The structure of laccase protein and its synthesis by the commercial mushroom Agaricus bisporus.
Agaricus bisporus secretes abundant laccase activity into the medium during mycelial growth. SDS-PAGE analysis of extracellular laccase protein, purified from compost extract, showed a predominant band of 65 kDa molecular mass, together with lesser amounts of smaller polypeptides. The main polypeptide was purified electrophoretically. Amino acid sequence analysis of the N-terminal region of the main polypeptide was used to specify the sequence of a 15-residue chemically synthesized peptide (N-terminal peptide). Rabbit antibodies were raised against pure laccase, electrophoretically purified main polypeptide and the synthetic N-terminal peptide. Electrophoretically purified main polypeptide antibody was further purified by affinity chromatography on laccase-CNBr-Sepharose. Western blot analysis showed that the antigenic behaviour of laccase in compost extract, culture filtrate from malt-extract culture, and the purified enzyme from both sources, differed. The patterns of bands revealed are most simply explained by generation of (proteolytically) partially cleaved enzyme molecules in the culture medium, possibly combined with differences in extent of glycosylation. [35S]Methionine incorporation and immunoprecipitation were used to follow laccase synthesis in cultures grown on malt extract. After short-term labelling, a single polypeptide of 68 kDa apparent molecular mass was immunoprecipitated from both mycelial extracts and the culture medium. When poly(A)-containing RNA from malt-extract-grown mycelium was translated in vitro in rabbit reticulocyte lysate, a single polypeptide of about 57 kDa molecular mass was immunoprecipitated, consistent with the previously measured carbohydrate content of 15% for the pure enzyme. After treatment with N-glycanase, the polypeptide showed an increase in mobility during SDS-PAGE consistent with a reduction in molecular mass of about 5 kDa, indicating about equal amounts of N- and O-linked carbohydrate. C-terminal labelling of pure laccase was attempted by transpeptidation with carboxypeptidase Y. Although some minor bands were labelled, the main polypeptide was not, indicating that the C-terminus of the enzyme may be blocked.
Perry
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CR
Microbial Physiology Research Group, King's College London, UK.
Matcham
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SE
Wood
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DA
Thurston
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CF
D000364:Agaricus / Q000201:enzymology* / Q000235:genetics; D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D002474:Cell-Free System; D002846:Chromatography, Affinity; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D006031:Glycosylation; D042845:Laccase; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D010446:Peptide Fragments / Q000276:immunology; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011061:Poly A / Q000378:metabolism; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism
D005656:Fungal Proteins; D006023:Glycoproteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D011061:Poly A; D010088:Oxidoreductases; D042845:Laccase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase