Abstract
BackgroundFloudas et al. (Science 336: 1715) established that lignin-degrading fungi appeared at the end of Carboniferous period associated with the production of the first ligninolytic peroxidases. Here, the subsequent evolution of these enzymes in Polyporales, where most wood-rotting fungi are included, is experimentally recreated using genomic information.ResultsWith this purpose, we analyzed the evolutionary pathway leading to the most efficient lignin-degrading peroxidases characterizing Polyporales species. After sequence reconstruction from 113 genes of ten sequenced genomes, the main enzyme intermediates were resurrected and characterized. Biochemical changes were analyzed together with predicted sequences and structures, to understand how these enzymes acquired the ability to degrade lignin and how this ability changed with time. The most probable first peroxidase in Polyporales would be a manganese peroxidase (Mn3+ oxidizing phenolic lignin) that did not change substantially until the appearance of an exposed tryptophan (oxidizing nonphenolic lignin) originating an ancestral versatile peroxidase. Later, a quick evolution, with loss of the Mn2+-binding site, generated the first lignin peroxidase that evolved to the extant form by improving the catalytic efficiency. Increased stability at acidic pH, which strongly increases the oxidizing power of these enzymes, was observed paralleling the appearance of the exposed catalytic tryptophan.ConclusionsWe show how the change in peroxidase catalytic activities meant an evolutionary exploration for more efficient ways of lignin degradation by fungi, a key step for carbon recycling in land ecosystems. The study provides ancestral enzymes with a potential biotechnological interest for the sustainable production of fuels and chemicals in a biomass-based economy.
Highlights
Floudas et al (Science 336: 1715) established that lignin-degrading fungi appeared at the end of Carboniferous period associated with the production of the first ligninolytic peroxidases
Reconstruction of ancestral sequences from Polyporales genomes From the information available in ten genomes of Polyporales sequenced at the Joint Genome Institute (JGI), a maximum likelihood (ML) phylogenetic tree of ligninolytic peroxidase and generic peroxidase (GP) sequences (Fig. 1) was constructed with RAxML [30]
In the path from the first peroxidase in Polyporales to LiPH8 (Fig. 1, red line), we focused on four proteins, whose most probable reconstructed sequences are shown in Fig. 2, because they are milestones in lignin peroxidase (LiP) appearance: common ancestor of Polyporales peroxidases (CaPo) is the Common ancestor of Polyporales peroxidases, common ancestor of cluster-D peroxidases (CaCD) represents the Common ancestor of Cluster D, ancestral versatile peroxidase (VP) (AVP) would be the most Ancestral VP in this evolutionary line, and ancestral LiP (ALiP) would be the most Ancestral LiP in Polyporales
Summary
Floudas et al (Science 336: 1715) established that lignin-degrading fungi appeared at the end of Carboniferous period associated with the production of the first ligninolytic peroxidases. The subsequent evolu‐ tion of these enzymes in Polyporales, where most wood-rotting fungi are included, is experimentally recreated using genomic information. The use of the tools provided by bioinformatics is mandatory [1]. To this point, ancestral sequence reconstruction can give us hints about ancient. Resurrected proteins are of interest because of the essential information about evolution that they can provide, and due to the biotechnological potential. Ancestral proteins often have higher stability [10] and, likely, new activities [11], which can make of them interesting biocatalysts. If we identify the elements that confer this stability or new activity we can improve extant enzymes by rational design. The famous James Gould [12] sentence “replaying the tape of life” will no longer be a metaphor if we evolve the ancient proteins in the laboratory [13, 14]
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