Isolation and partial nucleotide sequence of the laccase gene from Neurospora crassa: amino acid sequence homology of the protein to human ceruloplasmin.

Abstract

The laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) gene from Neurospora crassa was cloned and part of its nucleotide sequence corresponding to the carboxylterminal region of the protein has been determined. The gene was cloned by cDNA synthesis with a laccase-specific synthetic deoxyundecanucleotide as primer and poly(A) RNA isolated from cycloheximide-treated N. crassa cultures as template. Based on the nucleotide sequence of the cDNA obtained, a unique 21-mer was synthesized and used to screen a genomic DNA library from N. crassa. Five different positive clones were isolated and shown to share an overlapping DNA region with the same pattern of restriction sites. Sequence analysis of the common 1.36-kilobase Sal I fragment revealed an open reading frame of 726 nucleotides. The amino acid sequence deduced is in complete agreement with the primary structures of several tryptic peptides isolated previously from N. crassa laccase. The analyzed carboxyl-terminal region of laccase exhibits a striking sequence homology to the carboxyl-terminal part of the third homology unit of the multicopper oxidase ceruloplasmin and to a smaller extent, to the low molecular weight blue copper proteins plastocyanin and azurin. Based, on amino acid sequence comparison between these proteins, putative copper ligands ofN. crassa laccase are proposed. Moreover, these data further support the hypothesis that the small blue copper proteins and the multicopper oxidases have evolved from the same ancestral gene. Laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) belongs to the multicopper oxidases containing three distinct Cu centers known as type 1 or blue, type 2 or normal, and type 3 or coupled binuclear (1). The enzyme catalyzes the four-electron reduction of molecular oxygen to water with the concomitant oxidation of organic substrates. Laccases have been isolated from different sources such as microorganisms (2)(3)(4)(5)(6)(7)(8) and plants (9-11). Physical characterization of the proteins indicates a great variety in molecular weight, carbohydrate content, and quaternary structure. With the exception of the laccase from Agaricus bisporus and the tetrameric Podospora anserina laccase I, all native enzymes bind four copper ions per molecule, representing one type 1, one type 2, and one type 3 Cu center (12). In contrast, the other multicopper oxidases, ceruloplasmin and ascorbate oxidase, contain more than four copper ions per molecule (13-17). Hence, laccase provides the most simple system to study the active site structure and reactivity of multicopper oxidases.Neurospora crassa laccase is excreted into the culture medium after induction with low amounts of protein synthesis inhibitors (18). The enzyme has a molecular mass of 64 kDa, with a carbohydrate content of 11-12% (4, 5). In an attempt to determine the primary structure of N. crassa laccase and to study its molecular mechanisms of biosynthesis and excretion, we set out to clone the structural gene. Here we describe the strategy used to clone this gene a...

Laccases and Their Applications: A Patent Review

“…The first gene and/or cDNA sequences were recorded for laccase from the Ascomycete fungus, Neurospora crassa [98], and sequences were published from 1990…”

Tandem organization and highly disparate expression of the two laccase genes lcc1 and lcc2 in the cultivated mushroom Agaricus bisporus

“…Some fungal laccases are inducible by substrates such as 2,S-xylidene (see Bourbonnais et al, 1995, for an example). Some are ' induced ' by protein synthesis inhibitors such as cycloheximide (see for example Germann & Lerch, 1986), but the extent to which this is solely a consequence of laccase mRNA being more stable than average is very difficult to measure ; the alternative, that cycloheximide is a natural inducer, has yet t o be given a plausible explanation. Many fungal laccases are apparently constitutive, but whether they are mechanistically constitutive (no sequences for modulation of transcription present) or being induced by an unidentified product of metabolism of the fungus remains unknown.…”

“…During recent years fungal laccase gene and/or cDNA sequences have been reported from a number of sources, the first of which was the ascomycete Neurospora crassa M. S M I T H a n d OTHERS (Germann & Lerch, 1986;Germann et al, 1988).…”

Identification of two laccase genes in the cultivated mushroom Agaricus bisporus

“…During recent years the laccase gene and/or cDNA sequence has been described from four sources of which the first to be reported was the ascomycete fungus Neurospora crassa (Germann & Lerch, 1986 ;Germann et al, 1988). Subsequently the sequences from another ascomycete, Aspergillus nidulans (Aramayo & Timberlake, 1990) and two basidiomycetes, Coriolus hirsutus (Kojima et al, 1990) and Phlebia radiata (Saloheimo et al, 1991) have been published.…”