Browsing by Author "Salas, L"

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    Characterization of three new manganese peroxidase genes from the ligninolytic basidiomycete Ceriporiopsis subvermispora
    (2000) Tello, M; Corsini, G; Larrondo, LF; Salas, L; Lobos, S; Vicuña, R
    Three 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.
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    Cloning 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, R
    A 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.
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    Enzymology 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, R
    Lignin, 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.
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    Heterologous 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, R
    Analysis 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).