Evolutionary convergence in lignin-degrading enzymes Iván Ayuso-Fernándeza, Francisco J. Ruiz-Dueñasa,1, and Angel T. Martíneza,1 aCentro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, E-28040 Madrid, Spain Edited by C. Kevin Boyce, Stanford University, Stanford, CA, and accepted by Editorial Board Member David Jablonski May 7, 2018 (received for review February 12, 2018) The resurrection of ancestral enzymes of now-extinct organisms coal accumulation near the end of the Permo-Carboniferous (paleogenetics) is a developing field that allows the study of (16). However, geoclimatic factors would have also significantly evolutionary hypotheses otherwise impossible to be tested. In the contributed to coal formation under ever-wet tropical conditions, present study, we target fungal peroxidases that play a key role in and its decline could also be related to climatic shifts toward lignin degradation, an essential process in the carbon cycle and drier conditions (17, 18). Then, the expansion and diversification often a limiting step in biobased industries. Ligninolytic peroxidases of genes encoding ligninolytic peroxidases occurred leading to are secreted by wood-rotting fungi, the origin of which was recently the families existing today, as shown by genomic and evolution- established in the Carboniferous period associated with the appear- ary studies (16, 19, 20). The diversity and evolution of these ance of these enzymes. These first peroxidases were not able to enzymes have been studied particularly in the order Polyporales, degrade lignin directly and used diffusible metal cations to attack its where the lignicolous habitat is largely predominant, resulting in phenolic moiety. The phylogenetic analysis of the peroxidases of Polyporales, the order in which most extant wood-rotting fungi are the most efficient ligninolytic enzymes. Recent studies included included, suggests that later in evolution these enzymes would first analyses of the appearance and disappearance of relevant have acquired the ability to degrade nonphenolic lignin using a catalytic sites (20) and later sequence reconstruction, heterologous “ ” tryptophanyl radical interacting with the bulky polymer at the expression ( resurrection ), and experimental characterization of surface of the enzyme. Here, we track this powerful strategy for some ancestral enzymes (21). The evolutionary analysis of per- lignin degradation as a phenotypic trait in fungi and show that it is oxidases shows phylogenetically distant enzymes (corresponding to not an isolated event in the evolution of Polyporales. Using the above LiP and VP types) that would be a priori able to oxidize ancestral enzyme resurrection, we study the molecular changes nonphenolic lignin at the exposed catalytic tryptophan (11–15, 22). EVOLUTION that led to the appearance of the same surface oxidation site in two To determine if the LiP/VP distribution in the peroxidase phy- distant peroxidase lineages. By characterization of the resurrected logeny is due to duplication of an ancestor and maintenance of enzymes, we demonstrate convergent evolution at the amino acid function or to a convergent process, we first performed phyloge- level during the evolution of these fungi and track the different netic analyses and ancestral sequence reconstruction of Poly- changes leading to phylogenetically distant ligninolytic peroxidases porales peroxidases from sequenced genomes. This choice should from ancestors lacking the ability to degrade nonphenolic lignin. enable a precise description on the evolution of ligninolytic en- zymes within this order, although the reconstruction of enzymes convergent evolution | ancestral enzyme resurrection | lignin | predating Polyporales could be partially biased. Then we com- Polyporales | fungal peroxidases pared in the laboratory the previously described line leading to extant LiP (21) with a new independent evolutionary line leading egradation of lignin is essential for carbon recycling in land Decosystems and often represents a key step for the use of Significance biomass in the industry (1). The main organisms that are able to mineralize lignin are white-rot fungi, using an array of oxidative We analyze the molecular mechanisms that led to the rise of a enzymes (2). Three class II peroxidases of the peroxidase- powerful strategy for lignin degradation (i.e., the formation of a catalase superfamily (3) are involved in the initial attack on solvent-exposed tryptophanyl radical capable of oxidizing the lignin: (i) lignin peroxidases (LiP), which are able to oxidize its ii bulky lignin polymer) as a convergent trait in different species major nonphenolic moiety (4); ( )manganeseperoxidases(MnP) of fungi (order Polyporales). We use ancestral sequence recon- including short and long MnPs with slightly different properties 2+ 3+ struction and enzyme resurrection to obtain the ancestors of the (5) that oxidize Mn to Mn , the diffusible chelates of which two extant types of ligninolytic peroxidases—lignin peroxidase iii oxidize the minor phenolic moiety of lignin (6); and ( ) versatile (LiP) and versatile peroxidase (VP)—and compare their predicted peroxidases (VP), which combine the catalytic properties of LiP, molecular structures and catalytic properties after resurrection. MnP, and plant peroxidases (the latter oxidizing phenolic mono- The results presented demonstrate convergent evolution in dis- lignols) (7, 8). Also, white-rot fungi produce generic peroxidases tant LiP and VP lineages with the exposed tryptophan residue (GP), catalytically similar to plant peroxidases. The above four appearing twice, as two independent events, following different peroxidase types have been characterized, and their structural and molecular changes. kinetic properties are well known (9). Thereby, they can oxidize substrates at three sites: (i) the main heme access channel, where Author contributions: F.J.R.-D. and A.T.M. designed research; I.A.-F. performed research; low redox-potential compounds are oxidized (in all of them); (ii)a I.A.-F., F.J.R.-D., and A.T.M. analyzed data; and I.A.-F., F.J.R.-D., and A.T.M. wrote + Mn2 -oxidizing site, formed by three acidic residues near one of the the paper. heme propionates (10) (in MnP and VP); and (iii) a surface tryp- The authors declare no conflict of interest. tophan (11, 12) that is able to oxidize lignin directly (13, 14) via an This article is a PNAS Direct Submission. C.K.B. is a guest editor invited by the Editorial Board. aminoacyl radical and long-range electron transfer to heme (15) (in This open access article is distributed under Creative Commons Attribution-NonCommercial- VP and LiP). NoDerivatives License 4.0 (CC BY-NC-ND). In past years, there has been an increasing interest in the 1To whom correspondence may be addressed. Email: [email protected] or atmartinez@cib. evolution of wood-degrading organisms. The origin of lignin csic.es. degradation by fungi, associated with the appearance of the first This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ligninolytic peroxidases, has been estimated to have occurred 1073/pnas.1802555115/-/DCSupplemental. during the Carboniferous period, playing a role in the decline of www.pnas.org/cgi/doi/10.1073/pnas.1802555115 PNAS Latest Articles | 1of6 Downloaded by guest on September 25, 2021 to extant VP, and established their convergent evolution using 0.52 BJEAD-116816-LiP empirical analyses of resurrected enzymes. 1 BJEAD-157924-LiP 0.74 BJEAD-499082-LiP 1 BJEAD-172246-LiP BJEAD-184697-LiP Results BJEAD-323280-LiP BJEAD-120002-LiP 0.71 BJEAD-186344-LiP Ancestral Sequences of Polyporales Peroxidases. For reconstructing 1 BJEAD-269524-LiP 0.99 0.75 BJEAD-41982-LiP the ancestral sequences leading to the extant lignin-degrading BJEAD-269481-LiP BJEAD-121664-LiP 0.97 PHACH-2918435-LiP peroxidases in Polyporales, we built a phylogenetic tree with 0.80 PHACH-2918661-LiP 0.89 PHACH-1716776LiP RAxML (Fig. 1) after manual annotation of the complete set of 0.97 PHACH-1716042-LiP 0.85 PHACH-2989894-LiP peroxidase sequences (a total of 113) in the sequenced genomes 1 0.9 PHACH-3032425-LiP 0.74 1 PHACH-2910310-LiP Bjerkandera adusta Ceriporiopsis subvermispora Dichomitus 1 PHACH-3032409-LiP of , , ALiP PHACH-3043032-LiP squalens Fomitopsis pinicola Ganoderma Phlebia brevispora PHACH-1386770-LiP , , sp., , 1 PHLBR-129470-LiP 0.71 PHLBR-86283-LiP Phanerochaete chrysosporium, Postia placenta, Trametes versicolor, 1 PHLBR-150253-LiP PHLBR-116561-LiP and Wolfiporia cocos. For the present analysis, the tree was di- 0.50 PHLBR-150901-LiP 0.83 0.92 TRAVE-133918-LiP i ii TRAVE-134657-LiP vided into ( ) clade A containing GPs; ( ) clade B, including two 1 TRAVE-134250-LiP 0.98 TRAVE-133326-LiP subclades of short MnPs and VPs; (iii) clade C formed exclu- 0.88 TRAVE-52333-LiP 0.93 0.70 TRAVE-133731-LiP iv AVP-d 0.99 0.99 TRAVE-114944-LiP sively of long MnPs; and ( ) clade D containing short MnPs and TRAVE-43578-LiP 0.97 TRAVE-43576-LiP some VPs, together with the large LiP subclade. B and D are the 0.69 TRAVE-134226-LiP Clade D TRAVE-43289-VP only clades that contain enzymes that a priori would oxidize GANSP-116056-VP 0.89 TRAVE-51457-MnPshort nonphenolic lignin due to the presence of the exposed catalytic 0.52 TRAVE-51455-MnPshort 0.92 1 TRAVE-131080-MnPshort 0.89 TRAVE-130496-MnPshort tryptophan. To explain the presence of this residue in two phy- TRAVE-51451-MnPshort TRAVE-74179-MnPshort logenetically different peroxidase clades, horizontal gene trans- 0.73