Structural and phylogenetic analysis of laccases from Trichoderma: a bioinformatic approach.

The genus Trichoderma includes species of great biotechnological value, both for their mycoparasitic activities and for their ability to produce extracellular hydrolytic enzymes. Although activity of extracellular laccase has previously been reported in Trichoderma spp., the possible number of isoenzymes is still unknown, as are the structural and functional characteristics of both the genes and the putative proteins. In this study, the system of laccases sensu stricto in the Trichoderma species, the genomes of which are publicly available, were analyzed using bioinformatic tools. The intron/exon structure of the genes and the identification of specific motifs in the sequence of amino acids of the proteins generated in silico allow for clear differentiation between extracellular and intracellular enzymes. Phylogenetic analysis suggests that the common ancestor of the genus possessed a functional gene for each one of these enzymes, which is a characteristic preserved in T. atroviride and T. virens. This analysis also reveals that T. harzianum and T. reesei only retained the intracellular activity, whereas T. asperellum added an extracellular isoenzyme acquired through horizontal gene transfer during the mycoparasitic process. The evolutionary analysis shows that in general, extracellular laccases are subjected to purifying selection, and intracellular laccases show neutral evolution. The data provided by the present study will enable the generation of experimental approximations to better understand the physiological role of laccases in the genus Trichoderma and to increase their biotechnological potential

Identity (%) among protein sequences of the members of the <i>Trichoderma</i> laccase family.

<p>Proteins designated as the corresponding genes in the JGI GeneBank.</p

Phylogenetic analysis of laccases and of <i>Trichoderma</i> species.

<p> (<b>A</b>) Neighbor-joining tree of the deduced amino acid sequences of the five <i>Trichoderma</i> laccases and selected laccases of other ascomycetes, basidiomycetes and plants<b>.</b> The tree is calculated using the Jones-Taylor-Thornton (JTT) model in Mega Ver 5.05 based on a ClustalX alignment. Bootstrap values are from 1000 replications. The scale bar indicates a distance equivalent to 0.2 amino acid substitutions per site. Species and strains are indicated in the experimental procedures. Upward triangles indicate <i>Trichoderma</i> intacellular laccases and downward triangles extracellular ones. Green circles denote the laccases from <i>M. albomyces </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055295#pone.0055295-Kallio2" target="_blank">[44]</a> and <i>T. arenaria </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055295#pone.0055295-Kallio1" target="_blank">[42]</a>. (<b>B</b>) Phylogenetic tree of <i>Trichoderma</i> species showing gain, loss and retention of laccase genes.</p

Observed signature sequences in laccases.

<p>In the fungal laccase signature sequences L1–L4, an X represents an undefined residue, whereas the multiple residues within brackets represent a partially conserved residue.</p

Synonymous and non-synonymous substitution rates (%) between lacccase genes of <i>Trichoderma</i> spp.

<p>Genes designed as in the JGI GeneBank. ^aps, percentage of synonymous substitutions; pn, percentage of non-synonymous substitutions; pn/ps, ratio of synonymous, non synonymous substitutions.</p

Accession numbers and structural characteristics of laccases <i>sensu stricto</i> from <i>Trichoderma</i> spp.

a<p>ID, identification number in the <i>Trichoderma</i> species genome; ^bDenotes the location of signal peptide cleavage sites in amino acid sequences from <i>Trichoderma</i> laccases; nd, non detected. ^cMW, estimated molecular weight.^dp<i>I</i>, predicted isoelectric point.</p

Intron positions within the laccase genes of <i>Trichoderma</i> spp. define three gene subfamilies.

<p>The thin lines indicate intron positions and the black lines indicate the exons. The first subfamily (<b>A</b>) contains only one intron, the second subfamily (<b>B</b>) contains two introns and the final (<b>C</b>) subfamily contains seven introns.</p

Alignment of laccase sequences from <i>Trichoderma</i> spp.

<p>The alignment was constructed with the Clustal X multiple-sequence alignment program. The accession number of each sequence in the JGI GeneBank is indicated on the left of the alignment. An asterisk indicates that the residues at a position are identical in all sequences in the alignment; a colon indicates that conserved substitutions have been observed and a period indicates semiconserved substitutions. Putative signal sequences are indicated by italics and the mitochondrial targeting peptides are enclosed in boxes. The conserved residues involved in copper binding are in red, and the complete L1–L4 regions are indicated by a double line under the alignment. The sequences of potential substrate loops were identified based on loops I-IV of the rMaL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055295#pone.0055295-Kallio2" target="_blank">[44]</a> and TaLcc1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055295#pone.0055295-Kallio1" target="_blank">[42]</a>, laccases of <i>M. albomyces</i> and <i>T. arenaria</i>, respectively, and are underlined with a bold line. Amino acids shaded in yellow indicate residues in contact with the substrate. The residues forming the SDS gate are shaded in green in color, and the amino acid shaded in blue classified the laccases as class 1 (Met), class 2 (Leu) or class 3 (Phe). The conserved C-termini are in dark blue.</p