Peroxidase evolution in white-rot fungi follows wood lignin evolution in

Iván Ayuso-Fernándeza, Jorge Rencoretb, Ana Gutiérrezb, Francisco Javier Ruiz-Dueñasa, and Angel T. Martíneza,1 aCentro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, E-28040 Madrid, Spain; and bInstituto de Recursos Naturales y Agrobiología de Sevilla, Consejo Superior de Investigaciones Científicas E-41012 Seville, Spain

Edited by David M. Hillis, The University of Texas at Austin, Austin, TX, and approved July 25, 2019 (received for review March 23, 2019) A comparison of sequenced Agaricomycotina genomes suggests peroxidases and analyzed their evolution in Polyporales using that efficient degradation of wood lignin was associated with the high– and low–redox potential simple model substrates (11). appearance of secreted peroxidases with a solvent-exposed cata- These studies suggest that the most ancestral ligninolytic en- lytic tryptophan. This hypothesis is experimentally demonstrated zymes acted on phenolic lignin and later shaped their oxidation here by resurrecting ancestral fungal peroxidases, after sequence sites several times to become first VPs and later LiPs, the most reconstruction from genomes of extant white-rot Polyporales, and efficient lignin-degrading enzymes, as a convergent trait in fungal evaluating their oxidative attack on the lignin polymer by state-of- evolution (12). Also, ligninolytic peroxidases progressively in- the-art analytical techniques. Rapid stopped-flow estimation of the transient-state constants for the 2 successive one-electron transfers creased the redox potential of their reactive (13). Finally, their stability at acidic pH, where lignin decay takes place in from lignin to the peroxide-activated enzyme (k2app and k3app) showed a progressive increase during peroxidase evolution (up to nature, increased, and their catalytic tryptophan environment 50-fold higher values for the rate-limiting k3app). The above agreed became more negative to stabilize lignin cation radicals (11, 12). with 2-dimensional NMR analyses during steady-state treatments of Recent phylogenomic studies on over 1,000 Agaricomycetes hardwood lignin, showing that its degradation (estimated from the suggest that during evolution wood-rotting fungi became host normalized aromatic signals of lignin units compared with a control) specific depending on whether they were brown-rot fungi, often

and syringyl-to-guaiacyl ratio increased with the enzyme evolution- acting on conifers, or white-rot fungi, specializing on angiosperms EVOLUTION ary distance from the first peroxidase ancestor. More interestingly, (14). The evolution of host specialization is notably interesting the stopped-flow estimations of electron transfer rates also showed since conifer lignin basically consists of guaiacyl (G) units, while how the most recent peroxidase ancestors that already incorporated angiosperms introduce also syringyl (S) units, with minor amounts the exposed tryptophan into their molecular structure (as well as the of p-hydroxyphenyl (H) units in both lignins, as illustrated in Fig. 1 extant lignin peroxidase) were comparatively more efficient at oxi- (see SI Appendix,TableS1, for a comprehensive review of lignin dizing hardwood (angiosperm) lignin, while the most ancestral “tryptophanless” enzymes were more efficient at abstracting elec- composition). As shown in Fig. 1, S-type units were also incor- trons from softwood (conifer) lignin. A time calibration of the ances- porated by some nonconifer gymnosperms and a few ferns and try of Polyporales peroxidases localized the appearance of the first lycophytes as an example of convergent evolution discussed below. peroxidase with a solvent-exposed catalytic tryptophan to 194 ± 70 Lignin in conifers did not show detectable changes during evolu- Mya, coincident with the diversification of angiosperm plants char- tion (15). Then, the general inclusion of S units in angiosperms acterized by the appearance of dimethoxylated syringyl lignin units. Significance fungal evolution | lignin evolution | ancestral peroxidases | stopped-flow spectrophotometry | NMR spectroscopy We analyze the evolution of ligninolytic peroxidases from wood-rotting fungi using conifer and angiosperm lignin as ignin is an aromatic polymer formed by dehydrogenative representatives of 2 steps of lignin evolution. By enzyme res- Lcoupling of p-hydroxycinnamyl alcohols and other phenols (1) urrection, we show that during fungal evolution, these en- that confers resistance and other properties to vascular plants. zymes improved their activity and switched their degradative Due to lignin recalcitrance, its degradation is essential for carbon preferences with the rise of a surface tryptophan conferring on recycling in nature and often represents a limiting step for a them the ability to oxidize nonphenolic lignin. We calibrated biobased economy (2). The biological degradation of lignin is the peroxidase phylogeny and determined that this residue described as an enzymatic combustion (3) typically made by appeared coincident with angiosperm diversification, charac- Polyporales and other white-rot Agaricomycetes. It involves the terized by the synthesis of a more complex and less phenolic so-called manganese peroxidases (MnPs), versatile peroxidases lignin due to the general incorporation of a new unit in its (VPs), and lignin peroxidases (LiPs) together with auxiliary en- structure. This way, we show that fungal evolution followed zymes (4). MnPs oxidize the often minor phenolic moiety of that of lignin synthesis, pointing to a coevolution between + lignin via Mn3 chelates or directly at the heme cofactor (5). In fungal saprotrophs and their hosts. contrast, LiPs attack nonphenolic lignin directly using a solvent- exposed catalytic tryptophan forming a reactive radical, as shown Author contributions: F.J.R.-D. and A.T.M. designed research; I.A.-F. and J.R. performed research; A.G. contributed new reagents/analytic tools; I.A.-F., F.J.R.-D., and A.T.M. ana- using model compounds (6). Finally, VPs combine the structural lyzed data; I.A.-F. performed bioinformatic analysis; and I.A.-F., F.J.R.-D., and A.T.M. wrote and catalytic properties of MnPs and LiPs (7), including the the paper. surface tryptophan, whose role in direct oxidation of lignin was The authors declare no conflict of interest. recently demonstrated (8). This article is a PNAS Direct Submission. Fungal degradation of lignin via white rot arose around the This open access article is distributed under Creative Commons Attribution-NonCommercial- Carboniferous period, associated with the production of the first NoDerivatives License 4.0 (CC BY-NC-ND). ligninolytic peroxidases (9) and, along with geochemical factors, 1To whom correspondence may be addressed. Email: [email protected]. may have contributed to the decline of carbon sequestration This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (coal formation) toward the end of the Permo-Carboniferous 1073/pnas.1905040116/-/DCSupplemental. period (10). Recent works have resurrected ancestral ligninolytic www.pnas.org/cgi/doi/10.1073/pnas.1905040116 PNAS Latest Articles | 1of6 A B C D water solubility, a requisite for the above stopped-flow analyses. H H H H These preparations originate from sulfite pulping of wood, a S S S G process based on lignin sulfonation (19). While solubilizing lig- G G G nin, sulfonation scarcely affects peroxidase reactivity (18). Here we investigate how ancestral peroxidases modify lignin through their evolution and estimate the electron transfer rates from Fig. 1. Average lignin composition (H, G, and S molar content) in angio- sperms (A), conifers (B), other gymnosperms (C), and ferns and lycophytes softwood and hardwood lignosulfonates. With this purpose, we (D) from a total of 144 species (see SI Appendix, Table S1, for the complete use stopped-flow rapid spectrophotometry to calculate transient- inventory). state kinetic parameters and 2-dimensional (2D) NMR spec- troscopy to evaluate the polymer modification in extended steady-state treatments. resulted in a more complex lignin providing mechanical and physiological advantages to plants (16, 17). Results So far, the evolution of ancestral peroxidase has not been Evolution of Polyporales Lignin-Degrading Peroxidases. To investi- analyzed using the lignin polymer as a substrate since initial rates gate how the ability to oxidize the lignin polymer evolved in the of lignin oxidation cannot be obtained under steady-state con- ancestry of Polyporales peroxidases (20), we reconstructed with ditions. However, the electron transfer rates can be accurately PAML the lineage leading to the well-known isoenzyme H8 of estimated from the reduction of peroxide-activated enzymes by Phanerochaete chrysosporium LiP, corresponding to gene A in the lignin under stopped-flow conditions (8, 18). Among available sequenced genome (PC-LiPA) (21). Then, we resurrected by lignin preparations, lignosulfonates are unique because of their Escherichia coli expression (11) the main nodes in the evolutionary

BJEAD-LiP (12)

ALiP PC-LiPA PHACH-LiP (10)

PHLBR-LiP (5) AVPd TRAVE-LiP (10) Clade D TRAVE-43289-VP GANSP-116056-VP TRAVE-MnPshort (7)

DICSQ-MnPshort (2) GANSP-142454-MnPshort TRAVE-112835-MnPshort BJEAD-MnPshort (2) PHLBR-MnPshort (2) BJEAD-43095-VP BJEAD-306404-MnPshort GANSP-176788-MnPshort TRAVE-43477-MnPshort CERSU-124076-MnPshort CERSU-118677-LiP CaD CERSU-99382-VP BJEAD-173495-GP BJEAD-43329-MnPshort TRAVE-44897-MnPshort

CERSU-MnPlong (7)

DICSQ-MnPlong (4) CERSU-50297-MnPlong CERSU-143390-MnPlong CERSU-MnPlong (3) PHLBR-MnPlong (3) Clade C PHACH-2896529-MnPlong PHACH-MnPlong (2) PHACH-2907883-MnPlong PHACH-3589-MnPlong DICSQ-VP (3) GANSP-116446-VPatypical TRAVE-262396-VP TRAVE-28895-VPatypical PHACH-2911114-GP DICSQ-150431-MnPshort CaPo TRAVE-187228-MnPshort GANSP-MnPshort (2) DICSQ-141471-MnPshort GANSP-161386-MnPshort Clade B BJEAD-MnPshort (2) GANSP-155330-MnPshort DICSQ-108150-MnPshort TRAVE-74595-MnPshort POSPL-GP (3) FOMPI-GP (2) WOLCO-136670-GP Clade A CERSU-112162-GP STANO-GP (5) 0.3

Fig. 2. Evolution of Polyporales peroxidases from 10 sequenced genomes (12), including the Stagonospora nodorum generic peroxidases (GPs) for rooting the tree built by RAxML. Collapsed clusters are shown in green (LiPs), yellow (VPs), blue (MnPs) and purple (GPs). The path to the extant PC-LiPA is marked in green, including CaPo (common ancestor of Polyporales peroxidases), CaD (common ancestor of clade D), AVPd (ancestral VP in clade D), and ALiP (ancestral LiP). Labels start with the species (BJEAD, Bjerkandera adusta; CERSU, Ceriporiopsis subvermispora; DICSQ, Dichomitus squalens; FOMPI, Fomitopsis pinicola; GANSP, Ganoderma sp.; PHLBR Phlebia brevispora; PHACH, P. chrysosporium; POSPL, Postia placenta; STANO, S. nodorum; TRAVE, Trametes versicolor;and WOLCO, Wolfiporia cocos), followed by the JGI ID and the peroxidase type (GP, LiP, MnP short, MnP long, VP, or VP atypical).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1905040116 Ayuso-Fernández et al. Table 1. Kinetic constants for CI/CII and CII/RS reduction in ancestral (CaPo, CaD, AVPd, and ALiP) and extant (PC-LiPA) peroxidases by softwood and hardwood lignosulfonate (LS) Reduction Lignin type Constant CaPo CaD AVPd ALiP PC-LiPA

CI/CII Softwood LS KD2 (μM) 46 ± 357± 3.9 46 ± 3.3 44 ± 15 23 ± 1 −1 k2 (s )9± 020± 119± 142± 933± 2 −1 −1 k2app (s ·mM )197± 14 351 ± 34 413 ± 33 955 ± 385 1,440 ± 120

Hardwood LS KD2 (μM) —— 8 ± 140± 58± 2 −1 k2 (s ) ——18 ± 0.6 27 ± 1.4 25 ± 0.9 −1 −1 k2app (s ·mM )86± 18 202 ± 7 2,280 ± 280 674 ± 97 2,940 ± 740

CII/RS Softwood LS KD3 (μM) ——102 ± 12 — 95 ± 326 −1 k3 (s ) ——10 ± 1 — 25 ± 4 −1 −1 k3app (s ·mM )25± 878± 499± 14 331 ± 4263± 83 Hardwood LS KD3 (μM) —— 9 ± 258± 819± 2 −1 k3 (s ) —— 6 ± 029± 114± 0 −1 −1 K3app (s ·mM )16± 343± 1670± 145 500 ± 70 764 ± 86

Means and 95% confidence limits are from reactions at 25 °C in 0.1 M tartrate, with a pH of 3. A dash (—)

indicates no saturation was reached. KD2 and KD3, CI and CII dissociation constants; k2 and k3, CI and CII first-order rate constants; k2app, CI apparent second-order rate constant. line (Fig. 2) and characterized the degradative abilities of these (AVPd, ALiP, and PC-LiPA) (Fig. 4). For softwood lignin there ancestral enzymes using polymeric lignin as a substrate. is a progressive increase of the CII apparent second-order rate Previously, to calibrate the age of ancestral peroxidases in the constant (k3app) through evolution, but the differences between RAxML tree in Fig. 2, we built an ultrametric tree with PATHd8, enzymes do not exceed a 13-fold increase. However, for hard- which uses mean path lengths from the leaves of a tree with a wood lignin there is a huge boost in k3app, increasing nearly 50- correction of the deviation from a molecular clock by smoothing fold from CaPo to AVPd, when the solvent-exposed tryptophan EVOLUTION substitution rates locally (SI Appendix, Fig. S1). The ages appeared. These increments can be interpreted in terms of the obtained suggest that the evolution of ligninolytic peroxidases dissociation constant (KD3), indicating a greater affinity for the started 399 ± 140 Mya with a common ancestor of Polyporales polymer in the enzymes with the solvent-exposed tryptophan peroxidases (CaPo), which most likely was the ancestor of all (Fig. 3B). Therefore, a change in lignin preference was produced + Agaricomycetes class II peroxidases (9, 12). CaPo had a Mn2 - through peroxidase evolution: The ratio between hardwood and binding site for oxidation of this metal cation but also oxidized softwood lignosulfonate rate-limiting k3app is less than 1 for the phenolic substrates in direct contact with the heme (11). From enzymes lacking the catalytic tryptophan and greater (up to 7) CaPo, the lineage to extant LiPs did not experience significant for those incorporating that residue. changes for 200 million years (with the common ancestor of clade D peroxidases [CaD] dated to 259 ± 94 Mya still being a Steady-State Treatment of Lignins. To further explore the evolu- MnP-type enzyme) until the appearance of the catalytic tryp- tion of lignin degradation, the structural modification of G and tophan in ancestral VP in clade D (AVPd) 194 ± 70 Mya. AVPd S–G lignins from softwood and hardwood, respectively, was an- + retained the Mn2 oxidation site together with the new solvent- alyzed by heteronuclear single quantum correlation (HSQC) 2D exposed tryptophan, putatively responsible for nonphenolic lignin NMR spectroscopy. The signals of the main aromatic units and + oxidation. Then, a quick change, losing the Mn2 -binding site, side chain interunit linkages in hardwood (Fig. 5) and softwood occurred in a few million years, originating the first LiP of the (SI Appendix, Fig. S4) lignosulfonates included Cα-sulfonated phylogeny (ancestral LiP [ALiP], 178 ± 64 Mya) that further led to guaiacyl units (G) and Cα-sulfonated (S), nonsulfonated (S), and the extant LiPs maintaining the oxidation site architecture. This Cα-oxidized (S′) syringyl units, together with Cα-sulfonated (A) last evolutionary step resulted in a highly specialized enzyme whose and nonsulfonated (A) β-O-4′ substructures and less abundant efficient oxidation of nonphenolic lignin is not noncompetitively phenylcoumaran (B) and resinol (C) substructures. A semi- + inhibited by Mn2 , as in the case of VPs. quantitative analysis of these signals provided the lignin decrease (from aromatic signals), referred to the control without enzyme, Kinetics of Lignin Oxidation through Peroxidase Evolution. For better and S/G ratio and substructure composition referred to 100 ar- biological understanding of peroxidase evolution, we analyzed omatic units (SI Appendix, Table S2). the catalytic cycle of the resurrected ancestors with lignosulfo- Twenty-four-hour treatments of softwood lignosulfonate with nates as enzyme-reducing substrates. This cycle (18) includes 2- the ancestral enzymes and PC-LiPA did not reveal strong dif- electron activation of the resting state (RS) enzyme by H2O2 ferences between them or from the enzyme-free control (SI forming compound I (CI), which is reduced back to RS via a Appendix, Fig. S4). However, the same treatments on hardwood 1-electron oxidized compound II (CII) yielding 2 product mol- lignosulfonate resulted in significant spectroscopic changes (Fig. ecules (SI Appendix, Fig. S2). The different ultraviolet–visible 5 and SI Appendix, Table S2), a fact related to the peroxidase spectra of these intermediates make following the cycle reactions evolution acting on angiosperm lignin discussed below. By in- by stopped-flow rapid spectrophotometry possible. The CI/CII tegrating the aromatic signals in the hardwood lignosulfonate and CII/RS reduction kinetic curves were obtained, and the spectra, we observed a decrease through evolution being the corresponding transient-state constants for hardwood and soft- maximal change produced by ALiP (with a 59% decrease of S wood lignins were estimated (Table 1) with dissociation (KD) and units and an 81% decrease of G units). This is accompanied by first-order rate (k) constants being calculated when the pseudo- an overall increase (over 2-fold) of the S/G ratio. Concerning first-order rate constant kobs exhibited saturation behavior (as side chain signals, no strong changes in their relative abundance illustrated in Fig. 3 and SI Appendix, Fig. S3). (per 100 aromatic units) were observed. For all enzymes, CII/RS reduction is the rate-limiting step, The progressive decrease of lignin signals in the steady-state with the most ancestral CaPo and CaD having significantly treatments through peroxidase evolution supports the trend slower rates than the enzymes with the catalytic tryptophan observed in stopped-flow measurements. Interestingly, in both

Ayuso-Fernández et al. PNAS Latest Articles | 3of6 A 32% of the peroxidases from fungi with preference for angio- 30 sperm hosts versus only 15% in conifer-colonizing fungi (Fig. 6). CaPo Discussion CaD 25 Genomic studies from 2004 (21) demonstrated the presence of AVPd ligninolytic peroxidase (LiP, VP, and/or MnP) genes in all typical ALiP white-rot fungi sequenced (9, 20, 22), although some controversy ) 20

-1 has been raised regarding a few poor wood degraders (23). More (s PC-LiPA recently, several aspects of peroxidase evolution were addressed

obs 15 by ancestral sequence reconstruction and characterization of the k resurrected ancestors using simple substrates (11–13). Here, we 10 use softwood and hardwood lignin as substrates to analyze the evolution leading to the most efficient ligninolytic enzymes, Polyporales LiPs (4). 5 With both types of lignin, the efficiency of the rate-limiting electron transfer step (corresponding to CII reduction to RS) was higher for the enzymes containing the catalytic tryptophan 0 (AVPd, ALiP, and PC-LiPA). This is in agreement with the 0 20 40 60 80 100 decreased electron transfer rate observed for tryptophanless Softwood lignosulfonate (μM) variants of extant VP (18). As shown by stopped-flow spectro- photometry, uniquely interesting is the change in specificity when B the solvent-exposed tryptophan appeared in AVPd. These 20 analyses reveal a switch from better oxidation of softwood lignin by the oldest CaPo and CaD ancestors (although with low kinetic rates on both lignins) to better oxidation of hardwood lignin since then (by AVPd, ALiP, and PC-LiPA enzymes). Moreover,

15 enzymes using state-of-the-art 2D NMR (24), enzymatic modification of lignin was observed in steady-state treatments of lignosulfonates )

-1 with the peroxidase ancestors. Especially informative are the

(s results from hardwood lignosulfonates, revealing that the lignin- 10 obs

containing degrading ability increases as a result of evolution being higher in k the enzymes containing the solvent-exposed catalytic tryptophan,

Trp- in agreement with the stopped-flow measurements. During recent evolution of vascular plants, the route to S units 5 appeared before the diversification of angiosperms, which started 250 to 140 Mya (25), and remains present in all extant species (SI Appendix, Table S1). The presence of S-type lignin in some 0 nonangiosperm plants (SI Appendix, Table S1, including reports > 0 20 40 60 80 100 of S content 25% for 1 liverwort, 3 lycophytes, 1 fern, and 3 gnetophyte lignins) most likely represents evolutionary conver- Hardwood lignosulfonate (μM) gence events (26), as demonstrated for the lycophyte Selaginella Fig. 3. Kinetics of CII to RS reduction in ancestral (CaPo, CaD, AVPd, and ALiP) and extant (PC-LiPA) peroxidases by softwood (A) and hardwood (B) lignosulfonates. The lignosulfonate concentrations refer to the basic ) phenylpropanoid unit. Means and 95% confidence limits are shown. -1 400

More efficient enzymes containing a catalytic tryptophan residue are lignin 300 indicated in B. mM -1 200 (s 100 3app cases, the strongest change coincided with the evolutionary ap- Softwood k 0 pearance of the surface catalytic tryptophan. 100 200 Polyporales Specialization for Plant Substrates. Using the U.S. ) -1

Department of Agriculture (USDA) Fungus-Host Distribution lignin 300

Database (https://nt.ars-grin.gov/fungaldatabases/fungushost/ mM 400 -1 fungushost.cfm), we obtained a list of plant hosts for the 10 (s 500 Polyporales species analyzed. This information was compared

3app 600 Hardwood with the number and type of peroxidase genes in the genomes of k the same fungi, extracted from the Joint Genome Institute (JGI) 700 MycoCosm database (https://mycocosm.jgi.doe.gov/mycocosm/ 800 home)(SI Appendix, Table S3). With only the exception of P. 900 brevispora (with only 3 citations), a correlation could be found CaPo CaD AVPd ALiP PC-LiPA between the specialization toward angiosperm decay and the number of peroxidases with the solvent-exposed catalytic tryp- Fig. 4. Comparison of the rate-limiting step constant (k3app) in lignin stopped-flow transient-state kinetic experiments with ancestral and extant tophan (VPs or LiPs). When the analysis was extended to the 64 peroxidases, showing a shift from softwood to hardwood lignin preference Polyporales genomes currently available at JGI, the same ten- with the appearance of the catalytic tryptophan in AVPd (dashed line). dency was observed, with surface tryptophan being present in Means and 95% confidence limits are shown.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1905040116 Ayuso-Fernández et al. Cβ δ Cβ δ Cβ δ ControlMeO C CaPoMeO C CaD MeO C

Aγ Aγ Aγ 60 Aγ 60 Aγ Aγ 60

Aα Aα(S) Aα Aα(S) Aα(G) Aα(S) (G) (G) Aα(G) Aα(G) Aα(G) Aα(S) Aα(S) Aα(S)

Cγ Cγ Cγ Aβ(G) Aβ(G) Aβ(G) Aβ(S) 80 Aβ(S) 80 Aβ(S) 80

Cα Cα Cα Aβ(S) Aβ(S) Aβ(S)

S S2,6 100 S2,6 100 2,6 100 S'2,6 S'2,6 S'2,6 S S2,6 2,6 S2,6 G G2 G5 G2 G5 G2 5

120 120 120 G6 G decrease: 0% G6 G decrease: 17% G6 G decrease: 56% S decrease: 0% S decrease: 9% S decrease: 39%

7 654 δ 7 654 δ 7 654 δ H H H

δ δ δ AVPdMeO C ALiPMeO C PC-LiPA MeO C

Aγ Aγ 60 60 Aγ 60 Aγ Aγ Aγ

Aα(S) Aα(S) Aα(S) EVOLUTION

Aα(S) Aα(S) Aα(S)

Aβ(S) 80 Aβ(S) 80 Aβ(S) 80

Aβ(S) Aβ(S) Aβ(S)

S2,6 100 S2,6 100 S2,6 100 S'2,6 S'2,6 S'2,6 S2,6 S2,6 S2,6

G2 G G G5 2 2 G5

120 120 120 G decrease: 76% G decrease: 81% G decrease: 64% S decrease: 55% S decrease: 59% S decrease: 47%

7 654 δ 7 654 δ 7 654 δ H H H OMe O HO MeO HO MeO γ γ α O ′ SO H SO H OH R O γ (OMe) 3 3 HO3S HO α α α β 4′ β 4′ α α O α O β′ β (OMe) (OMe) γ 622 626 (MeO) ′ 6 2 α O 5 OMe MeO OMe MeO OMe MeO OMe (MeO) OMe (MeO) OMe O O O O O O O OMe A A C G S S S′

Fig. 5. HSQC NMR spectra of hardwood lignosulfonate treated for 24 h with ancestral peroxidases and extant PC-LiPA, and control without enzyme. The main substructures identified are included at the bottom, and the decrease in G and S units (with respect to the control) are indicated in each panel. Signals correspond to 1H–13C correlations at the different positions of Cα-sulfonated guaiacyl units (G); Cα-sulfonated, nonsulfonated, and Cα-oxidized syringyl units (S, S, and S′); Cα-sulfonated and nonsulfonated β-O-4′ substructures (A and A) and resinol (C) substructures; and methoxyls (MeO). moellendorffii (27). In a broader context, it has been suggested enzymatic degradability. Due to the apparent implication of the that S units emerged at least 5 times during plant evolution, in- surface tryptophan mentioned above in the oxidation of nonphenolic cluding “lignin-like” molecules in some nonvascular plants lignin, we calibrated here its evolutionary appearance. This time (28), although a stable and generalized presence was only pro- calibration suggests that AVPd, the first ancestral peroxidase with duced in flowering plants. Wood lignin containing S units has this exposed tryptophan, appeared 194 ± 70 Mya, concurrent with higher complexity (1) and lower phenolic content (29, 30), reducing angiosperm diversification.

Ayuso-Fernández et al. PNAS Latest Articles | 5of6 30 Materials and Methods The 118 peroxidase sequences were obtained from the genomes of 10 Polyporales (20) and the ascomycete S. nodorum sequenced at the De- 20 partment of Energy’s JGI and available from the JGI MycoCosm database (https://mycocosm.jgi.doe.gov/mycocosm/home). PAML 4.7 (32) was used for 10 ancestral sequence reconstruction, using the phylogeny from RAxML (33). PATHd8 (34) was used for time calibration of an ultrametric phylogenetic tree,

Surface tryptophan (%) using the diversification of Pezizomycotina (average 631 Mya), the internal 0 diversification of Stagonospora nodorum generic peroxidases (average 403 Hardwood Softwood All Mya), and the diversification of the Antrodia clade peroxidases (average 399 Preferred substrate (citation %) Mya) (9) as time constraints. Four reconstructed ancestral peroxidase sequences and extant PC-LIPA Fig. 6. Hardwood and softwood preference of Polyporales vs. the fre- quency of the catalytic surface tryptophan in peroxidases. The percentage of were expressed in E. coli and activated in vitro (11). Transient-state kinetic peroxidases with the surface catalytic tryptophan is from a survey of genes constants were estimated by stopped-flow rapid spectrophotometry (at (up to a total of 794) in 64 genomes sequenced at JGI. The substrate pref- 25 °C in 0.1 M tartrate, with a pH of 3) using softwood and hardwood lig- erence was defined from the number of angiosperm and conifer hosts in- nosulfonates, whose concentrations were referred to the basic phenyl- cluded in the USDA Fungus-Host Distribution Database. propanoid unit (260 and 290 Da, respectively). Lignosulfonate modification in steady-state treatments (24 h at 25 °C in 50 mM phosphate, with a pH of 5) were analyzed by HSQC 2D NMR after spectral normalization with respect

Our results show how saprotrophic fungi followed lignin evolu- to the δH/δC 2.49/39.5 ppm signal of the nondeuterated fraction of dimethyl tion in plants incorporating a catalytic tryptophan responsible for sulfoxide used as NMR solvent. See SI Appendix for details. The data sup- oxidation of the more complex and less phenolic lignin character- porting the conclusions of this article are included within the article and in izing angiosperm plants. This is in agreement with the apparent the SI Appendix. relationship between the number of Polyporales ligninolytic per- oxidases with the surface catalytic tryptophan (VPs and LiPs oxi- ACKNOWLEDGMENTS. This work was supported by the EnzOx2 (H2020-BBI- dizing nonphenolic lignin) and the frequency of angiosperms as PPP-2015-2-720297, https://www.enzox2.eu) European project and the their hosts. Recent character reconstruction basedonover5,000 BIO2017-86559-R and AGL2017-83036-R Spanish projects. The work conducted mushroom-forming species determined that the ancestor of Poly- by JGI was supported by the Office of Science of the US Department of Energy porales had mainly gymnosperms as hosts (31), and during fungal under Contract DE-AC02-05CH11231. We thank Esther Novo-Uzal (Gulbenkian evolution, white-rot fungi specialized in angiosperms, while brown- Institute, Oeiras, Portugal) for critical reading of the manuscript and comments on plant and lignin evolution, Guro E. Fredheim (Borregaard AS, Sarpsborg, rot fungi, without ligninolytic peroxidases, became more generalist Norway) for providing the lignosulfonate preparations, José M. Barrasa (Univer- (14). Our results analyzing ancestral ligninolytic peroxidases and sity of Alcalá, Spain) for helpful comments on Polyporales ecology and evolution, their behavior using real lignin substrates support these conclusions and Manuel Angulo from the Centro de Investigación Tecnológica e Innovación and point to a fascinating coevolution between fungi and plants. de la Universidad de Sevilla (CITIUS, Seville, Spain) for the NMR analyses.

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6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1905040116 Ayuso-Fernández et al. SI Appendix

Peroxidase evolution in white-rot fungi follows wood lignin evolution in plants

Iván Ayuso-Fernández‡, Jorge Rencoret§, Ana Gutiérrez§, Francisco Javier Ruiz-Dueñas‡ and Angel T. Martínez‡

‡Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain; and §Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, PO Box 1052, E-41080 Seville, Spain

The SI Appendix includes Supporting Materials and Methods, Tables S1-S3, Figures S1-S4, and Supporting References.

Supporting Materials and Methods

Phylogenetic analysis and ancestral enzyme reconstruction. Every class-II peroxidase (113 sequences) annotated in the genomes of the following Polyporales, available at the DOE JGI Mycocosm portal, were used in the present study: - B. adusta (http://genome.jgi.doe.gov/Bjead1_1/Bjead1_1.home.html) (1) - C. subvermispora (http://genome.jgi.doe.gov/Cersu1/Cersu1.home.html (2) - D. squalens (http://genome.jgi.doe.gov/Dicsq1/Dicsq1.home.html) (3) - F. pinicola (http://genome.jgi.doe.gov/Fompi3/Fompi3.home.html) (3) - Ganoderma sp. (http://genome.jgi.doe.gov/Gansp1/Gansp1.home.html) (1) - P. brevispora (http://genome.jgi.doe.gov/Phlbr1/Phlbr1.home.html) (1) - P. chrysosporium (http://genome.jgi.doe.gov/Phchr2/Phchr2.home.html) (4) - P. placenta (http://genome.jgi.doe.gov/Pospl1/Pospl1.home.html) (5) - T. versicolor (http://genome.jgi.doe.gov/Trave1/Trave1.home.html) (3) and - W. cocos (http://genome.jgi.doe.gov/Wolco1/Wolco1.home.html) (3). For tree rooting and time calibration, we used the GP sequences of the ascomycete S. nodorum available at JGI Mycocosm (http://genome.jgi.doe.gov/Stano2/Stano2.home.html) (3,6). The amino-acid sequences were aligned with MUSCLE as implemented in MEGA 7 (7). The sequences were tested with ProtTest (8) to determine the evolutionary model that best fits the data for maximum likelihood (ML) analysis, among 64 empirical models of evolution. A ML phylogeny was then constructed using RAxML (9), under the Whelan and Goldman (10) model of evolution using gamma-distributed rate of heterogeneity (gamma shape with 4 rates of categories = 1.33) with empirical amino-acid frequencies and invariant sites (proportion of invariant sites = 0.073) (WAG+I+G+F). The PAML 4.7 package (11) was used to obtain the most probable sequence at each node of the phylogeny and the posterior amino-acid probability per site in each ancestor. We used the WAG model of evolution, and the previously obtained ML phylogeny and MUSCLE alignment as inputs for the software. Both joint and marginal reconstructions were performed and the most probable sequences of the marginal reconstruction were selected and manually corrected for C-terminal and other insertions or deletions according to the sequences of the ancestor progeny. The genes were synthesized by ATG:biosynthetics (Merzhausen, Germany) using the most frequent codons for high expression in E. coli.

1 www.pnas.org/cgi/doi/10.1073/pnas.1905040116

Time calibration of ancestral nodes in Polyporales phylogeny. To determine the ages of the ancestral nodes, the peroxidase phylogenetic tree was converted in an ultrametric tree using the recognized non-parametric method implemented in PATHd8 (12). The algorithm in PATHd8 only considers the mean path lengths from a node to its descendants and addresses deviations from the molecular clock locally. We used as time constraints those obtained by Floudas et al. (3) using Bayesian methods corresponding to: i) the diversification of Pezizomycotina from the rest of the phylogeny (631 mya with 533-762 mya maximum deviations); ii) the internal diversification of S. nodorium GPs (403 mya with 276-533 mya maximum deviations); and iii) the diversification of the Antrodia clade peroxidases (formed by F. pinicola, P. placenta and W. cocos) from the rest of Polyporales peroxidases (399 mya with 446-207 mya maximum deviations).

E. coli expression and isolation of the resurrected enzymes. After gene synthesis, the coding DNA sequences of the selected ancestors (CaPo, CaD, AVPd and ALiP) and the extant PC-LiPA (JGI ID# 2989894) were cloned into pET23b(+) (Novagen). The resulting plasmids were transformed into BL21(DE3)pLysS (ancestors) or W3110 (PC-LiPA) as reported by Pérez-Boada et al (13). The apoenzymes accumulated in inclusion bodies and, after solubilization in 8 M urea, the in vitro activation conditions of the ancestral proteins were optimized - including 0.16 M urea, 5 mM CaCl2, 15 µM hemin, 0.4 mM oxidized glutathione, 0.1 mM dithiothreitol and 0.1 mg/mL of protein in 50 mM Tris-HCl, pH 9.5 - while those reported by Doyle and Smith (14) where used for PC-LiPA. The active enzymes were purified with a Resource-Q column (GE-Healthcare) using a 0- 400 mM NaCl salt gradient, 2 mL/min flow, in 10 mM sodium tartrate, pH 5.5, containing 1 mM of CaCl2.

Softwood and hardwood lignins. Water solubility is required for the transient kinetic studies, described below. Therefore, to represent natural lignin, two water-soluble sulfonated lignins were used: softwood (Picea abies) and hardwood (Eucalyptus grandis) lignosulfonates provided by G. E. Fredheim (Borregaard AS, Sapsborg, Norway). Lignosulfonates were prepared for reactions according to Sáez-Jiménez et al, (15) including dialysis in 10 mM EDTA, 50 mM Tris (pH 8) to remove Mn2+ traces, and finally in Milli-Q water twice.

Enzyme (transient-state) kinetics. The reduction of peroxidases CI (to CII) and CII (to RS) was evaluated using hardwood and softwood lignosulfonates as enzyme reducing substrates in a stopped-flow rapid spectrophotometry equipment (Bio-Logic, Claix, France) with a three syringe module (SFM300) synchronized to a diode array detector (J&M, Essingen, Germany), and BioKine software. All reactions were carried out in 0.1 M tartrate (pH 3). Specifically, the reduction of CI was analyzed by mixing the enzyme (1 µM final concentration) with H2O2 (1-8 molar equivalents, depending on the enzyme) for 3 s, observing total CI formation. Next, different concentrations of lignosulfonates in 0.1 M tartrate (pH3) were added, and the reactions were followed at 416 nm, the isosbestic point of CII and the resting state of peroxidases. CII reduction was studied by mixing a solution containing the enzyme and ferrocyanide (both at 1 µM final concentration) with H2O2 (1-8 molar equivalents, depending on the enzyme) to ensure the formation of CII after 6 s. After that, different concentrations of lignosulfonates in 0.1 M tartrate (pH 3) were added, following the reaction at 410 nm, the maximum of the resting state for the peroxidases analyzed. All the traces showed single-exponential character from which pseudo first-order rate constants (k2obs and k3obs for CI and CII reduction, respectively) were calculated. Plots of k2obs and k3obs vs substrate concentration fitted to linear, hyperbolic or sigmoid models. From those kinetics that fitted to a linear model, apparent second-order rate constants (k2app and k3app for CI and CII reduction, respectively) were obtained. Plots of kobs vs substrate concentration that fitted to a Michaelis- Menten or sigmoid models yielded dissociation constants of the CI-lignin and CII-lignin complexes (KD2 and KD3 respectively) and first-order rate constants (k2 and k3, respectively). The corresponding 2 apparent second-order rate constants, k2app (k2/KD2) and k3app (k3/KD3), were calculated with the equation: kobs = (k/KD)[S]/(1 + [S]/KD), where [S] indicates substrate concentration. Despite replicated experiments (> 5 replica) measurements of CI/CII reduction resulted in high error values due to the intrinsic instability of CI, together with the polydisperse nature of the lignosulfonate preparations (being higher in the softwood lignosulfonate). For comparison, the lignosulfonate concentrations in these and other experiments were referenced to the basic phenylpropanoid unit corresponding to 260 and 290 Da in the sulfonated softwood and hardwood lignins, respectively (16).

Lignin treatment under steady-state conditions. To analyze how ancestral peroxidases and extant PC-LiPA modify lignin, hardwood and softwood lignosulfonates (12 g L-1) were treated at 25 ºC over 24 h in 50 mM phosphate (pH 5). The concentration of enzymes were 1 µM, added in two doses, with a H2O2 final concentration of 10 mM, added continuously with a syringe pump. Control treatments were performed under the same conditions but without enzyme. It is important to note that although peroxidases exhibit their highest activity at pH 3 (used in stopped-flow measurements), reactions over 24 h were carried out at pH 5 to maintain the enzymes active.

NMR analyses. The treated lignosulfonates and the corresponding controls were freeze-dried for NMR analyses. Spectra were recorded at 25 ºC on a Bruker Avance-III 500 MHz instrument equipped with a cryogenically cooled 5 mm TCI gradient probe with inverse geometry. The lignosulfonate samples (40 mg initial weight, before treatments) were dissolved in 0.75 mL of deuterated dimethylsulfoxide (DMSO-d6). The central peak of residual non-deuterated DMSO was used as the internal reference (at δH/δC 2.49/39.5 ppm), and the spectra were normalized to the same intensity of the DMSO signals, since the same DMSO volume and initial amount of lignin was used in all the cases. The HSQC experiment used Bruker´s “hsqcetgpsisp.2” adiabatic pulse program with spectral widths from 0 to 10 ppm (5000 Hz) and from 0 to 165 ppm (20625 Hz) for the 1H and 13C 1 dimension. The JCH used was 145 Hz. Processing used typical matched Gaussian apodization in the 1H dimension and squared cosine-bell apodization in the 13C dimension. Prior to Fourier transformation, the data matrices were zero-filled to 1024 points in the 13C dimension. Signals were assigned by comparison with the literature (17-23). In the aromatic region of the spectrum, the H2-C2, H5-C5 and H6-C6 correlation signals were integrated to estimate the amount of lignins and the S/G ratio. In the aliphatic oxygenated region, the signals of methoxyls, and Hβ-Cβ (or Hα-Cα) correlations in the side chains of sulfonated and non-sulfonated β-O-4', phenylcoumaran and resinol substructures were integrated. The intensity corrections introduced by the adiabatic pulse program permits to refer the latter integrals to the previously obtained number of lignin units.

Supporting Tables and Figures

Information on lignin composition in 144 plant species (Table S1), changes in softwood and hardwood composition after peroxidase treatments (Table S2), host distribution and peroxidase type in the ten Polyporales analyzed (Table S3), ultrametric tree of the peroxidases analyzed (Fig. S1), catalytic cycle of ligninolytic peroxidases (Fig. S2), kinetics of CI reduction (Fig. S3), and HSQC spectra of softwood lignosulfonates (Fig. S4).

3

Table S1. Lignin composition (H:G:S molar ratio) in plants: A review of 144 species from 46 orders.a Order H G S Method Ref Angiosperms: Acacia mangium (acacia) Fabales 5 44 51 NMR (24) Acer rubrum (red maple) Sapindales 2 44 54 NMR (24) Acrocomia aculeata (macauba) endocarp Arecales 1 63 36 NMR (25) Aextoxicon punctatum (olivillo) Berberidopsidales 0 34 66 CO (26) Agave sisalana (sisal) Asparagales 0 20 80 NMR (22) Alnus rubra (red alder) Fagales 4 41 55 NMR (24) Ananas comosus (pineapple) Poales 0 21 79 DFRC (27) Arabidopsis thaliana (thale cress) Brassicales 8 68 24 Py (28) Aralia cordata (spikenard) Apiales 0 45 55 TA (29) Arundo donax (giant reed) stem Poales 1 61 38 NMR (30) Baloskion tetraphyllum (tassel cord rush) Poales 1 43 56 DFRC (27) Betula nigra (black birch) Fagales 0 29 71 NB (31) Betula papyrifera (paper birch) Fagales 0 29 71 NB (32) Betula pendula (silver birch) Fagales 3 24 73 NMR (24) Bischofia polycarpa (Zhong-yang Mu) Malpighiales 0 69 31 NB (32) Buxus sempervirens (European box) Buxales tr 67 33 TA (33) Cannabis sativa (hemp) Rosales 12 49 39 NMR (22) Carpinus betulus (European hornbeam) Fagales 1 19 80 Py (34) Cenchrus purpureus (elephant grass) cortex Poales 3 40 57 NMR (35) Cercidiphyllum japonicum (katsura) Saxifragales 0 26 74 NB (31) Chamaecytisus prolifer (tagasaste) Fabales 0 38 62 Py (36) Cocos nucifera (coconut) coir fiber Arecales 4 78 18 NMR (37) Copernicia prunifera (carnauba) endocarp Arecales 1 64 31 NMR (25) Corchorus capsularis (jute) Malvales 0 33 67 NMR (38) Cynara cardunculus (cardoon) stalk Asterales 0 58 42 NMR (39) Cyperus papyrus (Nile grass) Poales 4 63 33 DFRC (27) Dendrocalamus brandisii (Burma bamboo) Poales 4 49 47 NMR (40) Dichorisandra thyrsiflora (blue ginger) Commelinales 1 38 61 DFRC (27) Elegia capensis (Horsetail restio) Poales 0 22 78 DFRC (27) Eucalyptus dunnii (dunns white gum) Myrtales tr 27 73 NMR (41) Eucalyptus globulus (Tasmanian bluegum) Myrtales tr 26 74 NMR (41) Eucalyptus grandis (flooded gum) Myrtales tr 37 63 NMR (41) Eucalyptus nitens (shining gum) Myrtales tr 27 73 NMR (41) Eucryphia cordifolia (ulmo) Oxalidales tr 24 76 NMR (42) Fagus crenata (Japanese beech) Fagales tr 75 25 TA (43) Fagus sylvatica (European beech) Fagales 0 25 75 CO (26) Festuca arundinacea (tall fescue) stem Poales 3 42 55 TA (44) Gevuina avellana (canelo) Proteales 0 19 80 CO (26) gardnerianum (ginger lily) 0 32 68 DFRC (27) Hibiscus cannabinus (kenaf) bast fibers Malvales 1 15 83 Py (45) Juncus inflexus (hard rush) Poales 10 28 62 DFRC (27) Laureliopsis philipiana (tepa) Laurales 1 25 74 CO (26) Linum usitatissimum (flax) fibers Malpighiales 15 71 14 NMR (46) Liquidambar styraciflua (sweet gum) Saxifragales 2 38 60 NMR (24) Liriodendron chinense (Chinese tulip poplar) Magnoliales tr 29 71 CO (47) Liriodendron tulipifera (tulip tree) Magnoliales 1 29 70 CO (47) Magnolia grandiflora (southern magnolia) Magnoliales 2 38 60 CO (47) Magnolia kobus (kobus magnolia) Magnoliales tr 30 70 CO (47) Magnolia x soulangeana (hybrid) Magnoliales 0 54 46 CO (47) 4

Medicago sativa (alfalfa) Fabales 8 54 38 Py (28) Miscanthus x giganteus (hybrid) Poales 4 52 44 NMR (48) Musa acuminata (banana) leaf Zingiberales 7 43 50 NMR (49) Musa textilis (abaca) Zingiberales 0 10 90 NMR (22) Nicotiana tabacum cv Samsun (tobacco) Solanales 0 50 50 TA (50) Nothofagus dombeyi (coihue) Fagales 8 15 77 CO (26) Olea europaea (olive) 1 51 48 NMR (51) Oryza sativa (rice) straw Poales 4 52 44 TA (43) Panicum virgatum (switchgrass) Poales 8 51 41 NMR (52) fortunei (paulonia) Lamiales tr 60 40 NMR (53) (paulonia imperial) Lamiales 0 42 58 NB (31) Phoenix canariensis (Canary Island date palm) Arecales 1 19 80 DFRC (27) Phormium tenax ( flax) Asparagales 0 30 70 CO (47) Phyllostachys edulis (green bamboo) culm Poales 6 54 40 NMR (54) Populus deltoides (cottonwood) Malpighiales 8 38 54 NMR (24) Populus maximowiczii (Alamo japones) Malpighiales 0 34 66 TA (29) Populus tremuloides (aspen) Malpighiales 1 33 66 NMR (55) Populus x canadensis (Canadian poplar) hybrid Malpighiales 0 32 68 NB (31) Posidonia australis (fibreball weed) Alismatales 7 57 36 NMR (56) Posidonia oceanica (Neptune grass) Alismatales 3 81 16 NMR (56) Quercus falcata (red oak) Fagales 2 31 67 NMR (24) Quercus robur (common oak) Fagales 0 40 60 CO (47) Quercus suber (cork oak) xylem Fagales 1 45 55 NMR (57) Sabal palmetto (cabbage palm) Arecales 4 55 41 CO (47) Saccharum sp (hybrid sugarcane) straw Poales 4 68 28 NMR (58) Sarcandra glabra (herba sarcandrae) Chloranthales 0 93 7 NB (59) Strelitzia reginae (bird of paradise) Zingiberales 0 47 53 DFRC (27) Tectona grandis (teak) heartwood Lamiales 2 55 43 Py (60) Tilia x europaea (hybrid) Malvales tr 33 67 CO (47) Triticum aestivum (bread wheat) straw Poales 14 50 36 CO (26) Triticum turgidum ssp durum (durum wheat) straw Poales 6 46 30 NMR (61) Trochodendron aralioides (wheel tree) Trochodendrales 0 38 62 NB (59) Typha orientalis (bulrush) Poales 0 47 53 DFRC (27) Ulmus americana (American elm) Rosales 0 28 72 NB (31) Ulmus procera (common elm) Rosales 0 43 57 CO (47) Viscum album (European mistletoe) Santalales 0 37 63 NB (31) Zea mays (maize) stem Poales 3 44 53 TA (62) Conifers: Abies alba (silver fir) Pinales 1 99 tr CO (47) Araucaria araucana (Chilean pine) Araucariales 2 97 1 TA (63) Araucaria heterophylla (Norfolk pine) Araucariales 7 93 tr TA (63) Cryptomeria japonica (Japanese cedar) Cupressales 1 99 0 TA (43) Cupressus sempervirens (Mediterranean cypress) Cupressales 0 100 0 TA (63) Juniperus communis (common juniper) Cupressales 2 98 tr CO (47) Larix decidua (European larch) Pinales 2 97 1 CO (47) Picea abies (Norway spruce) Pinales 0 100 0 NMR (64) Picea glauca (white spruce) Pinales tr 100 tr NB (32) Pinus halepensis (Aleppo pine) Pinales 0 100 0 TA (63) Pinus pinaster (maritime pine) Pinales 6 94 0 Py (65) Pinus radiata (Monterey pine) Pinales 1 99 0 NMR (55) Pinus sylvestris (Scots pine) Pinales 2 98 0 CO (26) Pinus taeda (loblolly pine) Pinales 1 99 0 NMR (66) 5

Pinus thunbergii (Japanese black pine) Pinales tr 100 tr TA (67) Platycladus orientalis (Chinese thuja) Cupressales 7 93 0 TA (63) Podocarpus macrophyllus (yew plum pine) Araucariales 0 100 tr NB (59) Prumnopitys andina (Lleuque) Araucariales tr 99 1 CO (47) Pseudotsuga menziesii (Douglas fir) Pinales 0 100 0 NMR (68) Sciadopitys verticillata (Japanese umbrella-pine) Cupressales 2 98 0 CO (47) Sequoia sempervirens (coastal redwood) Cupressales tr 98 2 CO (47) Taxodium distichum (bald cypress) Cupressales 10 90 0 CO (47) Taxus baccata (English yew) Cupressales 3 97 tr TA (63) Other gymnosperms: Cycas revoluta (sago palm, cycad) Cycadales 0 100 tr TA (63) Encephalartos lebomboensis (Lebombo cycad) Cycadales 12 88 tr CO (47) Ephedra sinica (Chinese ephedra) Ephedrales 10 44 46 NB (59) Ephedra viridis (green Mormon tea, ephedra) Ephedrales 1 39 60 TA (63) Ginkgo biloba (maidenhair tree) Ginkgoales 3 97 tr TA (69) Gnetum gnemon (melinjo; gnetum) Gnetales 1 44 55 NB (70) Gnetum sp (gnetum) Gnetales 13 62 25 CO (47) Zamia furfuracea (carboard palm, cycad) Cycadales 7 87 5 CO (47) Ferns (Polypodiopsida) and lycopodes: Asplenium scolopendrium (hart's-tongue fern) Polypodiales tr 100 0 TA (71) Ceratopteris cornuta (fern) Polypodiales tr 52 48 TA (71) Ceratopteris thalictroides (Indian fern) Polypodiales 0 100 0 TA (71) Cibotium barometz (golden chicken fern) Cyatheales tr 100 0 CO (47) Cyathea arborea (West Indian treefern) Cyatheales 1 99 0 CO (47) Dennstaedtia bipinnata (cuplet fern) Polypodiales 1 83 16 CO (47) Dicksonia squarrosa (rough tree fern) Cyatheales 1 99 0 CO (47) Dryopteris affinis (scaly male fern) Polypodiales 3 97 0 TA (71) Equisetum fluviatile (water horsetail) Equisetales 30 56 14 CO (47) Equisetum telmateia (giant horsetail) Equisetales 0 100 0 TA (71) Isoetes fluitans (lycophyte) Isoetales 0 100 0 TA (71) Isoetes histrix (land qillwort, lycophyte) Isoetales 0 73 27 TA (71) Palhinhaea cernua (staghorn clubmoss) Lycopodiales 1 99 0 CO (47) Phlegmariurus squarrosus (rock tassel-fern) Lycopodiales 7 93 0 CO (47) Pteridium aquilinum (bracken, fern) Polypodiales 3 97 0 TA (71) Pteris podophylla (fern) Polypodiales 1 99 0 CO (47) Selaginella martensii (Martens's spikemoss) Selaginellales 5 24 71 TA (63) Selaginella myosurus (Dongolongo, lycophyte) Selaginellales 12 75 13 CO (47) Selaginella willdenowii (Willdenow's spikemoss) Selaginellales 35 20 45 CO (47) Bryophytes: Conocephalum conicum (great scented liverwort) Marchantiales 29 44 27 CO (47) Marchantia polymorpha (common liverwort) Marchantiales 14 80 6 TA (71) Plagiochila asplenioides (liverwort) Jungermanniales 25 75 tr CO (47) Polytrichum commune (common haircap, moss) Polytrichales 52 48 tr CO (47) Algae: Calliarthron cheilosporioides (red alga) Corallinales 40 60 tr DFRC (72) Coleochaete nitellarum (green alga) Coleochaetales 0 83 17 TA (73) Coleochaete orbicularis (green alga) Coleochaetales 0 60 40 TA (73) Coleochaete scutata (green alga) Coleochaetales 0 53 47 TA (73) a H:G:S ratio estimated by CO, DFRC, NB, NMR, Py or TA methods. The NCBI database (www.ncbi.nlm.nih.gov/taxonomy) is followed for species and order names. Abbreviations: CO, cupric oxide alkaline degradation; DFRC, derivatization followed by reductive cleavage, NB, nitrobenzene oxidation; NMR, nuclear magnetic resonance; Py, analytical pyrolysis; TA, thioacidolysis; tr, traces 6

Table S2. Softwood and hardwood lignin decrease by ancestral (CaPo, CaD, AVPd and ALiP) and extant (PC-LiPA) peroxidases referred to enzyme-free control treatment, composition including sulfonated (A) and nonsulfonated β-O-4' (A), phenylcoumaran (B) and resinol (C) substructures and oxidized syringyl units (S'), and S/G ratio (only in hardwood lignin). From integration of 2D-NMR signals in Figs 4 and S4. Control CaPo CaD AVPd ALiP PC-LiPA Softwood lignin Decrease a - 8% 13% 14% 25% 19% Composition: b - β-O-4' (A) 32 30 30 28 29 29 - β-O-4' (A) 3 3 3 3 3 3 - Phenylcoumaran (B) 4 4 4 4 4 4 - Resinol (C) 4 4 4 5 5 4 Hardwood lignin Decrease: a - G decrease - 17% 56% 76% 81% 64% - S decrease - 9% 39% 55% 59% 47% - Total - 11% 43% 61% 65% 51% Composition: b - β-O-4' (A) 20 20 20 18 19 19 - β-O-4' (A) 12 13 15 17 16 14 - Resinol (C) 10 11 10 9 9 8 - S' 3 6 10 11 10 10 S/G ratio 2.9 3.1 4.0 5.4 6.0 4.2 a From aromatic signals referred to the control (%).b Referred to 100 aromatic units in the sample.

Table S3. Distribution of plant host for the ten Polyporales analyzed (left) and number and types of peroxidases annotated in their genomes, including percentage of peroxidases with the surface catalytic tryptophan (right).a See Fig. 6 for general analysis of 64 genomes. Host (citations and %) Peroxidase type

Gymnosperms Angiosperms GP MnP VP LiP Surface Trp P. placenta 7 (78%) 2 (22%) 3 0 0 0 0 W. cocos 27 (59%) 19 (41%) 1 0 0 0 0 F. pinicola 126 (61%) 81 (39%) 2 0 0 0 0 C. subvermispora 8 (62%) 5 (38%) 1 13 1 1 13% D. squalens 46 (96%) 2 (4%) 0 9 3 0 25% Ganoderma sp. 2 (4%) 45 (96%) 0 6 2 0 25% T. versicolor 78 (17%) 376 (83%) 0 12 3 10 52% B. adusta 33 (16%) 176 (84%) 1 6 1 12 65% P. chrysosporium 0 5 (100%) 1 5 0 10 63% P. brevispora 3 (100%) 0 0 5 0 5 50% aFrom USDA Fungus-Host Distribution Database (https://nt.ars- grin.gov/fungaldatabases/fungushost/fungushost.cfm) and JGI Mycocosm (https://genome.jgi.doe.gov/mycocosm), respectively

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BJEAD-116816-LiP BJEAD-157924-LiP BJEAD-499082-LiP BJEAD-184697-LiP BJEAD-172246-LiP BJEAD-186344-LiP BJEAD-323280-LiP BJEAD-120002-LiP BJEAD-41982-LiP BJEAD-269524-LiP BJEAD-269481-LiP BJEAD-121664-LiP PHACH-2918661-LiP PHACH-2918435-LiP PHACH-1716776-LiP PHACH-1716042-LiP PHACH-2989894-LiP (178, 243-115) PHACH-3032425-LiP PHACH-2910310-LiP PHACH-3032409-LiP ALiP PHACH-3043032-LiP PHACH-1386770-LiP PHLBR-129470-LiP PHLBR-86283-LiP PHLBR-150253-LiP PHLBR-150901-LiP PHLBR-116561-LiP TRAVE-134250-LiP TRAVE-133326-LiP TRAVE-134657-LiP TRAVE-133918-LiP TRAVE-52333-LiP Clade D TRAVE-114944-LiP TRAVE-133731-LiP TRAVE-43578-LiP (194, 265-125) TRAVE-43576-LiP TRAVE-134226-LiP TRAVE-43289-VP AVPd GANSP-116056-VP TRAVE-51455-MnPshort TRAVE-51457-MnPshort TRAVE-130496-MnPshort TRAVE-131080-MnPshort TRAVE-51451-MnPshort TRAVE-74179-MnPshort TRAVE-51375-MnPshort DICSQ-50355-MnPshort DICSQ-169526-MnPshort GANSP-142454-MnPshort TRAVE-112835-MnPshort BJEAD-306863-MnPshort BJEAD-118718-MnPshort PHLBR-125598-MnPshort PHLBR-147108-MnPshort BJEAD-43095-VP BJEAD-306404-MnPshort GANSP-176788-MnPshort TRAVE-43477-MnPshort (259, 354-167) CERSU-124076-MnPshort CERSU-118677-LiP CaD CERSU-99382-VP BJEAD-173495-GP BJEAD-43329-MnPshort TRAVE-44897-MnPshort CERSU-139965-MnPlong CERSU-116608-MnPlong CERSU-157986-MnPlong CERSU-50686-MnPlong CERSU-114036-MnPlong CERSU-114076-MnPlong CERSU-105539-MnPlong DICSQ-169849-MnPlong DICSQ-169843-MnPlong DICSQ-70857-MnPlong DICSQ-59877-MnPlong CERSU-50297-MnPlong Clade C CERSU-143390-MnPlong CERSU-49863-MnPlong CERSU-94398-MnPlong CERSU-117436-MnPlong PHLBR-157664-MnPlong PHLBR-144001-MnPlong PHLBR-85963-MnPlong PHACH-2896529-MnPlong PHACH-8191-MnPlong PHACH-3118856-MnPlong PHACH-2907883-MnPlong PHACH-3589-MnPlong GANSP-176789-MnPshort GANSP-147340-MnPshort DICSQ-141471-MnPshort GANSP-161386-MnPshort BJEAD-175536-MnPshort BJEAD-172152-MnPshort GANSP-155330-MnPshort DICSQ-108150-MnPshort TRAVE-74595-MnPshort Clade B (399, 544-257) DICSQ-173638-VP DICSQ-147840-VP DICSQ-155734-VP CaPo GANSP-116446-VPatypical TRAVE-26239-VP TRAVE-28895-VPatypical PHACH-2911114-GP DICSQ-150431-MnPshort TRAVE-187228-MnPshort POSPL-50226-GP POSPL-134641-GP POSPL-44056-GP (631, 762-533) FOMPI-125354-GP FOMPI-281595-GP Clade A WOLCO-136670-GP CERSU-112162-GP STANO-8474-GP STANO-5910-GP (403, 538-276) STANO-4497-GP STANO-5940-GP STANO-6740-GP 531 485 443419 359 298 252 201 145 66 Today Cam Or Si Dev Car Per Tr Jr Cr Precambrian Paleozoic Mesozoic Cenozoic

Fig. S1. Ultrametric tree for calibration of the ancestral nodes in the present study. We used the phylogeny of Polyporales peroxidases rooted with S. nodorum GPs and the software PATHd8 with the time constraints (average and limits in mya) marked in bold. 8

Lignin· Lignin RS CII 3+ k B [Fe ] 3app [Fe3+ Trp·+]

H2O2 S·

S H2O

CIA CIIA [Fe4+=O P·+] 4+ S [Fe =O] S·

Lignin·

Lignin

CIB [Fe4+=O Trp·+]

Fig. S2. Catalytic cycle of ligninolytic peroxidases. The common cycle is initiated with the oxidation of the RS enzyme by H2O2 to form CI, and follows with two one-electron oxidations of substrate (S) leading to CII and RS, successively. The expanded cycle for VPs and LiPs is also shown, with the displacement of one oxidation equivalent to a solvent-exposed tryptophan able to attack nonphenolic lignin. k1app, k2app and k3app apparent second-order rate constants, analyzed with wood lignosulfonates in Table 1, are indicated.

9

30 A

25

20

)

1

- (s

obs 15 k

10

5

0 0 25 50 75 100 Softwood lignosulfonate (µM)

B 25 CaPo 20 CaD

AVPd

) 1 - ALiP

(s 15

PC-LiPA

obs k 10

5

0 0 25 50 75 100 Hardwood lignosulfonate (µM)

Fig. S3. Kinetics of CI to CII reduction in ancestral (CaPo, CaD, AVPd and ALiP) and extant (PC- LiPA) peroxidases by softwood (A) and hardwood (B) lignosulfonates. The lignosulfonates concentrations refer to the basis phenylpropanoid unit (260 and 290 Da for sulfonated softwood and hardwood lignins, respectively). Means and 95% confidence limits.

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Fig. S4. HSQC 2D-NMR spectra of softwood lignosulfonate treated for 24 h with ancestral peroxidases and extant PC-LiPA, and control without enzyme. The main structures identified are included. Signals correspond to 1H-13C correlations at different positions of Cα-sulfonated guaiacyl units (G), and Cα-sulfonated and non-sulfonated side chains in β‑O‑4′ (A and A) phenylcoumaran (B) and resinol (C) substructures, and methoxyls (MeO).

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