Browsing by Author "Vicuña, R"
Now showing 1 - 12 of 12
Results Per Page
Sort Options
- ItemCharacterization of three new manganese peroxidase genes from the ligninolytic basidiomycete Ceriporiopsis subvermispora(2000) Tello, M; Corsini, G; Larrondo, LF; Salas, L; Lobos, S; Vicuña, RThree new genes (Cs-mnp2A, Cs-mnp2B and Cs-mnp3) coding for manganese-dependent peroxidase (MnP) have been identified in the white-rot basidiomycete Ceriporiopsis subvermispora. The mature proteins contain 366 (MnP2A and MnP2B) and 364 (MnP3) amino acids, which are preceded by leader sequences of 21 and 24 amino acids, respectively. Cs-mnp2A and Cs-mnp2B appear to be alleles, since the corresponding protein sequences differ in only five residues, The upstream region of Cs-mnp2B contains a TATA box, AP-1 and AP-2 sites, as well as sites for transcription regulation by metals (two), cAMP (two) and xenobiotics (one). Some of these elements are also found in the regulatory region of Cs-MnP3, Transcription of Cs-mnp2A and Cs-mnp2B, but not that of Cs-mnp3, is activated by manganese. (C) 2000 Elsevier Science B.V. All rights reserved.
- ItemCloning and molecular analysis of a cDNA and the Cs-mnp1 gene encoding a manganese peroxidase isoenzyme from the lignin-degrading basidiomycete Ceriporiopsis subvermispora(1998) Lobos, S; Larrondo, L; Salas, L; Karahanian, E; Vicuña, RA cDNA (MnP13-1) and the Cs-mnp1 gene encoding for an isoenzyme of manganese peroxidase (MnP) from C. subvermispora were isolated separately and sequenced. The cDNA, identified in a library constructed in the vector Lambda ZIPLOX, contains 1285 nucleotides, excluding the poly(A) tail, and has a 63% G + C content. The deduced protein sequence shows a high degree of identity with MnPs from other fungi. The mature protein contains 364 amino acids, which are preceded by a 24-amino-acid leader sequence. Consistent with the peroxidase mechanism of MnP, the proximal histidine, the distal histidine and the distal arginine are conserved, although the aromatic binding site (L/V/I-P-X-P) is less hydrophilic than those of other peroxidases. A gene coding for the same protein (Cs-mnp1) was isolated from a genomic library constructed in Lambda GEM-11 vector using the cDNA MnP13-1 as a probe. A subcloned SacI fragment of 2.5 kb contained the complete sequence of the Cs-mnp1 gene, including 162 bp and 770 bp of the upstream and downstream regions, respectively. The Cs-mnp1 gene possesses seven short intervening sequences. The intron splice junction sequences as well as the putative internal lariat formation sites adhere to the GT-AG and CTRAY rules, respectively. To examine the structure of the regulatory region of the Cs-mnp1 gene further, a fragment of 1.9 kb was amplified using inverse PCR. A putative TATAA element was identified 5' of the translational start codon. Also, an inverted CCAAT element, SP-1 and AP-2 sites and several putative heat-shock and metal response elements were identified. (C) 1998 Elsevier Science B.V.
- ItemEnzymology and molecular genetics of the ligninolytic system of the basidiomycete Ceriporiopsis subvermispora(2001) Lobos, S; Tello, M; Polanco, R; Larrondo, LF; Manubens, A; Salas, L; Vicuña, RLignin, the most abundant renewable source of aromatic carbon on earth, consists in a highly irregular three-dimensional biopolymer of oxygenated phenylpropanoid units. In natural environments, lignin is only degraded efficiently by some fungi belonging to the group of basidiomycetes. These microorganisms secrete an array of oxidases and peroxidases for this purpose, which may be produced in various combinations. This article summarizes our studies on a particular strain called Ceriporiopsis subvermispora, a fungus which is highly agressive towards lignin when growing on wood.
- ItemHeterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns(2003) Larrondo, LF; Avila, M; Salas, L; Cullen, D; Vicuña, RAnalysis of genomic clones encoding a putative laccase in homokaryon strains of Ceriporiopsis subvermispora led to the identification of an allelic variant of the previously described Ics-1 gene. A cDNA clone corresponding to this gene was expressed in Aspergillus nidulans and in Aspergillus niger. Enzyme assays and Western blots showed that both hosts secreted active laccase. Relative to the isozymic forms of the native C. subvermispora enzyme, the A. niger-produced laccase had a higher molecular mass and gave a single band on IEF gels. In contrast, A. nidulans transformants secreted several isoforms remarkably similar to those of the native system. Considered together with previously reported Southern blots and protein sequencing, expression in A. nidulans supports the view that C. subvermispora has a single laccase gene and that multiple isoforms result from post-translational processes. In addition, several lines of evidence strongly suggest that under copper limitation, A. nidulans secretes apoprotein which can be reconstituted by a short incubation with Cu(I) and to a lesser extent with Cu(II).
- ItemIsoenzyme multiplicity and characterization of recombinant manganese peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium(2001) Larrondo, LF; Lobos, S; Stewart, P; Cullen, D; Vicuña, RWe expressed cDNAs coding for manganese peroxidases (MnPs! from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control of the alpha -amylase promoter from. Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar Levels and had molecular masses, both before and after deglycosylation, that were the same as those described For the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pls between 4.83 and 4.06, and one of these pls coincided with the pi described for H4 isolated from P. chrysosporium (pl 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium.
- ItemIsolation and characterization of homokaryotic strains from the ligninolytic basidiomycete Ceriporiopsis subvermispora(2001) Tello, M; Seelenfreund, D; Lobos, S; Gaskell, J; Cullen, D; Vicuña, RGenetic analyses of the lignin-degrading fungus Ceriporiopsis subvermispora is complicated by a dikaryotic nuclear condition and the absence of sport: forms. Previous investigations had identified a family of closely related sequences encoding manganese peroxidase (MnP), but the relationship between genes and allelic variants could not be experimentally established. Addressing this issue, homokaryotic derivatives of C. subvermipora strain FP105752 were isolated from regenerated protoplasts. Designated CsA and CsB. their homokaryotic nature was established by polymerase chain reaction amplification and sequence analysis of the allelic variants of three MnP genes. Isoelectrofocusing revealed fewer MnP isoenzymes in filtrates of homokaryon cultures relative to the parental strain. The homokaryotic strains will simplify genetic analyses, particularly the identification of neu genes. (C) 2001 Federation of European Microbiological Societies. published by Elsevier Science B.V. All rights reserved.
- ItemKinetics of Mn3+-oxalate formation and decay in reactions catalyzed by manganese peroxidase of Ceriporiopsis subvermispora(1998) Urzúa, U; Kersten, PJ; Vicuña, RThe kinetics of Mn3+-oxalate formation and decay were investigated in reactions catalyzed by manganese peroxidase (MnP) from the basiomycete Ceriporiopsis subvermispora in the absence of externally added hydrogen peroxide, A characteristic lag observed in the formation of this complex was shortened by glyoxylate or catalytic amounts of Mn3+ or hydrogen peroxide, MnP titers had a minor effect on this lag and did not influence the decay rate of the complex. In contrast, Mn2+ and oxalate drastically affected maximal concentrations of the Mn3+-oxalate complex formed, the decay of which was accelerated at high Mn2+ levels. The highest concentration of complex was obtained at pH 4.0, whereas an inverse relationship was found between the pH of the reaction and the decay rate of the complex with MnP present. In the absence of MnP, the best fit for the decay kinetics of the complex gave an order of 3/2 at concentrations in the range of 30-100 mu M, with a k(obs) = 0.012 min(-1) M-0.5 at pH 4.0. The rate constant increases at lower pH values and decreases at high oxalate concentrations. The physiological relevance of these findings is discussed. (C) 1998 Academic Press.
- ItemLigninolysis -: A very peculiar microbial process(2000) Vicuña, RLignin, the most abundant renewable source of aromatic carbon on earth, consists in a highly irregular three dimensional biopolymer of oxygenated phenylpropanoid units. Tn natural environments, lignin is only degraded efficiently by some basidioamycetes that secrete an array of enzymes for this purpose. Recent advances in our understanding of the mechanism of lignin breakdown have revealed several features that make this process highly unique from a biochemical standpoint. This article summarizes some of them.
- ItemManganese peroxidase dependent oxidation of glyoxylic and oxalic acids synthesized by Ceriporiopsis subvermispora produces extracellular hydrogen peroxide(1998) Urzúa, U; Kersten, PJ; Vicuña, RThe ligninolytic system of the basidiomycete Ceriporiopsis subvermispora is composed of manganese peroxidase (MnP) and laccase, In this work, the source of extracellular hydrogen peroxide required for MnP activity was investigated. Our attention was focused on the possibility that hydrogen peroxide might be generated by MnP itself through the oxidation of organic acids secreted by the fungus. Both oxalate and glyoxylate were found in the extracellular fluid of C. subvermispora cultures grown in chemically defined media, where MnP is also secreted, The in vivo oxidation of oxalate was measured; (CO2)-C-14 evolution was monitored after addition of exogenous [C-14]oxalate to cultures at constant specific activity. In standard cultures, evolution of CO2 from oxalate was maximal at day 6, although the MnP titers were highest at day 12, the oxalate concentration was maximal (2.5 mM) at day 10, and the glyoxylate concentration was maximal (0.24 mM) at day 5, However, in cultures containing low nitrogen levels, in which the pH is more stable, a better correlation between MnP titers and mineralization of oxalate was observed, Both MnP activity and oxidation of [C-14]oxalate were negligible in cultures lacking Mn(II). In vitro assays confirmed that Mn(II)-dependent oxidation of [C-14]oxalate by MnP occurs and that this reaction is stimulated by glyoxylate at the concentrations found in cultures, IG addition, both organic acids supported phenol red oxidation by MnP without added hydrogen peroxide, and glyoxylate was more reactive than oxalate in this reaction, Based on these results, a model is proposed for the extracellular production of hydrogen peroxide by C. subvermispora.
- ItemOxalate oxidase from Ceriporiopsis subvermispora(1999) Aguilar, C; Urzúa, U; Koenig, C; Vicuña, RThe enzyme oxalate oxidase was identified in mycelial extracts of the basidiomycete Ceriporiopsis subvermispora and thereafter purified to homogeneity. The purification procedure included only three steps: Q-Sepharose chromatography, precipitation at pH 3.0, and phosphocellulose chromatography. The enzyme is a 400-kDa homohexamer, as determined by gel permeation in Sephadex G-200 and SDS-polyacrylamide gel electrophoresis. Isoelectrofocusing revealed a pi of 4.2. Optimal activity was obtained at pH 3.5 and at 45 degrees C. The purified enzyme has K-m and k(cat) values of 0.1 mM and 88 s(-1), respectively. It is highly specific for oxalate, although it is inhibited at concentrations of this substrate above 2.5 mM. Hystochemistry studies conducted over mycelium slices showed reactions products in both endocellular and periplasmic associated elements. A possible connection between the intracellular metabolism of oxalate and the extracellular ligninolytic activity of the fungus is proposed. (C) 1999 Academic Press.
- ItemOxidation of kojic acid catalyzed by manganese peroxidase from Ceriporiopsis subvermispora in the absence of hydrogen peroxide(2002) Bastidas, F; Urzúa, U; Vicuña, RWe have previously reported the oxidation of kojic acid catalyzed by manganese peroxidase (MnP) from Ceriporiopsis subvermispora. This reaction is strictly dependent on Mn(II), although it does not require the addition of hydrogen peroxide. We have extended these studies because this reaction can be considered as a model system for the in situ generation of hydrogen peroxide in natural environments. We show here that oxidation of kojic acid with horseradish peroxidase (HRP) plus hydrogen peroxide or with manganic acetate rendered a product with identical chromatographic and spectral properties as the one obtained in the reaction catalyzed by MnP. The initial lag observed in the latter reaction decreased significantly upon UV irradiation of the substrate. On the other hand, ascorbic acid increased the lag and did not affect the yield of the reaction. The superoxide anion trapping agents glutathione, nitr blue tetrazolium, and superoxide dismutase markedly affected the reaction. In contrast, addition of the hydroxyl radical scavengers mannitol and salicylic acid had no effect. Based on these results, a mechanism for the MnP-catalyzed reaction is proposed.
- ItemStructure and expression of a laccase gene from the ligninolytic basidiomycete Ceriporiopsis subvermispora(1998) Karahanian, E; Corsini, G; Lobos, S; Vicuña, RA gene encoding laccase has been isolated from a genomic library of the white-rot basidiomycete Ceriporiopsis subvermispora constructed in Lambda GEM-ii. This gene (Cs-lcs1) contains an open reading frame of 2215 bp, encoding a mature protein of 499 amino acids with a 21-residue signal peptide. The protein sequence exhibits between 63 and 68% identity with laccases from other basidiomycetes and shares with all of them 10 conserved histidines and one cysteine involved in the coordination of copper atoms at the active site of the enzyme. The gene possesses ii introns, with splicing junctions and internal lariat formation sites adhering to the GT-AG and CTRAY rules, respectively. The upstream region of Cs-lcsl contains a TATA box, two CAAT sites, five putative metal response elements and a ACE1 element. In agreement with the presence of the latter element, transcription of Cs-lcsl is activated by copper and silver, as shown by Northern blot and reverse transcription followed by DNA amplification analyses. Based on Southern blot analysis, Cs-lcsl appears to be the only gene encoding laccase in C. subvermispora. (C) 1998 Elsevier Science B.V. All rights reserved.