Evolutionary convergence in lignin-degrading enzymes

Abstract

The resurrection of ancestral enzymes of now-extinct organisms (paleogenetics) is a developing field that allows the study of evolutionary hypotheses otherwise impossible to be tested. In the present study, we target fungal peroxidases that play a key role in lignin degradation, an essential process in the carbon cycle and often a limiting step in biobased industries. Ligninolytic peroxidases are secreted by wood-rotting fungi, the origin of which was recently established in the Carboniferous period associated with the appearance of these enzymes. These first peroxidases were not able to degrade lignin directly and used diffusible metal cations to attack its phenolic moiety. The phylogenetic analysis of the peroxidases of Polyporales, the order in which most extant wood-rotting fungi are included, suggests that later in evolution these enzymes would have acquired the ability to degrade nonphenolic lignin using a tryptophanyl radical interacting with the bulky polymer at the surface of the enzyme. Here, we track this powerful strategy for lignin degradation as a phenotypic trait in fungi and show that it is not an isolated event in the evolution of Polyporales. Using ancestral enzyme resurrection, we study the molecular changes that led to the appearance of the same surface oxidation site in two distant peroxidase lineages. By characterization of the resurrected enzymes, we demonstrate convergent evolution at the amino acid level during the evolution of these fungi and track the different changes leading to phylogenetically distant ligninolytic peroxidases from ancestors lacking the ability to degrade nonphenolic lignin.

Keywords: Polyporales; ancestral enzyme resurrection; convergent evolution; fungal peroxidases; lignin.

Fig. 1.

Phylogenetic tree of 113 peroxidases from 10 Polyporales genomes (sequences in Dataset S1; bootstrap values ≥0.5 are indicated). Clades A–D are shown. The paths to the extant LiPA of P. chrysosporium (JGI ID# 2989894) and VP2 of T. versicolor (JGI ID# 26239) are shown in red and blue, respectively. Also, the milestones in these evolutionary lines (CaPo for both lines; CaD, AVP-d, and ALiP in red line; and CaB and AVP-b in blue line) are marked. The circles show the characteristics of the oxidation sites present in each of these nodes (Left: catalytic tryptophan and homologous residues; Right: Mn²⁺ oxidation site). The sequence labels start with the species code (BJEAD, B. adusta; CERSU, C. subvermispora; DICSQ, D. squalens; FOMPI, F. pinicola; GANSP, Ganoderma sp; PHACH, P. chrysosporium; PHLBR, P. brevispora; POSPL, P. placenta; TRAVE, T. versicolor; and WOLCO, W. cocos) followed by the JGI ID# and the peroxidase type, including GP, LiP, MnP-short, MnP-long, VP, and VP-atypical.

Fig. 2.

Molecular model of ancestral CaPo and its main structural changes in evolution. The most relevant amino acids of the common ancestor (CaPo) are labeled, and only the main changes in the oxidation sites are represented for the other peroxidases (two structural Ca²⁺ ions are shown as gray spheres). The Mn²⁺-binding site, formed by three acidic residues, and Ala172, homologous to catalytic tryptophan, are circled in CaPo. Red line: changes in line D evolution, with the appearance of Trp172 in AVP-d and loss of the Mn²⁺-binding site due to the Asn183 appearance in ALiP. Blue line: changes in line B evolution, with the appearance of Asp170 in CaB that later changed to Trp170 for lignin oxidation by AVP-b. Aromatic residues (Phe206 and Trp252 in CaPo) involved in long-range electron transfer from the exposed tryptophan are conserved from the first ancestor.

Fig. 3.

Evolution of catalytic properties in the D (red line) and B (blue line) evolutionary pathways. Changes of catalytic efficiency (Upper bars) and k_cat (Lower bars) are shown for oxidation of Mn²⁺ (purple); DMP at low- and high-efficiency sites (light and dark brown, respectively); ABTS at low- and high-efficiency sites (light and dark green, respectively); RB5 (blue); and VA (gray) (means and 95% confidence limits). For each substrate, the maximum value was taken as 100% and referred to that for the other enzymes (see SI Appendix, Table S2 for absolute values). The circled W marks the point when the catalytic tryptophan appeared for the first time, and black circles represent the other nodes analyzed.

Fig. 4.

Changes in the electrostatic surface of the environment of the catalytic tryptophan and homologous residues (pink spheres in the center) in peroxidase evolution. In line B to TV-VP2 (blue line), the changes are more subtle, but in line D to PC-LiPA (red line), a clear increase in the negative charge (red) happened with time.

Fig. 5.

The pH and thermal stabilities in the D (red line) and B (blue line) evolutionary pathways. (A) Changes in residual activity after incubation at pH 3. (B) Changes in T₅₀. Means and 95% confidence limits are shown.