Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns.
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 lcs-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).
Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile and Instituto Milenio de Biología Fundamental y Aplicada, Santiago, Chile.
Luis F
LF
Larrondo
Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile and Instituto Milenio de Biología Fundamental y Aplicada, Santiago, Chile.
Marcela
M
Avila
Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile and Instituto Milenio de Biología Fundamental y Aplicada, Santiago, Chile.
Loreto
L
Salas
USDA Forest Products Laboratory, Madison, WI 53705, USA.
Dan
D
Cullen
Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile and Instituto Milenio de Biología Fundamental y Aplicada, Santiago, Chile.
Rafael
R
Vicuña
D001059:Apoproteins / Q000378:metabolism; D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D042845:Laccase; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D017422:Sequence Analysis, DNA
D001059:Apoproteins; D018076:DNA, Complementary; D020033:Protein Isoforms; D003300:Copper; D010088:Oxidoreductases; D042845:Laccase