Animal Peroxidases Research Articles

DyP-type peroxidases: a promising and versatile class of enzymes

DyP peroxidases comprise a novel superfamily of heme-containing peroxidases, which is unrelated to the superfamilies of plant and animal peroxidases. These enzymes have so far been identified in the genomes of fungi, bacteria, as well as archaea, although their physiological function is still unclear. DyPs are bifunctional enzymes displaying not only oxidative activity but also hydrolytic activity. Moreover, these enzymes are able to oxidize a variety of organic compounds of which some are poorly converted by established peroxidases, including dyes, β-carotene, and aromatic sulfides. Interestingly, accumulating evidence shows that microbial DyP peroxidases play a key role in the degradation of lignin. Owing to their unique properties, these enzymes are potentially interesting for a variety of biocatalytic applications. In this review, we deal with the biochemical and structural features of DyP-type peroxidases as well as their promising biotechnological potential.

DyP-type Peroxidases: A Promising and Versatile Class of Enzymes

Peroxidases (EC1.11.1.x) represent a large family of oxidoreductases that typically use hydrogen peroxide as an electron acceptor to catalyze the oxidation of substrate molecules. The vast majority of these enzymes contain heme as a cofactor [1], and are ubiquitously present in prokaryotes and eukaryotes. Peroxidases take center stage in a variety of biochemical processes, ranging from the biosynthesis of cell wall material to immunological host-defense responses [2,3]. Hemecontaining peroxidases were originally classified into two superfamilies: the plant peroxidases and animal peroxidases [4]. Remarkably, some members of the peroxidase superfamily have been studied for more than a century like, for example, Horseradish Peroxidase (HRP) [5], and in this respect, it was highly fascinating that the first member of a newly discovered peroxidase superfamily, the group of DyP-type peroxidases, was described in the late 90’s [6]. Here, we will discuss the biochemical and structural features of DyP-type peroxidases, as well as their promising biotechnological potential.

Peroxidasin Is Secreted and Incorporated into the Extracellular Matrix of Myofibroblasts and Fibrotic Kidney

Mammalian peroxidases are heme-containing enzymes that serve diverse biological roles, such as host defense and hormone biosynthesis. A mammalian homolog of Drosophila peroxidasin belongs to the peroxidase family; however, its function is currently unknown. In this study, we show that peroxidasin is present in the endoplasmic reticulum of human primary pulmonary and dermal fibroblasts, and the expression of this protein is increased during transforming growth factor-beta1-induced myofibroblast differentiation. Myofibroblasts secrete peroxidasin into the extracellular space where it becomes organized into a fibril-like network and colocalizes with fibronectin, thus helping to form the extracellular matrix. We also demonstrate that peroxidasin expression is increased in a murine model of kidney fibrosis and that peroxidasin localizes to the peritubular space in fibrotic kidneys. In addition, we show that this novel pathway of extracellular matrix formation is unlikely mediated by the peroxidase activity of the protein. Our data indicate that peroxidasin secretion represents a previously unknown pathway in extracellular matrix formation with a potentially important role in the physiological and pathological fibrogenic response.

Horseradish peroxidase: a valuable tool in biotechnology

Peroxidases have conquered a prominent position in biotechnology and associated research areas (enzymology, biochemistry, medicine, genetics, physiology, histo- and cytochemistry). They are one of the most extensively studied groups of enzymes and the literature is rich in research papers dating back from the 19th century. Nevertheless, peroxidases continue to be widely studied, with more than 2000 articles already published in 2002 (according to the Institute for Scientific Information). The importance of peroxidases is emphasised by their wide distribution among living organisms and by their multiple physiological roles. They have been divided into three superfamilies according to their source and mode of action: plant peroxidases, animal peroxidases and catalases. Among all peroxidases, horseradish peroxidase (HRP) has received a special attention and will be the focus of this review. A brief description of the three super-families is included in the first section of this review. In the second section, a comprehensive description of the present state of knowledge of the structure and catalytic action of HRP is presented. The physiological role of peroxidases in higher plants is described in the third section. And finally, the fourth section addresses the applications of peroxidases, especially HRP, in the environmental and health care sectors, and in the pharmaceutical, chemical and biotechnological industries.

Mutations Affecting the Calcium-Binding Site of Myeloperoxidase and Lactoperoxidase

Both myeloperoxidase (MPO) and lactoperoxidase (LPO) contain high affinity bound calcium, which has been suggested to play a structural role. Asp-96 in MPO, a residue next to the histidine distal from the heme prosthetic group, has been assigned to the calcium-binding site of the enzyme by X-ray crystallography. Multiple sequence alignment of known animal peroxidases has revealed that the calcium-binding site is highly conserved. In this study, we replaced Asp-96 in MPO and the counterpart Asp-227 in LPO both with Ala by site-directed mutagenesis. The level of peroxidase activity in insect cells infected with recombinant baculoviruses and their culture supernatants was reduced to virtually zero as a result of these mutations. Immunoblotting revealed that these mutant peroxidases were expressed in the cells but not secreted as effectively as the wild-type enzymes. Our findings suggest that a functional calcium-binding site is essential for the biosynthesis of active animal peroxidases.

Molecular evolution of thyroid peroxidase

Thyroid peroxidase is a member of a family of mammalian peroxidases that includes myeloperoxidase, lactoperoxidase, eosinophil peroxidase, and salivary peroxidase. Protein sequences showing a high degree of sequence similarity with mammalian peroxidases have recently been observed in several invertebrate species. A multiple sequence alignment prepared with five mammalian and six invertebrate peroxidases shows complete conservation of amino acid residues considered to be important in the formation of peroxidase compound 1. These include the distal and proximal histidines, a catalytic arginine residue, and an asparagine residue hydrogen bonded to the proximal histidine. TPO-2, an alternatively spliced form of TPO, lacks the essential asparagine (Asn 579). It is now possible to speak more broadly of the family of animal peroxidases, rather than mammalian peroxidases. The animal peroxidases comprise a group of homologous proteins that differ markedly from the plant/fungal/bacterial peroxidases in primary, secondary and tertiary structure, but which share with them a common function. Animal peroxidases probably arose independently of the plant/fungal/bacterial peroxidase superfamily and most likely belong to a different gene family. The relationship between animal and non-animal peroxidases probably represents an example of convergent evolution to a common enzymatic mechanism.

Molecular characterization of a novel peroxidase isolated from the ligninolytic fungus Pleurotus eryngii.

A haem peroxidase different from other microbial, plant and animal peroxidases is described. The enzyme is secreted as two isoforms by dikaryotic Pleurotus eryngii in peptone-containing liquid medium. The corresponding gene, which presents 15 introns and encodes a 361-amino-acid protein with a 30-amino-acid signal peptide, was isolated as two alleles corresponding to the two isoforms. The alleles differ in three amino acid residues and in a seven nucleotide deletion affecting a single metal response element in the promoter. When compared with Phanerochaete chrysosporium peroxidases, the new enzyme appears closer to lignin peroxidase (LiP) than to Mn-dependent peroxidase (MnP) isoenzymes (58-60% and 55% identity respectively). The molecular model built using crystal structures of three fungal peroxidases as templates, also showed high structural affinity with LiP (C alpha-distance 1.2 A). However, this peroxidase includes a Mn2+ binding site formed by three acidic residues (E36, E40 and D175) near the haem internal propionate, which accounts for the ability to oxidize Mn2+. Its capability to oxidize aromatic substrates could involve interactions with aromatic residues at the edge of the haem channel. Another possibility is long-range electron transfer, e.g. from W164, which occupies the same position of LiP W171 recently reported as involved in the catalytic cycle of LiP.

Proximal and distal histidines in thyroid peroxidase: relation to the alternatively spliced form, TPO-2.

The distal and proximal histidines in thyroid peroxidase (TPO), located by amino acid sequence alignment with their known counterparts in myeloperoxidase, are His 239 and His 494, respectively. These histidines lie outside the 57 amino acid peptide (residues 533-589) that is absent in the alternatively spliced form, TPO-2. However, asparagine 579, which very likely forms a stabilizing hydrogen bond with the proximal histidine in TPO, lies within the missing peptide region. The absence of Asn 579 from TPO-2 may be at least partially responsible for the reported lack of activity of this form of the enzyme. Formation of TPO compound I may also depend on Arg 396, based on analogy with the catalytic mechanism previously proposed for the more widely studied plant and fungal peroxidases. A multiple sequence alignment prepared with five mammalian and five invertebrate peroxidases shows complete conservation of Arg 396, as well as residues corresponding to His 239, His 494, and Asn 579 in TPO. The animal peroxidases comprise a family of homologous proteins that differ markedly from the plant/fungal/bacterial peroxidases in primary, secondary, and tertiary structure, yet share with them a common function. Animal peroxidases probably arose independently of the plant/fungal/bacterial peroxidase superfamily and most likely belong to a different gene family. The relation between animal and nonanimal peroxidases may represent an example of convergent evolution to a common enzymatic mechanism.

Peroxidase activity in the ink gland of Sepia officinalis and partial nucleotide sequence of a candidate cDNA encoding the enzyme

A peroxidase was partially purified from the ink gland of the cuttlefish Sepia officinalis. This enzyme acts on guaiacol and I − as substrates and is inhibited by cyanide and azide. By using the PCR technique we have isolated and sequenced a cDNA clone encoding a protein homologous to other animal peroxidases. These results are discussed in relation to the possible involvement of peroxidase in the biosynthesis of melanin.

Isolation of thyroid peroxidase from patients with graves' disease and comparison with animal peroxidases

1. 1. Human thyroid peroxidase (TPO) was isolated from 280-640 g of pooled thyroid tissue resected from patients with Graves' disease. 2. 2. Isolation was performed by an improved and simplified method. 3. 3. The Reinheit Zahl ( A 412/ A 280) of the final preparations was in the range of 0.16–0.32. 4. 4. The spectroscopic and enzymatic properties of Graves' TPO were compared with those of porcine TPO and bovine LPO, revealing closer resemblance to the former. 5. 5. Graves' TPO may provide a useful substitute for normal TPO, which is very difficult to isolate.

Relationships among amino acid sequences of animal, microbial and plant peroxidases

Relationships among 18 peroxidases amino acid sequences of animal, microbial and plant origin were examined using optimum alignment of all pairwise sequence combinations to generate a total distance matrix. The matrix was used to cluster the sequences with complete linkage (farthest neighbour) procedures. Specific distances were calculated from the total distances matrix. The patterns of specific distances for each sequence were compared to evaluate the relationships between sequences, check their significance and construct subgroups of related sequences. The results were compared with those from clustering and its resultant dendrogram; good agreement was achieved. The 18 sequences fell into two principal groups, plant peroxidases and animal/microbial peroxidases. Within the plant peroxidases four subgroups were detected; the animal/microbial peroxidases formed a fifth subgroup. Profiles were constructed for the subgroups from lists of matching amino acids generated by the alignment calculations. Superimposed lists were realigned to recognise conserved areas and elements. Individual subgroup profiles for the plant peroxidases were then combined into a single profile which in turn was combined with profiles from the animal/microbial peroxidases. The final profile suggested that numerous sequence features (motifs) were common to peroxidases of widely different function and origins.

Human myeloperoxidase and thyroid peroxidase, two enzymes with separate and distinct physiological functions, are evolutionarily related members of the same gene family

Human myeloperoxidase and human thyroid peroxidase nucleotide and amino acid sequences were compared. The global similarities of the nucleotide and amino acid sequences are 46% and 44%, respectively. These similarities are most evident within the coding sequence, especially that encoding the myeloperoxidase functional subunits. These results clearly indicate that myeloperoxidase and thyroid peroxidase are members of the same gene family and diverged from a common ancestral gene. The residues at 416 in myeloperoxidase and 407 in thyroid peroxidase were estimated as possible candidates for the proximal histidine residues that link to the iron centers of the enzymes. The primary structures around these histidine residues were compared with those of other known peroxidases. The similarity in this region between the two animal peroxidases (amino acid 396-418 in thyroid peroxidase and 405-427 in myeloperoxidase) is 74%; however, those between the animal peroxidases and other yeast and plant peroxidases are not significantly high, although several conserved features have been observed. The possible location of the distal histidine residues in myeloperoxidase and thyroid peroxidase amino acid sequences are also discussed.

Sea urchin sperm peroxidase is competitively inhibited by benzohydroxamic acid and phenylhydrazine.

Sea urchin sperm contain a phenylhydrazine-sensitive peroxidase that is believed to use hydrogen peroxide produced by the fertilized egg to reduce sperm fertility and thereby assist in the prevention of polyspermy. Strongylocentrotus purpuratus sperm were treated initially with hypotonic phosphate buffer (pH 7.0) to remove catalase and then extracted with 0.5% Triton X-100 in 0.5 M acetate buffer (pH 5.0). Peroxidase activity in this detergent extract was assayed using 3,3',5,5'-tetramethyl benzidine (TMB) as oxidizable substrate. Kinetic studies showed that the Km for TMB is 250 microM. Benzohydroxamic acid and phenylhydrazine are known to be competitive inhibitors of a variety of plant and animal peroxidases. These substances were found to competitively inhibit the sea urchin sperm peroxidase: for benzohydroxamic acid, Ki = 51.2 microM, mean inhibitory dose (ID50) = 146.7 microM; for phenylhydrazine, Ki = 201 nM, ID50 = 303 nM. These findings indicate that the biochemical properties of the sea urchin sperm peroxidase resembles those of peroxidases found in somatic tissues where oxygen radicals are produced by phagocytes to kill bacteria and support our hypothesis that the sperm peroxidase has a functional role in the prevention of polyspermy during fertilization.

Enzymatic reduction of 5-phenyl-4-pentenyl-hydroperoxide: Detection of peroxidases and identification of peroxidase reducing substrates

5-Phenyl-4-pentenyl-hydroperoxide (PPHP) is reduced to 5-phenyl-4-pentenyl-alcohol (PPA) by plant and animal peroxidases in the presence of reducing substrates. PPHP and PPA are rapidly isolated with solid phase extraction, separated by isocratic reverse-phase high-performance liquid chromatography, and quantitated with a fixed-wavelength ultraviolet detector. The procedure described is suitable for detecting peroxide-reducing enzymes, determining the kinetic properties of heme- and non-heme-containing peroxidases, and evaluating oxidizable compounds as reducing substrates for peroxidases. Horseradish peroxidase (HRP) and phenol reduce PPHP with a K m for phenol of 252 μM and a turnover number of 1.05 × 10 4 min −1. Under similar conditions, the K m of HRP for PPHP is 18 μM in the oxidation of guaiacol. A series of 21 compounds was evaluated for the ability to serve as reducing substrates for HRP. The results indicate that the procedure described can not only identify compounds that are reducing substrates but also rank them for relative activity. This may provide a new method with which to identify novel antithrombotic, antimetastatic, or anti-inflammatory drugs as well as to detect and characterize mammalian peroxidases.

Characterization of hog thyroid peroxidase.

Several fundamental properties of purified hog thyroid peroxidase (A413 nm/A280 nm = 0.55) were investigated in comparison with bovine lactoperoxidase. The Mr of thyroid peroxidase was 71,000. The prosthetic group of thyroid peroxidase was identified spectrophotometrically as protoheme IX after the enzyme was hydrolyzed with Pronase. Optical spectra of oxidized and reduced thyroid peroxidases and their complexes with azide and cyanide were very similar to lactoperoxidase, except that lactoperoxidase had two reduced forms with the Soret band either at 446 or 435 nm, and thyroid peroxidase lacked a reduced form having the 446-nm band. From comparison of their pyridine hemochrome spectra, epsilon mM at 413 nm of thyroid peroxidase was estimated to be 114, being the same as that of lactoperoxidase. The cyanide inhibition for the reaction of thyroid peroxidase was competitive with hydrogen peroxide and the inhibition constant was in rough accord with the dissociation constant of its cyanide complex measured from spectrophotometric titration. Azide inhibited the reaction with an inhibition constant which was about one one-thousandth of the dissociation constant for its spectrally discernible complex. The azide inhibition was not competitive with hydrogen peroxide and decreased as the reaction proceeded. Aminotriazole inhibited the reaction strongly, and the inhibition was augmented during the reaction. These inhibition patterns of azide and aminotriazole were more or less observed in the reaction of lactoperoxidase, but not in the case of horseradish peroxidase. Characteristics of animal peroxidases are discussed.

The participation of peroxidases in the formation of sulphoxides

Propionylpromazine, a sedative for animals, is in vitro oxidized completely by horse-reddish peroxidase to its sulphoxide. The sulphoxide was identified by its absorption and fluorescence spectra and by thin layer chromatography. At extremely small concentrations (<10−6 M) the oxidation does not proceed quantitatively. A two-step mechanism seems to be involved. It is suggested that at first a radicalion is formed by the loss of one electron and that from two radicalions the sulphoxide and the original compound are formed. Lacto-peroxidase, as an example for animal peroxidases, is also able to catalyse the sulphoxide formation.

The haem prosthetic groups of some animal peroxidases II. Myeloperoxidase

1. 1. The absorption spectra of the compounds formed from myeloperoxidase (donor: H 2O 2oxidoreductase, EC 1.11.1.7) with cyanide, azide and F − are given and the compound with H 2O 2, described by Agner, confirmed. The effect of low pH on the spectrum of the haemoprotein is studied and formation of an alkaline form described. 2. 2. The inability of reduced myeloperoxidase to react with CO, despite the availability of the haem iron atoms to hydrazoic acid, cyanide and H 2O 2 is confirmed over a wide pH range. 3. 3. Pyridine is shown to remove the unusual spectrum of the reduced enzyme to form a ferrohaemochrome with an α-band at 590 mμ and no β-band. The position of the α-band indicates strongly electrophilic substitution of the porphyrin ring; the absence of a β-band indicates that two electrophilic substituents exist on opposite pyrrole rings. 4. 4. The reaction of various reagents with myeloperoxidase pyridine haemochrome is described indicating that at least one of the conjugated electrophilic substituents of the haems is a carbonyl. 5. 5. Removal of the Fe atoms from the haems of myeloperoxidase gives a porphyrin-polypeptide with spectroscopic properties indicating electrophilic substitution of the porphyrin rings. 6. 6. Attempts to split the haem groups from the enzyme are described.

The haematin prosthetic groups 3f some animal peroxidases I. The preparation and properties of an ether-soluble haematin from milk peroxidase

A study of the pyridine haemochrome of milk peroxidase shows that the prosthetic haematin group contains a strongly electrophilic substituent conjugated to the porphyrin ring. This substituent is labile to strong alkali and to certain adsorbents. The haematin is co-valently bound to the apoenzyme through ester or amide bonds. This linkage can be broken by alkali and by either hydrogen iodide or hydrogen bromide in acetic acid. Only the latter reagent yields a haematin which retains the characteristic electrophilic group. The nature of this group is as yet unknown.