNA was successfully cloned in P. pastoris X-33 under the transcriptional control of the alcohol oxidase (AOX1) promoter. The heterologous production was greatly enhanced by the addition of hemin with a titer of 0.41 U mL(-1) peroxidase activity at the second day of incubation. The recombinant enzyme was purified to homogeneity (50 kDa) and characterized using a series of phenolic substrates that indicated similar characteristics with those of generic peroxidases. In addition, the enzyme was found thermostable, retaining its activity for temperatures up to 60 °C after eight hours incubation. Moreover, the enzyme displayed remarkable H2O2 stability, retaining more than 80% of its initial activity after 24h incubation in 5000-fold molar excess of H2O2. The ability of the peroxidase to polymerize catechol at high superoxide concentrations, together with its high thermostability and substrate specificity, indicate a potential commercial significance of the enzyme.

Fungal unspecific peroxygenases: heme-thiolate proteins that combine peroxidase and cytochrome p450 properties.

Eleven years ago, a secreted heme-thiolate peroxidase with promiscuity for oxygen transfer reactions was discovered in the basidiomycetous fungus, Agrocybe aegerita. The enzyme turned out to be a functional mono-peroxygenase that transferred an oxygen atom from hydrogen peroxide to diverse organic substrates (aromatics, heterocycles, linear and cyclic alkanes/alkenes, fatty acids, etc.). Later similar enzymes were found in other mushroom genera such as Coprinellus and Marasmius. Approximately one thousand putative peroxygenase sequences that form two large clusters can be found in genetic databases and fungal genomes, indicating the widespread occurrence of such enzymes in the whole fungal kingdom including all phyla of true fungi (Eumycota) and certain fungus-like heterokonts (Oomycota). This new enzyme type was classified as unspecific peroxygenase (UPO, EC 1.11.2.1) and placed in a separate peroxidase subclass. Furthermore, UPOs and related heme-thiolate peroxidases such as well-studied chloroperoxidase (CPO) represent a separate superfamily of heme proteins on the phylogenetic level. The reactions catalyzed by UPOs include hydroxylation, epoxidation, O- and N-dealkylation, aromatization, sulfoxidation, N-oxygenation, dechlorination and halide oxidation. In many cases, the product patterns of UPOs resemble those of human cytochrome P450 (P450) monooxygenases and, in fact, combine the catalytic cycle of heme peroxidases with the "peroxide shunt" of P450s. Here, an overview on UPOs is provided with focus on their molecular and catalytic properties.

Systematic analysis of maize class III peroxidase gene family reveals a conserved subfamily involved in abiotic stress response.

Class III peroxidases (PRXs) are plant-specific enzymes that play key roles in the responses to biotic and abiotic stress during plant growth and development. In this study, we identified 119 nonredundant PRX genes (designated ZmPRXs). These PRX genes were divided into 18 groups based on their phylogenetic relationships. We performed systematic bioinformatics analysis of the PRX genes, including analysis of gene structures, conserved motifs, phylogenetic relationships and gene expression profiles. The ZmPRXs are unevenly distributed on the 10 maize chromosomes. In addition, these genes have undergone 16 segmental duplication and 12 tandem duplication events, indicating that both segmental and tandem duplication were the main contributors to the expansion of the maize PRX family. Ka/Ks analysis suggested that most duplicated ZmPRXs experienced purifying selection, with limited functional divergence during the duplication events, and comparative analysis among maize, sorghum and rice revealed that there were independent duplication events besides the whole-genome duplication of the maize genome. Furthermore, microarray analysis indicated that most highly expressed genes might play significant roles in root. We examined the expression of five candidate ZmPRXs under H2O2, SA, NaCl and PEG stress conditions using quantitative real-time PCR (qRT-PCR), revealing differential expression patterns. This study provides useful information for further functional analysis of the PRX gene family in maize.

Molecular cloning, sequence analysis, prokaryotic expression, and function prediction of foot-specific peroxidase in Hydra magnipapillata Chinese strain.

The cDNA sequence of foot-specific peroxidase PPOD1 from the Chinese strain of Hydra magnipapillata was cloned by reverse transcription-polymerase chain reaction. The cDNA sequence contained a coding region with an 873-bp open reading frame, a 31-bp 5'-untranslated region, and a 36-bp 3'-untranslated region. The structure prediction results showed that PPOD1 contains 10.34% of α-helix, 38.62% of extended strand, 12.41% of ß-turn, and 38.62% of random coil. The structural core was α-helix at the N terminus. The GenBank protein blast server showed that PPOD1 contains 2 fascin-like domains. In addition, high-level PPOD1 activity was only present in the ectodermal epithelial cells located on the edge of the adhesive face of the basal disc, and that these cells extended lamellipodia and filopodia when the basal disc was tightly attached to a glass slide. The fascin-like domains of Hydra PPOD1 might contribute to the bundling of the actin filament of these cells, and hence, the formation of filopodia. In conclusion, these cells might play an important role in strengthening the adsorbability of the basal disc to substrates.

Turning points in the evolution of peroxidase-catalase superfamily: molecular phylogeny of hybrid heme peroxidases.

Heme peroxidases and catalases are key enzymes of hydrogen peroxide metabolism and signaling. Here, the reconstruction of the molecular evolution of the peroxidase-catalase superfamily (annotated in pfam as PF00141) based on experimentally verified as well as numerous newly available genomic sequences is presented. The robust phylogenetic tree of this large enzyme superfamily was obtained from 490 full-length protein sequences. Besides already well-known families of heme b peroxidases arranged in three main structural classes, completely new (hybrid type) peroxidase families are described being located at the border of these classes as well as forming (so far missing) links between them. Hybrid-type A peroxidases represent a minor eukaryotic subfamily from Excavates, Stramenopiles and Rhizaria sharing enzymatic and structural features of ascorbate and cytochrome c peroxidases. Hybrid-type B peroxidases are shown to be spread exclusively among various fungi and evolved in parallel with peroxidases in land plants. In some ascomycetous hybrid-type B peroxidases, the peroxidase domain is fused to a carbohydrate binding (WSC) domain. Both here described hybrid-type peroxidase families represent important turning points in the complex evolution of the whole peroxidase-catalase superfamily. We present and discuss their phylogeny, sequence signatures and putative biological function.

Peroxidase gene discovery from the horseradish transcriptome.

BACKGROUND: Horseradish peroxidases (HRPs) from Armoracia rusticana have long been utilized as reporters in various diagnostic assays and histochemical stainings. Regardless of their increasing importance in the field of life sciences and suggested uses in medical applications, chemical synthesis and other industrial applications, the HRP isoenzymes, their substrate specificities and enzymatic properties are poorly characterized. Due to lacking sequence information of natural isoenzymes and the low levels of HRP expression in heterologous hosts, commercially available HRP is still extracted as a mixture of isoenzymes from the roots of A. rusticana. RESULTS: In this study, a normalized, size-selected A. rusticana transcriptome library was sequenced using 454 Titanium technology. The resulting reads were assembled into 14871 isotigs with an average length of 1133 bp. Sequence databases, ORF finding and ORF characterization were utilized to identify peroxidase genes from the 14871 isotigs generated by de novo assembly. The sequences were manually reviewed and verified with Sanger sequencing of PCR amplified genomic fragments, resulting in the discovery of 28 secretory peroxidases, 23 of them previously unknown. A total of 22 isoenzymes including allelic variants were successfully expressed in Pichia pastoris and showed peroxidase activity with at least one of the substrates tested, thus enabling their development into commercial pure isoenzymes. CONCLUSIONS: This study demonstrates that transcriptome sequencing combined with sequence motif search is a powerful concept for the discovery and quick supply of new enzymes and isoenzymes from any plant or other eukaryotic organisms. Identification and manual verification of the sequences of 28 HRP isoenzymes do not only contribute a set of peroxidases for industrial, biological and biomedical applications, but also provide valuable information on the reliability of the approach in identifying and characterizing a large group of isoenzymes.

Nomenclature and classification of vasculitis: lessons learned from granulomatosis with polyangiitis (Wegener's granulomatosis).

Names influence how something is perceived. Diagnostic terms (diagnoses) are the names of diseases that are usually derived either from some distinctive characteristic of the disease or include an eponym recognizing someone who elucidated the disease. No matter how logical and appropriate a name may be, if it is not usable and used it is of no lasting value. This brief commentary focuses on the nomenclature of systemic vasculitides, and uses as a prime example Wegener's granulomatosis, which has been renamed recently 'granulomatosis with polyangiitis', in part because of concerns about the suitability of Friedrich Wegener as the source of an eponym. The most distinctive pathological feature of Wegener's granulomatosis is multi-focal necrotizing inflammation that has long been called granulomatosis. The systemic variant of Wegener's granulomatosis also is characterized by inflammation in many different vessels or different types, i.e. polyangiitis. Thus, granulomatosis with polyangiitis is a very appropriate alternative term for Wegener's granulomatosis. This term also is in accord with the name for a closely related vasculitis, i.e. microscopic polyangiitis. Terms that indicate aetiology and pathogenesis, when known, are useful to include in names for diseases (diagnoses). Anti-neutrophil cytoplasmic autoantibodies specific for myeloperoxidase (MPO-ANCA) or proteinase 3 (PR3-ANCA) are implicated in the cause of granulomatosis with polyangiitis and thus also should be specified in the diagnosis (e.g. PR3-ANCA-positive granulomatosis with polyangiitis or MPO-ANCA-positive microscopic polyangiitis). As our understanding of the clinical manifestations, pathogenesis and aetiology of vasculitides change over time, the names and approaches for diagnosing these diseases will change accordingly.

Actinobacterial peroxidases: an unexplored resource for biocatalysis.

Peroxidases are redox enzymes that can be found in all forms of life where they play diverse roles. It is therefore not surprising that they can also be applied in a wide range of industrial applications. Peroxidases have been extensively studied with particular emphasis on those isolated from fungi and plants. In general, peroxidases can be grouped into haem-containing and non-haem-containing peroxidases, each containing protein families that share sequence similarity. The order Actinomycetales comprises a large group of bacteria that are often exploited for their diverse metabolic capabilities, and with recent increases in the number of sequenced genomes, it has become clear that this metabolically diverse group of organisms also represents a large resource for redox enzymes. It is therefore surprising that, to date, no review article has been written on the wide range of peroxidases found within the actinobacteria. In this review article, we focus on the different types of peroxidases found in actinobacteria, their natural role in these organisms and how they compare with the more well-described peroxidases. Finally, we also focus on work remaining to be done in this research field in order for peroxidases from actinobacteria to be applied in industrial processes.

Cloning, characterization and localization of a novel basic peroxidase gene from Catharanthus roseus.

Catharanthus roseus (L.) G. Don produces a number of biologically active terpenoid indole alkaloids via a complex terpenoid indole alkaloid biosynthetic pathway. The final dimerization step of this pathway, leading to the synthesis of a dimeric alkaloid, vinblastine, was demonstrated to be catalyzed by a basic peroxidase. However, reports of the gene encoding this enzyme are scarce for C. roseus. We report here for the first time the cloning, characterization and localization of a novel basic peroxidase, CrPrx, from C. roseus. A 394 bp partial peroxidase cDNA (CrInt1) was initially amplified from the internodal stem tissue, using degenerate oligonucleotide primers, and cloned. The full-length coding region of CrPrx cDNA was isolated by screening a leaf-specific cDNA library with CrInt1 as probe. The CrPrx nucleotide sequence encodes a deduced translation product of 330 amino acids with a 21 amino acid signal peptide, suggesting that CrPrx is secretory in nature. The molecular mass of this unprocessed and unmodified deduced protein is estimated to be 37.43 kDa, and the pI value is 8.68. CrPrx was found to belong to a 'three intron' category of gene that encodes a class III basic secretory peroxidase. CrPrx protein and mRNA were found to be present in specific organs and were regulated by different stress treatments. Using a beta-glucuronidase-green fluorescent protein fusion of CrPrx protein, we demonstrated that the fused protein is localized in leaf epidermal and guard cell walls of transiently transformed tobacco. We propose that CrPrx is involved in cell wall synthesis, and also that the gene is induced under methyl jasmonate treatment. Its potential involvement in the terpenoid indole alkaloid biosynthetic pathway is discussed.

Isolation and characterization of the first putative peroxidase gene from oilseed rape (Brassica napus) which is highly homologous to HRPC.

A new gene, designated as BnPrx (GenBank Accession No. DQ078754), was isolated from oilseed rape (Brassica napus) by SMART Rapid Amplification of cDNA Ends (RACE). The full-length cDNA is 1307 bp long and contains a 1062 bp open reading frame (ORF), which encodes a 354 amino acid peroxidase precursor, with a 31 aa N-terminal signal peptide and a 15 aa C-terminal propeptide. The putative protein has a molecular weight of 38.86 kDa and a calculated pI of 5.85. BnPrx shares high identity with HRPC (89%). BnPrx possesses all active residues and two Ca(2+) sites present in Horseradish peroxidase isoenzymes C (HRPC) as well as six N-glycosylation sites. The predicted 3-D structure of BnPrx is very similar to that of HRPC. Assisted by genomic walking technology, the genomic DNA of BnPrx was also cloned, consisting of 3 introns and 4 exons. Thirty-two TATA boxes, 18 CAAT boxes and many cis-elements, such as WUN, MeJR, were found in its promoter region. Southern blot analysis indicated that BnPrx belonged to a small gene family. Northern blot analysis revealed that BnPrx was constitutively expressed in all tested tissues, including roots, stems and leaves, with the high expression in leaves and stems. The expression of BnPrx could be induced by methyl jasmonate (MeJA), salicylic acid (SA), cold and H(2)O(2). The cloning and characterizing of BnPrx might not only help us understand the physiological function and molecular evolution of the large peroxidase gene family more comprehensively, but also provide an alternative way of seeking a more effective and economical substitute for HRPC.

Molecular and biochemical markers associated with leaffolder (Cnaphalocrocis medinalis G.) resistance in rice (Oryza sativa L.).

Association of molecular markers namely isozymes and simple sequence repeats (SSRs) and various biochemical markers to leaffolder (Cnaphalocrocis medinalis G., a predominant insect pest of rice) resistance were studied in rice (Oryza sativa L.). Recombinant inbred lines (RILs) of F8 generation obtained by crossing IR36 (susceptible parent) and TNAULFR831311 (moderately resistant parent) were used in this study. Soluble protein content, protein profile, and peroxidase and phenylalanine ammonia lyase (PAL) activities were the various biochemical markers studied. Decrease in soluble leaf protein content was observed in all lines, due to insect infestation. Protein profiling revealed an enhanced expression of a high molecular mass (> 97 kDa) protein in all the infested lines. Besides, there was an increased induction of a 38 kDa protein in infested resistant parent and resistant RILs. A significant increase in peroxidase and PAL activities was observed after infestation. In peroxidase isozyme analysis, carried out after infestation, "isoform 1" was found to be more prominent in the susceptible lines and "isoform 2" in the resistant lines. Bulk segregant analysis (BSA) with twenty-five rice microsatellites (RM) resulted in identification of three polymorphic markers between bulks RM11 and RM432 located on chromosome 7 and RM271 on chromosome 10 of rice. These markers may be associated with leaffolder resistance in rice and can be used in marker-assisted selection for leaffolder resistance in rice.

Phylogenetic relationships in class I of the superfamily of bacterial, fungal, and plant peroxidases.

Molecular phylogeny among catalase-peroxidases, cytochrome c peroxidases, and ascorbate peroxidases was analysed. Sixty representative sequences covering all known subgroups of class I of the superfamily of bacterial, fungal, and plant heme peroxidases were selected. Each sequence analysed contained the typical peroxidase motifs evolved to bind effectively the prosthetic heme group, enabling peroxidatic activity. The N-terminal and C-terminal domains of catalase-peroxidases matching the ancestral tandem gene duplication event were treated separately in the phylogenetic analysis to reveal their specific evolutionary history. The inferred unrooted phylogenetic tree obtained by three different methods revealed the existence of four clearly separated clades (C-terminal and N-terminal domains of catalase-peroxidases, ascorbate peroxidases, and cytochrome c peroxidases) which were segregated early in the evolution of this superfamily. From the results, it is obvious that the duplication event in the gene for catalase-peroxidase occurred in the later phase of evolution, in which the individual specificities of the peroxidase families distinguished were already formed. Evidence is presented that class I of the heme peroxidase superfamily is spread among prokaryotes and eukaryotes, obeying the birth-and-death process of multigene family evolution.

Identification of a novel class of endoperoxides from arachidonate autoxidation.

Free radical-initiated lipid autoxidation in low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. Oxidation of the lipid components of LDL leads to a complex mixture of hydroperoxides, bicyclic endoperoxides, monocyclic peroxides, and serial cyclic peroxides. The oxidation compounds and/or their decomposition products can modify protein components, which may lead to various diseases. A novel class of peroxides (termed dioxolane-isoprostanes) having a bicyclic endoperoxide moiety characteristic of the isoprostanes and a dioxolane peroxide functionality in the same molecule was identified in the product mixture formed from in vitro autoxidation of cholesteryl arachidonate. The same products are also detected in in vitro oxidized LDL. Various mass spectrometric techniques have been applied to characterize these new peroxides. The structure of these compounds has also been confirmed by independent synthesis. We reason, based on the free radical mechanism of the transformation, that only the 12- and 8-peroxyl radicals (those leading to 12-HPETE and 8-HPETE) of arachidonate can form these new peroxides. We also suggest that the formation of these peroxides provides a rationale to explain the fact that 5- and 15-series isoprostanes are formed in preference to 8- and 12-series. Furthermore, series of other isoprostanes, such as dioxolane A(2), D(2), E(2), etc., can be derived from the dioxolane-isoprostane peroxides. These findings offer further insights into the oxidation products of arachidonate and the opportunity to study their potential biological relevance.

Molecular evolution of the myeloperoxidase family.

Animal myeloperoxidase and its relatives constitute a diverse protein family, which includes myeloperoxidase, eosinophil peroxidase, thyroid peroxidase, salivary peroxidase, lactoperoxidase, ovoperoxidase, peroxidasin, peroxinectin, cyclooxygenase, and others. The members of this protein family share a catalytic domain of about 500 amino acid residues in length, although some members have distinctive mosaic structures. To investigate the evolution of the protein family, we performed a comparative analysis of its members, using the amino acid sequences and the coordinate data available today. The results obtained in this study are as follows: (1) 60 amino acid sequences belonging to this family were collected by database searching. We found a new member of the myeloperoxidase family derived from a bacterium. This is the first report of a bacterial member of this family. (2) An unrooted phylogenetic tree of the family was constructed according to the alignment. Considering the branching pattern in the obtained phylogenetic tree, together with the mosaic features in the primary structures, 60 members of the myeloperoxidase family were classified into 16 subfamilies. (3) We found two molecular features that distinguish cyclooxygenase from the other members of the protein family. (4) Several structurally deviated segments were identified by a structural comparison between cyclooxygenase and myeloperoxidase. Some of the segments seemed to be associated with the functional and/or structural differences between the enzymes.

Complement activation by myeloperoxidase products released from stimulated human polymorphonuclear leukocytes.

Purified human myeloperoxidase (MPO) converted human C5 to an activated form, i.e. the C5 protein adopted a configuration expressing a binding site for C6; the resulting C56 complex then reacted with C7, C8 and C9 forming a hemolytic C5-9 complex. For the activation by myeloperoxidase chloride and hydrogen peroxide were essential. This indicates that the peroxidase acted through the generation of HOCl which had been shown earlier to oxidize and activate C5. Human polymorphonuclear leukocytes (PMN) were stimulated in vitro by incubation with opsonized zymosan; thereafter the supernatants were tested for C5 activating potency. Stimulated PMN release H2O2 and MPO that produces hypochlorite and secondarily various chloramines. As a trap for the labile hypochlorite generated excess taurine was added to the PMN suspensions during the incubation. Hypochlorite is then stoichiometrically converted to the relatively stable taurine chloramine. In order to rule out interfering activities of proteolytic enzymes released from the PMN and known to attack C5, the supernatants were ultracentrifuged, and the ultrafiltrates, containing only low molecular weight compounds, were used for the further studies. They contained taurine chloramine, estimated photometrically, and they activated C5 upon incubation, assayed functionally by reactive lysis. Azide, an inhibitor of myeloperoxidase, and catalase which destroys H2O2, essential for MPO-catalyzed oxidations, prevented the generation of C5 activating potency and of chloramines. Unstimulated PMN produced neither oxidants nor C5 activating potency. When taurine was omitted from the PMN suspensions during stimulations much less oxidant was found in the supernatants and less C5 activating potency. These findings indicate that the C5 activating agent was produced by stimulated PMN through MPO-generated hypochlorite, trapped as taurine chloramine. In the absence of added taurine the hypochlorite formed by MPO oxidized endogenous amines that also activated C5. Further studies suggested that among these was some monochloramine derived from endogenous ammonia. Activation of the terminal complement reaction sequence by MPO released from stimulated PMN may represent a third pathway to complement activation contributing to and reinforcing complement and PMN functions at the site of inflammation or tissue injury.

Killing of Actinobacillus actinomycetemcomitans by the human neutrophil myeloperoxidase-hydrogen peroxide-chloride system.

Actinobacillus actinomycetemcomitans is a facultative gram-negative coccobacillus associated with periodontal disease and nonoral infections. This organism is resistant to serum bactericidal mechanisms but is nevertheless killed by human neutrophils under aerobic and anaerobic conditions. Most of the killing attributable to oxidative mechanisms is inhibited by sodium cyanide, which suggests that the myeloperoxidase-hydrogen peroxide-chloride (MPO-H2O2-Cl-) system may be a key factor in the oxidative killing process. In this report, we examine whether the isolated MPO-H2O2-Cl- system is bactericidal against A. actinomycetemcomitans. We found that three major chromatographic forms of MPO were capable of killing A. actinomycetemcomitans at sublethal concentrations of H2O2 and that both catalase-positive and catalase-negative strains of this organism were sensitive to killing by the MPO-H2O2-Cl- system. We conclude that the isolated MPO-H2O2-Cl- system is bactericidal for A. actinomycetemcomitans independent of other neutrophil granule constituents and may be an important component of the oxygen-dependent bactericidal activity of the neutrophil with respect to this periodontopathic organism.