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Functional characterization of a yellow laccase from Leucoagaricus gongylophorus.

Ike PT, Moreira AC, de Almeida FG, Ferreira D, Birolli WG, Porto AL, Souza DH - Springerplus (2015)

Bottom Line: Lac1Lg presented kcat/Km almost threefold higher for syringaldazine than for ABTS, showing a higher catalytic efficiency of Lac1Lg for syringaldazine.Lac1Lg did not absorb at 600 nm, which is a characteristic of so-called yellow laccases.Our Lac1Lg three-dimensional molecular model, constructed using homology modeling, showed that the Lac1Lg catalytic site is very closed to blue laccases.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP Brazil.

ABSTRACT
In this work we have identified, using mass spectrometry, two laccases produced by Leucoagaricus gongylophorus. One of them, Lac1Lg, was isolated, purified and characterized. Lac1Lg, a monomeric enzyme, was studied using ABTS and syringaldazine substrates. Lac1Lg presented kcat/Km almost threefold higher for syringaldazine than for ABTS, showing a higher catalytic efficiency of Lac1Lg for syringaldazine. The interference of several metal ions and substances in the laccase activity were evaluated. Lac1Lg did not absorb at 600 nm, which is a characteristic of so-called yellow laccases. Lac1Lg also was able to oxidize non-phenolic substrate (anthracene) in the absence of an exogenous mediator, showing that the enzyme has potential to explore in biotechnological processes. Our Lac1Lg three-dimensional molecular model, constructed using homology modeling, showed that the Lac1Lg catalytic site is very closed to blue laccases.

No MeSH data available.


Related in: MedlinePlus

Sequential alignment of yellow laccases Lac1Lg and Yel1p (Ducros, et al. 1998) using ClustalW2 program (McWilliam et al. 2013). Asterisk (*) represents identical residue in both sequences, colon represent high similarity residues in the sequences and dot represent residues with medium similarity. In yellow histidines which could bind to type-2,3 and 4; in blue amino acids which could bind to copper type-1. Leucine residue close to Cu type-1 is underlined
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Fig3: Sequential alignment of yellow laccases Lac1Lg and Yel1p (Ducros, et al. 1998) using ClustalW2 program (McWilliam et al. 2013). Asterisk (*) represents identical residue in both sequences, colon represent high similarity residues in the sequences and dot represent residues with medium similarity. In yellow histidines which could bind to type-2,3 and 4; in blue amino acids which could bind to copper type-1. Leucine residue close to Cu type-1 is underlined

Mentions: To sequentially identify the Lac1Lg, the sample was applied to a 1D SDS-PAGE gel; next, the band was excised from the gel, treated with trypsin and the peptides were analyzed by on line LC–MS nanoflow. Databases with different numbers of sequences were used to increase the protein identification confidence. By searching the nucleotide and protein databases, we identified a laccase sequence (accession number NCBI 409151740) in the published sequence of L. gongylophorus strain Ae322 so-called LgLcc6 (De Fine Licht et al. 2013). The analysis showed 13 peptides with about 27 % of sequence coverage (Table 4). The Lac1Lg (corresponding to LgLcc6) sequence is shown in Fig. 3 and the peptides are in bold in the sequence. This figure shows also a sequential alignment of Lca1Lg and a yellow laccase from Stropharia aeruginosa (discussed below).Table 4


Functional characterization of a yellow laccase from Leucoagaricus gongylophorus.

Ike PT, Moreira AC, de Almeida FG, Ferreira D, Birolli WG, Porto AL, Souza DH - Springerplus (2015)

Sequential alignment of yellow laccases Lac1Lg and Yel1p (Ducros, et al. 1998) using ClustalW2 program (McWilliam et al. 2013). Asterisk (*) represents identical residue in both sequences, colon represent high similarity residues in the sequences and dot represent residues with medium similarity. In yellow histidines which could bind to type-2,3 and 4; in blue amino acids which could bind to copper type-1. Leucine residue close to Cu type-1 is underlined
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4628026&req=5

Fig3: Sequential alignment of yellow laccases Lac1Lg and Yel1p (Ducros, et al. 1998) using ClustalW2 program (McWilliam et al. 2013). Asterisk (*) represents identical residue in both sequences, colon represent high similarity residues in the sequences and dot represent residues with medium similarity. In yellow histidines which could bind to type-2,3 and 4; in blue amino acids which could bind to copper type-1. Leucine residue close to Cu type-1 is underlined
Mentions: To sequentially identify the Lac1Lg, the sample was applied to a 1D SDS-PAGE gel; next, the band was excised from the gel, treated with trypsin and the peptides were analyzed by on line LC–MS nanoflow. Databases with different numbers of sequences were used to increase the protein identification confidence. By searching the nucleotide and protein databases, we identified a laccase sequence (accession number NCBI 409151740) in the published sequence of L. gongylophorus strain Ae322 so-called LgLcc6 (De Fine Licht et al. 2013). The analysis showed 13 peptides with about 27 % of sequence coverage (Table 4). The Lac1Lg (corresponding to LgLcc6) sequence is shown in Fig. 3 and the peptides are in bold in the sequence. This figure shows also a sequential alignment of Lca1Lg and a yellow laccase from Stropharia aeruginosa (discussed below).Table 4

Bottom Line: Lac1Lg presented kcat/Km almost threefold higher for syringaldazine than for ABTS, showing a higher catalytic efficiency of Lac1Lg for syringaldazine.Lac1Lg did not absorb at 600 nm, which is a characteristic of so-called yellow laccases.Our Lac1Lg three-dimensional molecular model, constructed using homology modeling, showed that the Lac1Lg catalytic site is very closed to blue laccases.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP Brazil.

ABSTRACT
In this work we have identified, using mass spectrometry, two laccases produced by Leucoagaricus gongylophorus. One of them, Lac1Lg, was isolated, purified and characterized. Lac1Lg, a monomeric enzyme, was studied using ABTS and syringaldazine substrates. Lac1Lg presented kcat/Km almost threefold higher for syringaldazine than for ABTS, showing a higher catalytic efficiency of Lac1Lg for syringaldazine. The interference of several metal ions and substances in the laccase activity were evaluated. Lac1Lg did not absorb at 600 nm, which is a characteristic of so-called yellow laccases. Lac1Lg also was able to oxidize non-phenolic substrate (anthracene) in the absence of an exogenous mediator, showing that the enzyme has potential to explore in biotechnological processes. Our Lac1Lg three-dimensional molecular model, constructed using homology modeling, showed that the Lac1Lg catalytic site is very closed to blue laccases.

No MeSH data available.


Related in: MedlinePlus