Modeling the 3-D Structure of a Recombinant Laccase from Trametes trogii Active at a pH Close to Neutrality

Abstract

A cDNA encoding a novel laccase from the white-rot fungus Trametes trogii was cloned and expressed in Pichia pastoris. The recombinant protein (Lcc2) exhibited kinetic parameters for both phenolic and non phenolic substrates that were different from the previously described Lcc1, the main laccase isoform expressed by T. trogii; in addition, the pH/activity profiles for phenolic substrates of Lcc2 were shifted upward by 1-1.5 pH units towards neutrality as compared to Lcc1. Comparative modeling of the two laccases (69.2% identity) showed that the overall fold of Lcc2 is very similar to Lcc1 and other laccases. The substrate cavity of Lcc2 contains the Asp residue which is thought to mediate the laccase activity at acidic pHs, whereas two hydrophobic residues (Phe, Ile) on the cavity orifice of Lcc2 replace the two polar residues (Thr, Ser) of Lcc1. These structural differences may be responsible for the unique kinetic performances of Lcc2.

Heterologous production, characterization and dye decolorization ability of a novel thermostable laccase isoenzyme from Trametes trogii BAFC 463

“…In this work we used a PCR-based screening to clone four laccase coding sequences of T. trogii BAFC 463. Of these, sequences lcc1 and lcc2 encoded proteins with 100% and 99% identity with laccases LCC1 and LCC2 previously reported for T. trogii 201 [22,23] while lcc3 and lcc4 encoded two novel isoenzymes. Cloning and expression of sequence lcc3 in K. pastoris showed that it encodes a laccase with a functional signal peptide, capable of oxidizing the substrates ABTS and DMP with characteristic pH activity profiles and kinetic constants commonly observed for other fungal laccases, with higher catalytic efficiency for ABTS that for the phenolic substrate DMP (Fig.…”

“…Sequence lcc1 (GenBank KU055621) encodes a protein with 100% amino acid identity with previously reported LCC1 of T. trogii strain 201 (GenBank CAC13040, [22]). Sequence lcc2 of T. trogii BAFC 463 (GenBank KU055622) encodes a protein with 99% amino acid identity with the reported sequence for LCC2 of T. trogii 201 (GenBank CAL23367, [23]), with 4 amino acid substitutions in mature translated protein sequence: D14N, I186L, A251 V, T433A (Fig. S1).…”

“…Production of manganese peroxidase (MnP), lignin peroxidase (LiP) and auxiliary H 2 O 2 -producing oxidases such as glyoxal oxidase was described in this fungus; nevertheless the more outstanding property of T. trogii is its ability to express high levels of laccases [16][17][18][19][20][21]. In T. trogii, two different laccase coding genes have been cloned from strain 201 and the presence of at least five isoenzymes has been proposed, suggesting that an extensive number of laccases is present in this species [22,23]. Studies performed in T. trogii BAFC 463 have shown the expression of at least two laccase isoenzymes and its ability to degrade wood lignin and organic pollutants such as nitrobenzene and anthracene as well as anthraquinonic dyes and polychlorinated biphenyls, demonstrating its potential for detoxification of xenobiotics [20,[24][25][26].…”

Engineering Platforms for Directed Evolution of Laccase from Pycnoporus cinnabarinus

“…However, the affinity for phenolic substrates decreased (being notably lower at this pH than at pH 5.0), with K m values being up to 4.2-and 4.7-fold higher for DMP and sinapic acid, respectively. At pH 5.0, deprotonation of a highly conserved aspartate residue (Asp206; side chain pK a , 3.9) that interacts with the reducing substrate at the T1 Cu site (9) may have contributed to this effect (19).…”