Publications

• The presence of sinapyl lignin in Ginkgo biloba cell cultures changes our views of the evolution of lignin biosynthesis.

  Novo Uzal E., Gomez Ros L.V., Pomar F., Bernal M.A., Paradela A., Albar J.P., Ros Barcelo A.

  Suspension cell cultures (SCCs) from one of the oldest seed plants, Ginkgo biloba, show unpredictable alterations in the nature of the lignins, such as is the recruitment of sinapyl alcohol for lignin biosynthesis, compared with the woody tissues of the same species, which lack syringyl (S) lignin ... >> More

  Suspension cell cultures (SCCs) from one of the oldest seed plants, Ginkgo biloba, show unpredictable alterations in the nature of the lignins, such as is the recruitment of sinapyl alcohol for lignin biosynthesis, compared with the woody tissues of the same species, which lack syringyl (S) lignins. These results show that, in this gymnosperm, the genes involved in sinapyl alcohol biosynthesis are latent and that their regulatory regions respond, by initiating gene expression, to the developmental signals and the environmental clues, which condition its in vitro culture. G. biloba SCCs not only synthesize S lignins but also their extracellular proteome contains both class III peroxidases capable of oxidizing sinapyl alcohol and enzymes involved in H2O2 production, observation which suggests that the peroxidase branch for the oxidative coupling of sinapyl alcohol units into lignins is operative. The incomplete knowledge of the G. biloba peroxidase-encoding genes led us to purify, characterize and partially sequence the peroxidase responsible for monolignol oxidation. When the major peroxidase from G. biloba SCCs (GbPrx) was purified to homogeneity, it showed absorption maxima in the visible region at 414 (Soret band), and at 543 and 570 nm, which calls to mind those shown by low-spin ferric peroxidases. However, the results also showed that the paraperoxidase-like character of GbPrx is not an obstacle for oxidizing the three monolignols compared with high-spin ferric peroxidases. Taken together, these results mean that the time at which the evolutionary gain of the segment of the route that leads to the biosynthesis of S lignins took place in seed plants needs to be revised. << Less

  Physiol. Plantarum 135:196-213(2009) [PubMed] [EuropePMC]

• Purification, cloning and characterization of a novel peroxidase isozyme from sweetpotatoes (Ipomoea batatas).

  Rompel A., Albers M., Naseri J.I., Gerdemann C., Bueldt-Karentzopoulos K.B., Jasper B., Krebs B.

  An anionic peroxidase from sweetpotato tubers is purified and characterized. The isozyme ibPrx15 is purified to homogeneity by affinity chromatography using a concanavalin A column. The isoelectric point was determined to pI 4.9. MALDI-MS detected a singly charged molecule with a mass of 42029 Da. ... >> More

  An anionic peroxidase from sweetpotato tubers is purified and characterized. The isozyme ibPrx15 is purified to homogeneity by affinity chromatography using a concanavalin A column. The isoelectric point was determined to pI 4.9. MALDI-MS detected a singly charged molecule with a mass of 42029 Da. Absorption spectra of ibPrx15 compounds I, II and III were obtained after treatment with H(2)O(2) at room temperature. Comparative data of ibPrx15 on substrate specificity to tobacco anionic peroxidase (TOP) and horseradish peroxidase (HRP) reveal similar specific activity towards a series of conventional substrates except for iodide, which is a two-electron donor interacting directly with the compound I derivative in the catalytic cycle. ibPrx15 exhibits a high specific activity towards iodide about 10(3)-fold to that of tobacco peroxidase. The amino acid sequence of the main isozyme ibPrx15 was determined by Edman degradation and by sequencing the amplified cDNA fragments. ibPrx15 has 86% identity to another Ipomoea sequence ibPrx05 and 72% identity with a sequence from Populus trichocarpa (PtPrx72). << Less

  Biochim. Biophys. Acta 1774:1422-1430(2007) [PubMed] [EuropePMC]

• Identification and characterization of a multifunctional dye peroxidase from a lignin-reactive bacterium.

  Brown M.E., Barros T., Chang M.C.

  Plant biomass represents a renewable feedstock that has not yet been fully tapped because of the difficulty in accessing the carbon in its structural biopolymers. Lignin is an especially challenging substrate, but select microbes have evolved complex systems of enzymes for its breakdown through a ... >> More

  Plant biomass represents a renewable feedstock that has not yet been fully tapped because of the difficulty in accessing the carbon in its structural biopolymers. Lignin is an especially challenging substrate, but select microbes have evolved complex systems of enzymes for its breakdown through a radical-mediated oxidation process. Fungal systems are well-characterized for their ability to depolymerize lignin, but the ability of bacteria to react with this substrate remains elusive. We have therefore focused on elucidating strategies used by lignin-reactive soil bacteria and describing their oxidative enzyme systems. We now report the identification and characterization of an unusual C-type dye-decolorizing peroxidase from Amycolatopsis sp. 75iv2 (DyP2), which belongs to a family of heme peroxidases reported to be involved in bacterial lignin degradation. Biochemical studies indicate that DyP2 has novel function for this family, with versatile and high activity both as a peroxidase and Mn peroxidase (k(cat)/K(M) ≈ 10(5)-10(6) M(-1) s(-1)). It also has a Mn-dependent oxidase mode of action that expands its substrate scope. Crystallographic studies of DyP2 at 2.25 Å resolution show the existence of a Mn binding pocket and support its key role in catalysis. << Less

  ACS Chem. Biol. 7:2074-2081(2012) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Combined extracellular matrix cross-linking activity of the peroxidase MLT-7 and the dual oxidase BLI-3 is critical for post-embryonic viability in Caenorhabditis elegans.

  Thein M.C., Winter A.D., Stepek G., McCormack G., Stapleton G., Johnstone I.L., Page A.P.

  The nematode cuticle is a protective collagenous extracellular matrix that is modified, cross-linked, and processed by a number of key enzymes. This Ecdysozoan-specific structure is synthesized repeatedly and allows growth and development in a linked degradative and biosynthetic process known as m ... >> More

  The nematode cuticle is a protective collagenous extracellular matrix that is modified, cross-linked, and processed by a number of key enzymes. This Ecdysozoan-specific structure is synthesized repeatedly and allows growth and development in a linked degradative and biosynthetic process known as molting. A targeted RNA interference screen using a cuticle collagen marker has been employed to identify components of the cuticle biosynthetic pathway. We have characterized an essential peroxidase, MoLT-7 (MLT-7), that is responsible for proper cuticle molting and re-synthesis. MLT-7 is an active, inhibitable peroxidase that is expressed in the cuticle-synthesizing hypodermis coincident with each larval molt. mlt-7 mutants show a range of body morphology defects, most notably molt, dumpy, and early larval stage arrest phenotypes that can all be complemented with a wild type copy of mlt-7. The cuticles of these mutants lacks di-tyrosine cross-links, becomes permeable to dye and accessible to tyrosine iodination, and have aberrant collagen protein expression patterns. Overexpression of MLT-7 causes mutant phenotypes further supporting its proposed enzymatic role. In combination with BLI-3, an H2O2-generating NADPH dual oxidase, MLT-7 is essential for post-embryonic development. Disruption of mlt-7, and particularly bli-3, via RNA interference also causes dramatic changes to the in vivo cross-linking patterns of the cuticle collagens DPY-13 and COL-12. This points toward a functionally cooperative relationship for these two hypodermally expressed proteins that is essential for collagen cross-linking and proper extracellular matrix formation. << Less

  J. Biol. Chem. 284:17549-17563(2009) [PubMed] [EuropePMC]

• Structural evidence of substrate specificity in mammalian peroxidases: structure of the thiocyanate complex with lactoperoxidase and its interactions at 2.4 A resolution.

  Sheikh I.A., Singh A.K., Singh N., Sinha M., Singh S.B., Bhushan A., Kaur P., Srinivasan A., Sharma S., Singh T.P.

  The crystal structure of the complex of lactoperoxidase (LPO) with its physiological substrate thiocyanate (SCN(-)) has been determined at 2.4A resolution. It revealed that the SCN(-) ion is bound to LPO in the distal heme cavity. The observed orientation of the SCN(-) ion shows that the sulfur at ... >> More

  The crystal structure of the complex of lactoperoxidase (LPO) with its physiological substrate thiocyanate (SCN(-)) has been determined at 2.4A resolution. It revealed that the SCN(-) ion is bound to LPO in the distal heme cavity. The observed orientation of the SCN(-) ion shows that the sulfur atom is closer to the heme iron than the nitrogen atom. The nitrogen atom of SCN(-) forms a hydrogen bond with a water (Wat) molecule at position 6'. This water molecule is stabilized by two hydrogen bonds with Gln(423) N(epsilon2) and Phe(422) oxygen. In contrast, the placement of the SCN(-) ion in the structure of myeloperoxidase (MPO) occurs with an opposite orientation, in which the nitrogen atom is closer to the heme iron than the sulfur atom. The site corresponding to the positions of Gln(423), Phe(422) oxygen, and Wat(6)' in LPO is occupied primarily by the side chain of Phe(407) in MPO due to an entirely different conformation of the loop corresponding to the segment Arg(418)-Phe(431) of LPO. This arrangement in MPO does not favor a similar orientation of the SCN(-) ion. The orientation of the catalytic product OSCN(-) as reported in the structure of LPO.OSCN(-) is similar to the orientation of SCN(-) in the structure of LPO.SCN(-). Similarly, in the structure of LPO.SCN(-).CN(-), in which CN(-) binds at Wat(1), the position and orientation of the SCN(-) ion are also identical to that observed in the structure of LPO.SCN. << Less

  J. Biol. Chem. 284:14849-14856(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification of lactoperoxidase from creek-water buffalo milk and investigation of kinetic and antibacterial properties.

  Ozdemir H., Hacibeyoglu H.I., Uslu H.

  Water buffalo lactoperoxidase (WBLP) was purified with Amberlite CG 50 H+ resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from skim milk. All purification steps of the WBLP were shown with SDS-PAGE and Rz (A412/A280) controlled the purification ... >> More

  Water buffalo lactoperoxidase (WBLP) was purified with Amberlite CG 50 H+ resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from skim milk. All purification steps of the WBLP were shown with SDS-PAGE and Rz (A412/A280) controlled the purification degree of the enzyme. Rz value for the purified WBLP was 0.8. To determine purification steps and kinetic properties, the activity of enzyme was measured by using 2,2-azino-bis-(3-ethylbenzthiazoline-6 sulfonic acid) diammonium salt (ABTS) as a choromogenic substrate at pH=6. Km, Vmax, optimum pH, and optimum temperature for the WBLP were found by means of graphics for ABTS as substrates. Optimum pH and optimum temperature of the WBLP were 6 and 60 degrees C, respectively. Km value at optimum pH and optimum temperature for the WBLP was 0.82 mM. Vmax value at optimum pH and optimum temperature was 13.7 micromol/mL x min. Km value at optimum pH and 25 degrees C for the WBLP was 0.77 mM. Vmax value at optimum pH and 25 degrees C was 4.83 micromol/mL x min. The purified WBLP was found to have high antibacterial activity in a thiocynate-H2O2 medium for some pathogenic bacteria, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginose, Shigella sonnei, Staphylococcus saphrophyticus, Staphylococcus epidermidis, and Shigella dysenteriae and compared with well known antibacterial substances such as tetracycline, penicillin, and netilmicine. << Less

  Prep. Biochem. Biotechnol. 32:143-155(2002) [PubMed] [EuropePMC]

• Cloning and molecular characterization of the basic peroxidase isoenzyme from Zinnia elegans, an enzyme involved in lignin biosynthesis.

  Gabaldon C., Lopez-Serrano M., Pedreno M.A., Barcelo A.R.

  The major basic peroxidase from Zinnia elegans (ZePrx) suspension cell cultures was purified and cloned, and its properties and organ expression were characterized. The ZePrx was composed of two isoforms with a M(r) (determined by matrix-assisted laser-desorption ionization time of flight) of 34,7 ... >> More

  The major basic peroxidase from Zinnia elegans (ZePrx) suspension cell cultures was purified and cloned, and its properties and organ expression were characterized. The ZePrx was composed of two isoforms with a M(r) (determined by matrix-assisted laser-desorption ionization time of flight) of 34,700 (ZePrx34.70) and a M(r) of 33,440 (ZePrx33.44). Both isoforms showed absorption maxima at 403 (Soret band), 500, and 640 nm, suggesting that both are high-spin ferric secretory class III peroxidases. M(r) differences between them were due to the glycan moieties, and were confirmed from the total similarity of the N-terminal sequences (LSTTFYDTT) and by the 99.9% similarity of the tryptic fragment fingerprints obtained by reverse-phase nano-liquid chromatography. Four full-length cDNAs coding for these peroxidases were cloned. They only differ in the 5'-untranslated region. These differences probably indicate different ways in mRNA transport, stability, and regulation. According to the k(cat) and apparent K(m)(RH) values shown by both peroxidases for the three monolignols, sinapyl alcohol was the best substrate, the endwise polymerization of sinapyl alcohol by both ZePrxs yielding highly polymerized lignins with polymerization degrees > or =87. Western blots using anti-ZePrx34.70 IgGs showed that ZePrx33.44 was expressed in tracheary elements, roots, and hypocotyls, while ZePrx34.70 was only expressed in roots and young hypocotyls. None of the ZePrx isoforms was significantly expressed in either leaves or cotyledons. A neighbor-joining tree constructed for the four full-length cDNAs suggests that the four putative paralogous genes encoding the four cDNAs result from duplication of a previously duplicated ancestral gene, as may be deduced from the conserved nature and conserved position of the introns. << Less

  Plant Physiol. 139:1138-1154(2005) [PubMed] [EuropePMC]

• Biochemical characterization of the major sorghum grain peroxidase.

  Dicko M.H., Gruppen H., Hilhorst R., Voragen A.G.J., van Berkel W.J.H.

  The major cationic peroxidase in sorghum grain (SPC4) , which is ubiquitously present in all sorghum varieties was purified to apparent homogeneity, and found to be a highly basic protein (pI approximately 11). MS analysis showed that SPC4 consists of two glycoforms with molecular masses of 34,227 ... >> More

  The major cationic peroxidase in sorghum grain (SPC4) , which is ubiquitously present in all sorghum varieties was purified to apparent homogeneity, and found to be a highly basic protein (pI approximately 11). MS analysis showed that SPC4 consists of two glycoforms with molecular masses of 34,227 and 35,629 Da and it contains a type-b heme. Chemical deglycosylation allowed to estimate sugar contents of 3.0% and 6.7% (w/w) in glycoform I and II, respectively, and a mass of the apoprotein of 33,246 Da. High performance anion exchange chromatography allowed to determine the carbohydrate constituents of the polysaccharide chains. The N-terminal sequence of SPC4 is not blocked by pyroglutamate. MS analysis showed that six peptides, including the N-terminal sequence of SPC4 matched with the predicted tryptic peptides of gene indice TC102191 of sorghum chromosome 1, indicating that TC102191 codes for the N-terminal part of the sequence of SPC4, including a signal peptide of 31 amino acids. The N-terminal fragment of SPC4 (213 amino acids) has a high sequence identity with barley BP1 (85%), rice Prx23 (90%), wheat WSP1 (82%) and maize peroxidase (58%), indicative for a common ancestor. SPC4 is activated by calcium ions. Ca2+ binding increased the protein conformational stability by raising the melting temperature (Tm) from 67 to 82 degrees C. SPC4 catalyzed the oxidation of a wide range of aromatic substrates, being catalytically more efficient with hydroxycinnamates than with tyrosine derivatives. In spite of the conserved active sites, SPC4 differs from BP1 in being active with aromatic compounds above pH 5. << Less

  FEBS J. 273:2293-2307(2006) [PubMed] [EuropePMC]

• Isolation of tobacco isoperoxidases accumulated in cell-suspension culture medium and characterization of activities related to cell wall metabolism.

  de Marco A., Guzzardi P., Jamet E.

  All of the most important guaiacol-type peroxidase (POX) isoforms accumulated in the culture medium of BY-2 tobacco (Nicotiana tabacum L. cv Bright Yellow 2) cells have been isolated. Five basic and two acidic isoforms were found. The four major isoforms (B2, B3, P1, and P2), all strongly basic, h ... >> More

  All of the most important guaiacol-type peroxidase (POX) isoforms accumulated in the culture medium of BY-2 tobacco (Nicotiana tabacum L. cv Bright Yellow 2) cells have been isolated. Five basic and two acidic isoforms were found. The four major isoforms (B2, B3, P1, and P2), all strongly basic, have been purified to homogeneity and partially sequenced. B2 and B3 are new isoforms showing high homology to only one POX isolated so far. Amino acid sequencing and specific activities indicated that basic isoPOXs constitute two pairs of strictly related isoforms (P1/P2 and B2/B3). Their specific activities measured in the presence of different substrates, as monolignols and NAD(P)H, indicated possible specialized functions in cell wall metabolism. Only P-type POXs were able to oxidize indoleacetic acid. Variations in pH could play a regulatory role by changing the relative contribution of different isoforms to total POX activity. Apart from cell culture medium, polyclonal antibodies obtained against P1 and P2 detected P1 in roots and in lower parts of stems. Immunocytochemical labeling indicated that P-type POXs were expressed in stem phloem and in phloem and epidermal cells of roots. << Less

  Plant Physiol. 120:371-382(1999) [PubMed] [EuropePMC]

• Identification and characterization of VPO1, a new animal heme-containing peroxidase.

  Cheng G., Salerno J.C., Cao Z., Pagano P.J., Lambeth J.D.

  Animal heme-containing peroxidases play roles in innate immunity, hormone biosynthesis, and the pathogenesis of inflammatory diseases. Using the peroxidase-like domain of Duox1 as a query, we carried out homology searching of the National Center for Biotechnology Information database. Two novel he ... >> More

  Animal heme-containing peroxidases play roles in innate immunity, hormone biosynthesis, and the pathogenesis of inflammatory diseases. Using the peroxidase-like domain of Duox1 as a query, we carried out homology searching of the National Center for Biotechnology Information database. Two novel heme-containing peroxidases were identified in humans and mice. One, termed VPO1 for vascular peroxidase 1, exhibits its highest tissue expression in heart and vascular wall. A second, VPO2, present in humans but not in mice, is 63% identical to VPO1 and is highly expressed in heart. The peroxidase homology region of VPO1 shows 42% identity to myeloperoxidase and 57% identity to the insect peroxidase peroxidasin. A molecular model of the VPO1 peroxidase region reveals a structure very similar to that of known peroxidases, including a conserved heme binding cavity, critical catalytic residues, and a calcium binding site. The absorbance spectra of VPO1 are similar to those of lactoperoxidase, and covalent attachment of the heme to VPO1 protein was demonstrated by chemiluminescent heme staining. VPO1 purified from heart or expressed in HEK cells is catalytically active, with a K(m) for H(2)O(2) of 1.5 mM. When co-expressed in cells, VPO1 can use H(2)O(2) produced by NADPH oxidase enzymes. VPO1 is likely to carry out peroxidative reactions previously attributed exclusively to myeloperoxidase in the vascular system. << Less

  Free Radic. Biol. Med. 45:1682-1694(2008) [PubMed] [EuropePMC]