www.nature.com/scientificreports

OPEN The tree of life of polyamine oxidases Daniele Salvi1 & Paraskevi Tavladoraki2*

Polyamine oxidases (PAOs) are characterized by a broad variability in catalytic properties and subcellular localization, and impact key cellular processes in diverse organisms. In the present study, a comprehensive phylogenetic analysis was performed to understand the evolution of PAOs across the three domains of life and particularly within . Phylogenetic trees show that PAO-like sequences of bacteria, archaea, and eukaryotes form three distinct clades, with the exception of a few procaryotes that probably acquired a PAO through horizontal transfer from a eukaryotic donor. Results strongly support a common origin for archaeal PAO-like and eukaryotic PAOs, as well as a shared origin between PAOs and monoamine oxidases. Within eukaryotes, four main lineages were identifed that likely originated from an ancestral eukaryotic PAO before the split of the main superphyla, followed by specifc gene losses in each superphylum. PAOs show the highest diversity within eukaryotes and belong to three distinct clades that underwent to multiple events of gene duplication and gene loss. Peptide deletion along the evolution of plant PAOs of Clade I accounted for further diversifcation of function and subcellular localization. This study provides a reference for future structure–function studies and emphasizes the importance of extending comparisons among PAO subfamilies across multiple eukaryotic superphyla.

Polyamines are small organic polycations with primary and secondary amino groups. Tey are widely present in all organisms and are involved in a variety of biological processes. Te relative abundance of the diferent polyamines depends on species and varies in a tissue-specifc way. Te most common polyamines are the diamine putrescine (Put), triamine (Spd), and tetraamine (Spm), although a large number of algal, fungal and bacterial species do not contain Spm­ 1–3. In some organisms a wider variety of polyamines has been observed, such as 1,3-diaminopropane (Dap), cadaverine, agmatine, thermospermine (T-Spm), norspermine (Nor-Spm), norspermidine, homospermidine, various long-chain and branched polyamines, as well as conjugated forms and acetylated derivatives of polyamines­ 4–9. Polyamine biosynthetic pathway is an ancient and well conserved metabolic pathway. Most eukaryotes syn- thesize Put from ornithine through ornithine decarboxylase (ODC), whereas in and bacteria there is an additional pathway to Put which involves arginine decarboxylase (ADC). It has been suggested that eukaryotic ODC was inherited from an α-proteobacterial ODC progenitor and that the ADC pathway has been acquired by plants from the cyanobacterial precursor of the chloroplast­ 10. Transfer of an aminopropyl group to Put by sper- midine synthase (SPDS) results to the production of Spd, while spermine synthase (SPMS) and (TSPMS) incorporate a new aminopropyl group at the N8-(aminobutyl)- and N1-(aminopropyl)-end of Spd to synthesize Spm and T-Spm, respectively. Additional triamines, tetramines, as well as long-chain and branched-chain polyamines may be also formed by transfer of aminopropyl or aminobutyl groups to diferent polyamines­ 8,11–15. Phylogenetic studies have suggested that the SPDS of the various organisms derive from a common ancestor preceding the separation between prokaryotes and eukaryotes and that they have been the origin of SPMS and TSPMS activities through gene duplication and/or ­neofunctionalization16. Moreover, it has been hypothesized that plants acquired TSPMS early during evolution by horizontal gene transfer from archaea or ­bacteria16–18. Polyamine oxidases (PAOs) have an important role in polyamine metabolism and contribute to several physiological processes through regulation of polyamine levels and reaction products. PAOs are characterized by a broad variability in specifcity, catalytic mechanism and subcellular localization. Tey are FAD- dependent catalyzing the oxidation of the free, and/or acetylated form, of polyamines at the second- ary amino groups­ 19–21. In mammals, the peroxisomal PAOs (PAOXs) preferentially oxidize N1-acetyl-Spm, N1- acetyl-Spd, and N1,N12-bisacetyl-Spm through an exo-mode to produce Spd, Put, and N1-acetyl-Spd, respectively, 3,20,22 in addition to 3-acetamidopropanal and H­ 2O2 . Moreover, the mammalian spermine oxidases (SMOXs), which present cytosolic/nuclear localization, preferentially oxidize Spm to produce Spd, 3-aminopropanal and

1Department of Health, Life and Environmental Sciences, University of L’Aquila, 67100 L’Aquila, Italy. 2Department of Science, University ‘Roma Tre’, 00146 Rome, Italy. *email: [email protected]

Scientifc Reports | (2020) 10:17858 | https://doi.org/10.1038/s41598-020-74708-3 1 Vol.:(0123456789) www.nature.com/scientificreports/

19,23,24 ­H2O2 . Unlike PAOXs and SMOXs, Saccharomyces cerevisiae PAO (FMS1) catalyzes the oxidation of both acetylated and non-acetylated polyamines­ 25,26. In plants, the intracellular PAOs (e.g., the Arabidopsis thaliana AtPAO1 with a putative cytosolic localization and the three peroxisomal AtPAO2, AtPAO3, AtPAO4) preferentially oxidize the free form of Spd, Spm, T-Spm 27–32 or Nor-Spm to produce 3-aminopropanal, H­ 2O2 and Put or Spd­ . Te cytosolic AtPAO5 (which has a higher activity as dehydrogenase than as oxidase) and its rice orthologue oxidize also N1-acetyl-Spm9,32–34. In contrast to the intracellular and plant PAOs, the extracellular Zea mays PAO (ZmPAO1, previously ­ZmPAO35,36) and its orthologues in Oryza sativa, Avena sativa and Hordeum vulgare oxidize the carbon at the endo-side of 4 32,37 the N -nitrogen of the free forms of Spd and Spm with the production of Dap, H­ 2O2 and an ­aminoaldehyde . In some bacterial species, such as Pseudomonas aeruginosa, Citrobacter freundii and Serratia marcesens, Spd is oxidized by spermidine dehydrogenases with FAD and/or as prosthetic groups­ 38–40. Reaction products of these enzymes with Spd are Dap and 4-aminobutanal, indicating cleavage at the endo-side of the N4-nitrogen. Te P. aerugonosa (SpdH) oxidizes also Spm through an exo-mode producing Spd and 3-aminopropa- nal38–41. In several bacterial species, an FAD-dependent classifed as putrescine oxidase (PuO) is also present. PuO additionally catalyses Spd oxidation, though less efciently than Put ­oxidation42–45. However, being active at the primary amino group, this enzyme cannot be considered as a PAO. Unlike the well charac- terized polyamine catabolic pathways in eukaryotes and bacteria, nothing is known about polyamine catabo- lism in archaea which possess distinct polyamine biosynthetic pathways and produce long-chain and branched ­polyamines15,46,47. While several studies have been performed on the evolutionary pathways of genes involved in polyamine ­biosynthesis10,11,16–18,48, comparatively less is known about the evolutionary history of genes involved in polyamine catabolism. Most studies have focused on the genomic identifcation and biochemical characterization of PAO isoforms from single species and therefore they used phylogenetic methods mainly for delimiting clusters and subfamilies to which to assign PAO isoforms­ 49–51. Only a few studies considered a wide taxonomic representa- tion of PAO sequences in large groups of organisms and enabled the elucidation of evolutionary relationships among PAO subfamilies and the processes underlying their functional and structural diversity. For example, the phylogenetic analyses of animal PAOs clarifed that SMOX and PAOX subfamilies originate from a duplication event preceding the diversifcation of vertebrates and that subsequently SMOX and PAOX enzymes acquired dif- ferences in substrate specifcity through divergent evolution and functional specialization­ 52,53. Likewise, a recent phylogenetic study on plant PAOs identifed four main subfamilies and two main duplication events preceding angiosperm ­diversifcation54. However, it is still unclear whether all plant PAO gene subfamilies originate from a common ancestral gene along the lineage. Te same applies for metazoan PAO gene subfamilies, whereas phylogenetic studies on fungal PAO subfamilies have not been performed at all. At a more general level, the phylogenetic origin of the extensive diversity of eukaryotic PAOs and their evolutionary relationships with the few putative PAOs recently identifed in bacteria­ 55 are still unknown. In this study, a phylogenetic framework was developed to explore the relationships between eukaryotic PAOs and related proteins from bacteria and archaea, and to better understand the evolutionary root of eukaryotic PAO subfamilies, with a special focus on plants that show the highest PAO diversity. Analysis of gene structure and amino acid residues of the putative catalytic sites was also performed to better understand the evolutionary processes that led to functional diver- sifcation of PAO genes. Results and discussion Early origin of PAO‑like proteins within the three domains of life. To investigate the evolution of PAOs, we assembled, through extensive iterative sequence similarities searches, a set of 428 sequences from bac- teria, archaea, and diferent groups of eukaryotes including , amoebozoans, , excavates, fungi, , haptista, land plants, metazoans, , rhizarians, and stramenopiles (Supplementary Table S1). Searches targeting genomic data of Asgard archaea and , , and returned no hits (Supplementary Table S2). Phylogenetic analyses including 300 PAO and PAO-like sequences of bacteria, archaea, and eukaryotes showed three main clades (Fig. 1): the ‘Bacteria clade’ including proteins from bacteria (Bootstrap support, BS = 100), the ‘Archaea clade’ (BS = 100) including 13 archaeal PAO-like proteins, one bacterial putrescine oxi- dase (Rhodococcus erythropolis PuO, coded as Bat-Re) and two eukaryotic monoamine oxidases (Mus musculus MAO-A and MAO-B, coded as Mm-MmMAO-A and Mm-MmMAO-B), and the ‘Eukaryota clade’ (BS = 91) including all eukaryotic PAOs plus two small clades of bacterial (N = 7) and archaeal (N = 3) proteins. Terefore, besides a few exceptions, each of life has specifc PAO-like proteins that evolved from distinct ancestral proteins (Fig. 1). Proteins of the Archaea clade are recovered as sister to the Eukaryota clade (BS = 100; Fig. 1) using either the midpoint or the MAD rooting methods, thus suggesting a shared evolutionary history between archaeal PAO-like proteins and eukaryotic PAOs. Moreover, Archaeal PAO-like proteins show a close phylogenetic relationship with the mammalian Mm-MmMAO-A and Mm-MmMAO-B56 and the bacterial PuO­ 45, thus suggesting a common origin between PAOs and MAOs, likely from an oxidase carried by the common ancestor of archaea and ­eukaryotes57. A common origin between PAOs and MAOs is further supported by their signifcant structural similarity­ 35,58,59. While biochemical information for archaeal PAO-like sequences is not available, their close evolutionary relationship with eukaryotic MAOs and bacterial PuO raises the question of whether the archaeal PAO-like proteins have catalytic properties more similar to MAOs and PuO than to PAOs. Most of the bacterial PAOs are included in the well-supported Bacteria clade. Tis clade includes PAOs from beta- and gamma-proteobacterial species, as well as PAOs from some alpha- and epsilon-proteobacteria, acido- bacteria, actinobacteria and Deinococcus-Termus. Tese PAOs have a sequence identity ranging between 30

Scientifc Reports | (2020) 10:17858 | https://doi.org/10.1038/s41598-020-74708-3 2 Vol:.(1234567890) www.nature.com/scientificreports/

Pd P Pd Pd Pd Pd Pd Pm Pd Pm 3 Pm d 4 6 2 Pm 5 3 Pm - - c3 5 t - - 5 -

- C Pm Gm6 - - 6 Gm B At - C M Pr3

- P Zm Pd Os4 M -Sb a Pd - c Sb - - Ma -R 4 -Vv r6 - - M a4 - Os 3 - t3 -Zm Pd 4 4

S d - a4 M - Os3 -Ma bacteria Pd - 5 - Hv a 7 - Pd Vv5 b Pd P Gm Pd 6 Pd 3 Pm 2 Pm Sm Pd -Ca Pm t 2 2 Pm - Sm Pm Ct t - Pm l - -Pp1 Pd - - M 4 Pm - C t1 Gm3 Ca Pm b - C Cp Pd - 4 P 1 Gm5 Pl - P - t2 2 P h - a Pd - Hh -Gt2 b P V a - C 3 H P - Pd d Cc Hh - - s4 SD d M - P CR H - t y Pd C AMm - r 3 y E S Pd R s3 R - - Pd - 4 AM Ca1 P c Bc - a Pd - 2 y - P t4 Bp -Rc rchaea Pd Br5 t3 Bcy Pd - Bc -Ha -Oc - At Pd At Bg -Vc s1 - 3 Bg T r Pd Br lo - -Br 2 Bcy o C M - 2 C - Mi i B Cl lo 1 2 - r 3 Cs2 Mi - Dm 4 C Cv Ea a1 Ph Ct- - - Mac - -E a3 Mc Am c Ct Re E A - Ma - - Re 2 Mm DrABf A Re -XlA Cv Mm- -Me A t- -Pr2s1 M C -P Mm-Mmm Eq A SO Pi 2 - SO - Hs A -Ps 3 Ma A SO Mac SO -Pr 1 Mm -Xl A -Pr - S SO Mm - DrS SO -Pu 4 Me SO -Fc 2 Mm -Eq S D Pt Mm - S - h1 H S SD -D -M sS y Ald mS Fa y-Dh2 -Sm Fa -Ps Rc Ct Rp -G Fay - -Pop s Fay -Cp CR ay 2 -Gt31 F -Cb Ct- Fa -Cb1 Cos y Ct- Fa -Ss Ct Te Fay Pa -Ap - Ct- Fay -FMS1 Ct Cs -Cv31 Fay-Bf1 Ct-Mc Fa -Sc Pl-Sm Fa Zt Eukaryota clade 6 Fa- Pl-Sm5 -Af2 Pm- Fa Ma7 Fa-Ao Pm-Si -An4 Pm 5 Fa 3 -Zm4 Fa-Nf Pm-Sb -Nfu2 Pm 5 Fa 1 -Os1 Fa-Ac Pm-Hv -Cf 5 Fb 1 Pm-Bd2 Fb-Cn Pd-At5 Fb-Cn2 Pd-Br8 Fa-Ch Pd-Br7 Fa-Co -Cg Pd-Bj1 91 Fa Pd-Cs1 Fby-Ta Pd-Cs2 Fb-Gt1 Pd-Sl2 100 Fb-Um1 Pd-Sl3 Fb-Uh Pd-Sl4 Fb-Sr Pd-Vv7 Fby-Rg Pd-Pr1 Fby-Rt - 5 Fb-Ml1 Pd Cc F -Rc5 b-Ml2 Pd 10 Fb- Pd-Pt6 0 Pg Fa-Ff1 Pd-Gm8 RH- -Gm9 Pb2 Pd t4 Rp-P Pd-M SD op2 -Ca1 -To3 Pd 4 SD-To SD-Pt SD 2 -Ng -To Eh SD- 1 -Tt1 SD Fc1 ALc 4 -Fc c-Tt AR 2 AL -Tt2 A e-Ni3 ALc -ka Re- Rf ARe Ni1 -Cc A -Hn Rc Pu Re- - ARe Ng Rb -Ph A - Rb Re Na z-Rd AR -N F c1 A e- o -M Re N Fz -Hi A - s1 Fb 1 Re Ns An AR -Hv 2 - 1 A e- Fa -Aro 0 R N 0 e c Fa -Ag AR -N Fa -Tr A e- m Archaea clade a t M Re Ni F -T m - 2 Fa Mm - Ha -Te 3 M Fa Ba - mMAO -Fo 1 B t MmMAO-B p -Re Fa -Fo d Bp -Ye -V 1 B -A Fa p -Ha r Fa -Nh 2 B - Mus musculus o Bp p Cs Fa -F f2 -S PUTRESCINE OXIDASE -F B -S o Rodococcus Fa -Pt Bp p Fa Pn -Ah p Fa - B - -At 3 B p A Fa n Bp p -Eb s Fa - -A 2 B E B p -Yo n Fa -Nfu1Nf -Sf - o B p Fa -Mas B p -Cm r Fa f1 Bp p - Fa -A 2 Hd -A Bp - a f1 B - Pf l F An 2 Pp N Fa - N c B p - a - Bp p - Sp 1 F A Pd Bp A - - Pc Bp - Fa a - - Cv l d Bp Cn F Fa B - H Eukaryota Bp -H Cp Fa Sm1 - Bp p Ps1 - -Sm2- B - L T Pl t1 B - C Bp a s CC Pl Gm1 Bat d - NOR5 - Gm2 M 1 P c N l - - P Ca5 l1 Pm - - - Nt1 P m - M n - 2 P m Kv 1 Pm Rg Pd -St S Pm Pm m - Pd - Pm - Pd d P m s Pm S Pm Pm Archaea Cs5 P Pm - Pr v4 2 O Pd d - c1 t2 - Pp P - 1 Os 1 m - r 1 Os2 Pd Md V r1 d - 3 - Hv1 s P - - R P - S - Z Bacteria clade - c1 -

- v1 - Cc B v - Si1 6 At - - Si - Pd - Si Pt V 7 Pd - - Si b - Hv2 m1 Bd Ah Hv3 Ma - C Rc4 V - 1 Pd - v Pd - V - - 4 Pd d 3 Pd 2

2

Pd Pd d Pd P 1

1 Pd Pd Pd Bacteria Pd P

0.4

Figure 1. Maximum likelihood tree depicting the phylogenetic relationships between eukaryote PAOs and related PAO-like enzymes in bacteria and archaea; the tree is drawn to scale, with branch lengths measured in number of substitutions per site. Bootstrap support values (ultrafast bootstrap approximation) over 1000 replicates are reported in correspondence of the main nodes. Te analysis involved 300 amino acidic sequences (see Supplementary Table S1). Sequences are coloured according to the three main domains of life: eukaryote = green, archaea = blue, and bacteria = red. Te three main clades are named as follow: Eukaryota clade, Archaea clade, Bacteria clade; prokaryotic sequences clustering within the Eukaryota clade and non- archaean sequences clustering within the Archaea clade are indicated by black arrows.

to 75% with Pseudomonas aeruginosa spermidine dehydrogenase (SpdH) which oxidizes both Spd and Spm­ 40. On the other hand, a few PAOs of cyanobacteria (Bcy-Oc, Bcy-Ma, Bcy-Sy, and Bcy-Ca1), chlorobacteria (Bg-Rc and Bg-Ha), and of the proteobacterium Edwardsiella tarda (Bp-Et) form a monophyletic clade nested within the Eukaryota clade (Fig. 1), and in particular within the PAO Clade IV (Fig. 2). Tis latter clade includes vari- ous eukaryotic lineages (amoebozoans, criptista, green algae, haptista, land plants, , and stramenopiles), as well as a small clade of three archaeal proteins. Tis phylogenetic pattern suggests that the PAOs of these prokaryotes have been probably acquired through horizontal transfer from an eukaryotic ­lineage60. However, further data on bacterial and archaeal PAO diversity, including detailed biochemical information, are required to corroborate this preliminary hypothesis.

Four main clades of eukaryotic PAOs. PAO sequences of eukaryotes exhibit signifcant diversity. Phy- logenetic trees show that all eukaryotic PAOs evolved from a common ancestor (Fig. 1) and represent four main evolutionary lineages referred to as Clade I-IV (Fig. 2). Te four main clades of eukaryotic PAOs and their sublineages are strongly supported by bootstrap approximation and/or SH-like approximate likelihood ratio test values ≥ 90 (Fig. 2). Te midpoint rooting method recovered a close relationship between Clades I and II (plus a few sequences from stramenopiles, rhizarians and red algae), as well as between Clades III and IV (plus a small clade of stramenopiles); however, the placement of the root is not consistent between the midpoint and the MAD methods (result not shown).

Scientifc Reports | (2020) 10:17858 | https://doi.org/10.1038/s41598-020-74708-3 3 Vol.:(0123456789) www.nature.com/scientificreports/

Pd-At1 Pd-Br1 Pd-Cc2 Pd-Pt2 Pd-Rc1 Pd-Vv4 Pd-Md1 Pd-Pr2 Pd-Cs5 Pd-Sl1 Pd-St1 Pd-Nt1 100 Pd-Ca5 Pd-Mt1 100 Pd-Gm2 Pd-Gm1 Pp-Ps1 Pd-Cc1 Pd-Rc4 95 100 Pd-Pt1 Land Plants 94 100 Pd-Ah1 Pd-Vv2 Pd-Vv3 96 Pd-Vv1 ZmPAO1/AtPAO1-like Pm-Sb1 94 Pm-Zm1 Pm-Si1 Pm-Hv1 100 100 Pm-Os2 Pm-Os6 100 100 Pm-Os7 Pm-Si4 Pm-Si3 Pm-Si2 Pm-Hv2 94 Pm-Bd1 94 Pm-Hv3 Pm-Ma1 Pl-Sm2 Pl-Sm1 Fa-An1 Fa-Te Fa-Tt Fa-Tr Fa-Ag Fa-Aro 99 Fa-Ff2 Fa-Fo2 100 Fa-Nh1 Fa-Vd Fa-Fo1 Fa-Fo3 Fa-Pc Fa-Pd Fa-Ac2 Fa-Nf1 Fa-An2 Fungi Fa-Af1 100 100 Fa-Aso − 100 Fa-Nf2 100 C I Fa-Nfu1 Fa-An3 83 Fa-At 84 Fa-Ma 100 Fa-Pn Fa-Pt 100 Fb-Hi 100 Fz-Mc1 100 Fz-Rd 100 Rb-Ph 100 100 Rb-Pu 100 100 Rc-Cc 100 Rf-ka Red algae Mm-MmA Mm-HsA Mm-EqA 100 Mm-MeA Ma-XlA 100 Mac-DrA 79 Mm-MmS 76 Mm-HsS Mm-EqS 79 100 Mm-MeS 79 100 Mac-DrS 92 Ma-XlS Mc-Bf 92 Mi-Am 100 Mi-Dm 100 Mi-Phc Pd-Ca1 Pd-Mt4 Pd-Gm9 Pd-Gm8 Pd-Pr1 Pd-Cs1 Pd-Cs2 Pd-Vv7 Pd-Pt6 Pd-Rc5 Pd-Cc5 100 Pd-Sl4 100 Pd-Sl3 100 Pd-Sl2 100 Pd-Bj1 Pd-Br7 Land Plants Pd-Br8 99 Pd-At5 Pm-Bd2 100 Pm-Hv5 AtPAO5-like Pm-Os1 96 100 Pm-Sb5 98 Pm-Zm4 96 100 Pm-Si5 90 99 Pm-Ma7 − 100 Pl-Sm5 100 Pl-Sm6 90 C II 95 Ct-Cs1 95 Ct-Cv3 Ct-Mc 100 Ct-Ap Ct-Cos Green algae 87 100 Ct-Te 75 96 Rc-Gs 90 Rp-Pop1 ALd-Sm Red algae 100 ALc-Tt2 ALc-Tt4 99 100 ALc-Tt1 Alveolata 70 92 Eh-Ng SD-Pt4 Stramenopiles + SD-To3 SD-To2 99 SD-To1 100 SD-Fc1 Stramenopiles SD-Fc2 94 RH-Pb2 94 Rp-Pop2 Rhizaria + Red algae Fa-Ff1 Fb-Cf Fb-Cn1 Fb-Cn2 FaCh Fa-Co 95 Fa-Cg Fby-Ta 98 Fb-Gt1 Fb-Um1 Fb-Uh Fb-Sr 99 Fby-Rg 97 100 Fby-Rt Fb-Ml1 98 100 Fb-Ml2 100 Fb-Pg 91 Fay-FMS1 − Fay-Dh1 99 Fay-Dh2 Fungi C III Fay-Ps 100 Fay-Ct Fay-Cp 98 Fay-Cb2 Fay-Cb1 100 Fay-Ss Fay-Pa 100 Fa-Bf1 81 Fa-Sc 100 Fa-Zt 88 Fa-Af2 Fa-Ao Fa-An4 100 Fa-Nf3 100 Fa-Nfu2 Fa-Ac1 Pd-Br4 Pd-Br3 Pd-Br2 Pd-At2 Pd-At3 Pd-Br5 Pd-Pt3 Pd-Pt4 Pd-Rc2 Pd-Cs3 Pd-Pr4 Pd-Cs4 Pd-Gm5 Pd-Gm3 Pd-Ca3 Pd-Mt2 90 Pd-Ca2 Pd-Gm4 90 Pd-Cc3 90 Pd-Vv5 Pm-Hv4 81 Pm-Os3 100 Pm-Sb2 70 100 Pm-Ma2 82 Pm-Ma3 Pm-Ma4 96 Pl-Sm4 Land Plants 100 95 Pl-Sm3 95 Pp-Pp1 Pd-Gm7 Pd-Gm6 AtPAO2,3,4-like 100 Pd-Ca4 Pd-Mt3 100 Pd-At4 Pd-Br6 Pd-Cc4 100 Pd-Pt5 Pd-Rc3 100 Pd-Vv6 Pd-Pr3 97 Pm-Sb4 Pm-Zm3 98 Pm-Os4 Pm-Sb3 Pm-Zm2 Pm-Os5 Pm-Ma5 Pm-Ma6 83 SD-Pt1 96 80 CR-Gt2 Stramenopiles + Cryptista 97 88 RH-Pb1 99 79 AMm-Cp AMd-Va Rhizaria + 100 Hh-Ct5 70 − Hh-Ct6 CIV 100 Hh-Ct2 Haptista 100 Ct-Cs2 96 100 Ct-Cv1 88 Clo-Ts1 100 73 Clo-Vc Green algae 90 100 Clo-Cr 76 98 ARe-Ea1 ARe-Ea2 89 ARe-Ea3 Bp-Et Archaeobacteria Bcy-Ma Bcy-Sy Bcy-Ca1 98 Bg-Rc Bacteria Bg-Ha 95 Bcy-Oc SO-Pr2 SO-Ps1 SO-Pi SO-Ps2 100 SO-Pr3 99 100 SO-Pr1 SO-Pu Stramenopiles 98 100 SD-Fc4 100 SD-Pt2

0.4

Figure 2. Phylogeny of eukaryote PAOs based on the 253 amino acidic sequences of the Eukaryota clade of Fig. 1. Te tree shown was estimated with the Maximum likelihood method under the WAG + I + G model of amino acid replacement. Nodal support (> 70) is reported along the main branches: above, ultrafast bootstrap approximation (1000 replicates); below, SH-like approximate likelihood ratio test (1000 replicates). Main clades and eukaryote groups are indicated.

Scientifc Reports | (2020) 10:17858 | https://doi.org/10.1038/s41598-020-74708-3 4 Vol:.(1234567890) www.nature.com/scientificreports/

Clade I includes three main subclades, one represented by plant PAOs that have high similarity (33–93%) to A. thaliana PAO isoform 1 (AtPAO1) and Z. mays ZmPAO1, another one consisting of fungal PAOs (mainly from ascomycetes) and a third one, sister to the other two, consisting of red algae PAOs. Clade II includes three subclades: one composed of animal PAOs (including vertebrate SMOXs and PAOXs) that is sister to an sub-clade including land plant AtPAO5-like PAOs, green algae and red algae PAOs; a third subclade includes PAOs from alveolates, stramenopiles and excavata. Clade III consists exclusively of fungal PAOs grouped into two main subclades, both including yeast forms: one subclade is composed of ascomycete PAOs including S. cerevisiae ­FMS125 and the other is composed of basidiomycetes PAOs, including Ustilago maydis PAO (UmPAO; Fb-Um1 in Fig. 2)2. Clade IV includes one subclade of plant PAOs with high sequence similarity to the AtPAO2, AtPAO3 and AtPAO4 (AtPAO2,3,4-like PAOs), as well as PAOs from green algae and various (amoebozoans, cryptista, rhizarians and stramenopiles) and a few prokaryotic PAOs that have been discussed above. In addition to these four main clades, putative PAOs of stramenopiles form two small clades, one including diatom proteins and the other one including both diatom and proteins (Fig. 2). PAO-like sequences of these two stramenopile clades have sequence identity of 21–31% to AtPAO1-AtPAO5, MmSMOX, MmPAOX and FMS1. Te phylogenetic distribution of PAOs of each main clade in multiple eukaryotic superphyla and the lack of monophyly of PAO isoforms of plants (Clades I, II, and IV), fungi (Clades I and III), green algae (Clades II and IV), red algae (Clades I and II) and stramenopiles (various clades), suggest a birth-and-death scenario, with the origin of the main lineages arising from the ancestral eukaryotic PAO before the split of the main superphyla followed by specifc gene losses in each superphylum. According to this scenario PAO genes of Clade I would have been lost, for example, in animals and PAO genes of Clade II in fungi, whereas PAO of Clade III would have been retained only in fungi and those of Clade IV only in green plants and some protists. On the other hand, the low number of PAO-like sequences of protists available in the databases suggests caution with the interpretation of the absence of PAO genes, of one or more PAO lineages, in these groups. Te four unique groups of homologous PAO sequences identifed within the broad phylogenetic framework used in this study provide a crucial reference for future structure–function studies and emphasize the importance of extending the comparisons among PAO subfamilies across multiple eukaryotic superphyla. Tis is particularly true, for example, for the plant PAO subfamilies ZmPAO1/AtPAO1-like and AtPAO5-like that show a closer relationship to either fungal or animal PAOs rather than to the plant AtPAO2,3,4-like PAOs subfamily. Tis fnd- ing is in agreement with previous studies showing that Arabidopsis AtPAO5 is more similar to animal PAOXs/ SMOXs in terms of amino acid sequence (including amino acids of the catalytic site) and substrate specifcity (specifcity for N1-acetyl-Spm), than to plant AtPAO1-49. By analogy, the close phylogenetic relationships between plant ZmPAO1/AtPAO1-like proteins and ascomycete PAOs of Clade I provide directions for future comparative biochemical analyses and suggest that the available ZmPAO1 crystal structure may be a valuable resource for homology modelling and function prediction of related fungal proteins. In the following subsections, we discuss in detail phylogenetic relationships between and within eukaryotic PAO clades in conjunction with gene structure, subcellular localization, substrate specifcity and amino acid residues of the catalytic site.

Clade I: ZmPAO1/AtPAO1‑like PAOs of plants and fungi. Plant PAOs of Clade I (Fig. 2) have high amino acid sequence identity to each other (from 40 to 55%). A phylogenetic analysis based on an extended dataset, including 81 ZmPAO1/AtPAO1-like PAOs from bryophytes, lycopodiophytes, pinophytes, angiosperms (eudicots and monocots) and their sister lineage Amborella trichopoda (Fig. 3) shows that plant PAOs of Clade I belong to two distinct groups (Fig. 3, box a). One includes ZmPAO1-like PAOs characterized by an extracellular localization (possessing a N-terminal signal peptide for secretion to the apoplast) and an endo-mode of substrate oxidation; whereas the second includes AtPAO1-like PAOs characterized by putative cytosolic localization (lack- ing any known targeting sequence to a specifc subcellular compartment) and an exo-mode of substrate oxida- tion. ZmPAO1-like PAOs are widespread across Land Plants lineages (thought in eudicot angiosperms they are only present in a few species), whereas AtPAO1-like PAOs are exclusive of eudicot angiosperms (Fig. 3). Te phylogenetic tree indicates that bryophyte and lycopodiophyte ZmPAO1-like PAOs are sister to seed plant PAOs (BS = 100, node a, Fig. 3). Among the latter, two large clades are present, one including ZmPAO1-like isoforms of monocots, Vitales eudicots and the A. trichopoda Pa-Amt2 (Fig. 3; BS = 77, node c) and the other one being exclusively composed of AtPAO1-like isoforms of eudicots plus the A. trichopoda Pa-Amt1 (BS = 100, node f). Two smaller clades of extracellular PAOs were also recovered that have a close relationship with the AtPAO1- like clade (BS = 100, node b): one is composed of ZmPAO1-like isoforms found in some eudicots (BS = 98, node d), and the other of a third isoform of A. trichopoda (Pa-Amt3) and two PAOs from gymnosperms (BS = 98, node e). Overall, these results are consistent with an origin of the AtPAO1-like clade from a gene duplication event occurring on a ZmPAO1-like gene ancestor before angiosperm ­diversifcation54. Tis duplication would have been followed by AtPAO1-like gene extinction in monocots and ZmPAO1-like gene extinction in several eudicots. However, the close relationship between the AtPAO1-like clade and the two newly discovered clades including ZmPAO1-like PAOs found in some eudicots, Amborella (Pa-Amt3) and two gymnosperms (BS = 93, node b) suggests an additional duplication event, followed by a gene loss in monocots, that preceded the origin of the AtPAO1-like clade (see the scheme in Fig. 3, box a). Indeed, albeit phylogenetic relationships among the AtPAO1-like clade and these two small ZmPAO1-like clades are not well resolved, the latter two have a closer afnity with the AtPAO1-like clade than with the other ZmPAO1-like clades (node c), strongly indicating that a single duplication event does not explain well the phylogenetic diversity of plant PAOs of Clade I. Moreover, the addition of representatives of gymnosperm PAOs compared to previous studies suggests that duplication

Scientifc Reports | (2020) 10:17858 | https://doi.org/10.1038/s41598-020-74708-3 5 Vol.:(0123456789) www.nature.com/scientificreports/

pA Sp 100 Pd-Rc1 a) Pd-Jc1 pA Sp Eudicots - - X X Pd-Pt2 X AtPAO1-like Pd-Cc2 f Amborella Amt-1 - - Pd-Gb1 ? Gymnosperm 100 Pd-Gh1 GD b 100 Pd-Gh2 Gymnosperm + + Pd-Tc1

100 Pd-Mt1 X Amborella Amt-3 + + d-e 100 Pd-Mt5 GD a 100 Pd-Ca5 Eudicots + + 96 Pd-Pv1 Pd-Va1 100 Pd-Gm2 Eudicots + + ZmPAO1-like 100 Pd-Cs5 Monocots Pd-Cm1 c + + Amborella Amt-2 + + Pd-Vv4 ? Gymnosperm 100 Pd-Pr2 100 Pd-Pm1 Pd-Md1 Lycopodiophyta + + Pd-Mn1 Pd-Gm1 Bryophyta Eudicots + + 100 Pd-Eg2 Pd-Eg1 Pd-Cms1 100 Pd-Cms3 89 Pd-Cms2 81 Pd-At1 100 Pd-Es1 78 Pd-Br1 98 Pd-Bn2 100 Pd-Bn1 100 Pd-Th2 Pd-Th1 78 Pd-St1 100 Pd-Sl1 100 100 Pd-Cpa1 Pd-Nt1 100 Pd-Ns1 100 f Pd-Cfc1 Pd-Sei1 Pd-Ga1 Pd-Nn1 Pa-Amt1 100 Pp-Pg1 Amborellales e Pp-Ps1 Pinophyta b 98 Pa-Amt3 Amborellales 100 Pd-Cc1 93 Pd-Cts1 99 Pd-Pt1 95 Pd-Jc2 98 d 100 Pd-Jc3 Eudicots Pd-Rc4 100 Pd-Ah1 Pd-Bv1 95 Pd-Vv3 70 Pd-Vv2 Eudicots Pd-Vv1 100 99 Pm-Zm1 a 81 Pm-Sb1 97 Pm-Si1 Pm-Hv1 90 94 92 Pm-Os6 Pm-Os7 Pm-Os2 100 Pm-Hv2 Monocots 77 Pm-Ae1 99 100 Pm-Si2 Pm-Bd1 75 Pm-Hv3 91 Pm-Ma8 c Pm-Pd1 77 Pm-Ma1 Pa-Amt2 Amborellales Pl-Sm1 Pl-Sm2 Lycopodiophyta 100 93 Ph-Mp1 99 Ph-Mp2 77 Ph-Mp3 Bryophyta 100 Pb-Pp3 Pb-Pp2

0.3

Figure 3. Maximum likelihood phylogeny of plant PAOs of the ZmPAO1/AtPAO1-like clade (see Fig. 2) based on 81 amino acidic sequences. Main clades are labelled from a to f; bootstrap values of support (BS) are reported along the branches (BS > 70). Box (a) illustrates the evolutionary model for the AtPAO1-like proteins with two gene duplications (GD) in correspondence of node a and b (giving rise to the AtPAO1 subclade), and the loss of peptide A (pA) and of the signal peptide (sP) in correspondence of node f. Putative gene extinctions are indicated with ‘x’, whereas ‘?’ denote missing information on the Gymnosperm group.

events within this clade might have been even older than previously thought, likely before the diversifcation of seed plants. Comparative analysis of the available genomic and amino acid sequences showed three main diferences between ZmPAO1-like and AtPAO1-like PAOs of Clade I. All ZmPAO1-like PAOs, including PAOs of the early divergent land plants, pinophytes and two isoforms of Amborella (Pa-Amt2 and Pa-Amt3), share a common struc- ture with (i) 8 introns at highly conserved positions (plus an additional intron in the two Selaginella moellendorfi

Scientifc Reports | (2020) 10:17858 | https://doi.org/10.1038/s41598-020-74708-3 6 Vol:.(1234567890) www.nature.com/scientificreports/

PAOs and Pa-Amt3), (ii) a domain of 9 amino acids (peptide A, indicated as pA in Fig. 3, box a) close to Glu170 residue of ZmPAO1 catalytic site­ 36,58 (aa174-aa182; numbering of mature ZmPAO1; Supplementary Figure S1), and (iii) the previously mentioned signal peptide for extracellular localization (indicated as Sp in Fig. 3, box a). In contrast, all AtPAO1-like PAOs, including A. trichopoda Pa-Amt1, show an additional intron at a position corresponding to the highly conserved amino acid residue Glu173 (numbering of mature ZmPAO1) (Supple- mentary Figure S1) and lack the peptide A and a signal peptide (given their intracellular localization). Terefore, these two peptides were lost afer the gene duplication that gave rise to the AtPAO1-like clade (Fig. 3, box a). Te fungal PAO group of Clade I comprises mainly ascomycete PAOs with the single basidiomycete Fb-Hi PAO and the two zygomycete PAOs as sister to this clade (Fig. 2). Amino acid sequence identity between fungal and plant PAOs of Clade I is high (32–40%), including the amino acids of the catalytic site (see below Table 1). Most fungal PAOs (23 out of 29) have a predicted cleavable signal peptide suggesting extracellular localization; only three of them do not have typical features of the cleavable signal peptide in the N-terminal extension, whereas for the remaining three fungal PAOs only partial sequence data were available. Similarly to the extra- cellular ZmPAO1-like plant PAOs, the fungal PAOs possess a domain corresponding to the ZmPAO1 region aa174-aa182 (numbering of mature ZmPAO1), though with low sequence similarity. Analysis of the available genomic sequences revealed that the ascomycete PAOs share some common intron positions, which however are diferent from those of the AtPAO1-like and ZmPAO1-like plant PAO genes, as well from those of Fb-Hi PAO. Within Clade I, sister to plant and fungal PAOs, there is a sub-clade of four red algae PAOs (Fig. 2), that have amino acid sequence identity of 21–30% with plant and fungal PAOs. Two of the four red algae PAOs have a putative signal peptide for extracellular localization, while for the other two only partial sequence were available thus preventing the identifcation of a signal peptide. Furthermore, similarly to the fungal PAOs, the four red algae PAOs possess a domain corresponding to the characteristic ZmPAO1 region aa174-aa182. Te identifcation of a putative signal peptide for extracellular localization in both the red algae and the fungal PAOs, as well as in most groups of plant PAOs of Clade I (except in the derived clade AtPAO1-like proteins), suggests that the ancestral PAO of this clade was extracellular and that it appeared early in the evolution of the eukaryotes.

Clade II: AtPAO5‑like PAOs of plants and animal PAOs. Clade II includes the two reciprocally mono- phyletic sub-clades of animal PAOXs/SMOXs and of Archaeplastida AtPAO5-like PAOs (Fig. 2) with amino acid sequence identity among them in the range of 25–37%. Clade II also includes PAOs from two alveolates (Tetrahymena thermophila and Symbiodinium microadriaticum), a stramenopile (Phaeodactylum tricornutum) and an excavate heteroloboseans (Naegleria gruberi) which present amino acid sequence identity with plant and animal PAOs of Clade II in the range of 18–31%. In agreement with the detailed study on animal PAOs by Polticelli et al.52, vertebrate PAOs consist of two sub- families, SMOXs and PAOXs, with diferent substrate specifcity (free and acetylated form of Spm, respectively) and subcellular localization (cytosolic/nuclear and peroxisomal localization, respectively), probably derived from a duplication event followed by divergent evolution and functional specialization­ 52. A recent study deter- mined that the two PAO proteins of the cephalochordate amphioxus also show the same substrate specifcity as vertebrate SMOXs and PAOXs, suggesting that gene duplication and functional specialization predates the diversifcation of ­chordates61. As shown by a phylogenetic analysis based on an extended dataset (Fig. 4), phylogenetic relationships among plant PAO group of Clade II mirror the phylogenetic relationships among land plants and include three main sublineages, with the PAOs of Marchantia polymorpha, S. moellendorfi and Selaginella lepidophylla having a sister relationship to the clade formed by Amborella Pa-Amt6 and the groups of eudicot and monocot PAOs. Terefore, in contrasts to the vertebrate PAOs, AtPAO5-like PAOs comprise a relatively homogeneous group of proteins that have orthologous relationships, except a few plant species that have multiple copies as a result of recent gene duplications. Plant AtPAO5-like enzymes have cytosolic localization (all lacking a targeting sequence to a specifc subcellular compartment) and broad substrate specifcity. Indeed, AtPAO5 and the AtPAO5-like enzymes of O. sativa and S. lepidophylla are able to oxidize the two substrates of the animal SMOXs/PAOXs (Spm and acetylated Spm) in addition to T-Spm and Nor-Spm9,34,62,63. Furthermore, plant AtPAO5-like PAOs share a very simple gene structure with no intron­ 9, with the exceptions of PAO genes in Malus domestica and Tarenaya hassleriana (Pd-Md6 and Pd-T7, respectively) that have a single intron and in S. moellendorfi (Sl-Sm5 and Sl-Sm6) that have two introns. In contrast, animal PAO genes consist of 4 to 7 exons interspaced by 3 to 6 introns. Among animals, only Trichoplax adhaerens and Nematostella vectensis, representing early divergent animal phyla of and echinoderms, have intron-less PAOs. Tese data indicate that animal and plant PAOs of Clade II experienced a very diferent evolutionary history from their common ancestor.

Clade III: a fungal‑specifc PAO clade. Clade III PAOs are exclusively from fungi and include one clade of ascomycete PAOs and another of PAOs found predominantly in basidiomycetes and a few ascomycetes (Fig. 2). Both clades include also yeast forms, such as FMS1 (Fay-FMS1) and UmPAO (Fb-Um1). Sequence identity among the PAOs of the ascomycete and basidiomycete PAO clades is relatively low, ranging from 19 to 28%. Moreover, available genomic sequences showed signifcant diferences in gene structure between these two clades of fungal PAOs. In particular, ascomycete PAO genes possess from 2 to 4 introns at conserved positions, with the exception of yeast forms that have intron-less genes. Basidiomycete PAO genes have 1 to 3 introns at conserved positions, but diferent to the intron positions of ascomycete PAOs. Overall, the intron position of fungal PAOs of Clade III are diferent to those of PAOs of Clade I, II, and IV. Sequence analyses suggest intracel- lular localization of all fungal PAOs of Clade III in contrasts to the fungal PAOs of Clade I which have extracel- lular localization.

Scientifc Reports | (2020) 10:17858 | https://doi.org/10.1038/s41598-020-74708-3 7 Vol.:(0123456789) www.nature.com/scientificreports/

Clade E62 E170 Y298 K300 T402 F403 Y439

Land plant AtPAO1-like (42) A34, ­Q1, ­V7 E Y K S Y Y

Land plant ZmPAO1-like (36) E25, ­H1, ­I1, ­F3, ­S1, ­N2 E34, ­T2 Y35, ­F1 K S23, ­T14, ­C1, ­A1 F20, ­Y16 Y Clade I Fungi (29) Q13, ­E11, ­H4, ­S1 E27, ­G1, ­D1 Y K S F28, ­Y11 F18, ­Y11

Red algae (4) Q E Y K S3, ­A1 Y3, ­F1 Y3, ­F1

Land plant AtPAO5-like (35) H E12, ­Q20, ­H2, ­N1 V K S Y T Animal SMOX-like (38) H E T K S Y T

Animal PAOX-like (24) H E23, ­T1 N21, ­S1, ­C2 K S Y T

Clade II Animal PAOX/SMOX (12) H E11, ­A1 V8, ­A1, ­I1, ­T1, ­L1 K S10, ­A2 Y T

Green Algae (6) H Q5, ­T1 V K S Y T5, ­C1 Red Algae (2) H E V K S Y T

Alveolates, diatoms, Excavates (6) H ND G4, ­V1, ­A1 K S1, ­T1, ­A1, ­N2, ­G1 Y T

Clade III Fungi (36) H ND L K S21, ­A15 Y22, ­T14 C15, ­T6, ­S13, ­R1, ­M1

Land plant AtPAO2,3,4-like (102) H E E K S64, ­C37, ­A1 Y S83, ­T19

Green algae (4) H E2, ­Q2 Y2, ­L2 K S Y T Diatoms, amoebozoans cryptista, H4, ­V1 E L K S4, ­A1 Y4, ­F1 T3, ­S1, ­C1 Clade IV rhizarians (5) Haptista (3) H E V K A Y T

Bacteria (7) H6, ­Q1 E L K S6, ­A1 Y T

Archaea (3) H A2, ­S1 A K2, ­V1 A1, ­G1, ­S1 Y T2, ­A1

Archaea (9) G ND V K G7, ­C1, ­A1 Y F5, ­Y3, ­H1, Archaea Clade RePuO (1) S ND V K A Y H Mm-MmMAO (2) G ND V K C Y Y

Diatom Clade* Diatoms (5) Y ND M1, ­V1, ­S1, ­F1, ­L1 K A3, ­S1, ­V1 Y4, ­H1 T4, ­N1

Oomycetes (7) H E Y4, ­C3 K A4 Y4 A5 Stramenopile Clade Diatoms (2) H E Y K S1, ­A1 Y S

Table 1. Amino acid residues of the catalytic site of the various PAOs. Amino acid numbering refers to ZmPAO1 mature ­protein35. Numbers in parentheses indicate the number of PAOs analyzed. Subscript numbers indicate the number of PAOs sequence with each amino acid residue at the corresponding position. ND: not determined, due to the presence of gaps and regions of low . *SD-To1,2,3 and SD-Fc1,2.

Clade IV: AtPAO2,3,4‑like PAOs from plants, green algae and photosynthetic bacteria. Land plant PAOs of Clade IV have high sequence identity (55–93%) to AtPAO2, AtPAO3 and AtPAO4. Furthermore, within the Clade IV (Fig. 2), PAO-like sequences from amoebozoans, cryptista, green algae, haptista, rhizarians, stramenopiles (diatoms; SD-Pt1), and prokaryotes (archaea and bacteria) have sequence identity to AtPAO2, AtPAO3 and AtPAO4 in the range of 25–38%. Phylogenetic analysis based on an extended dataset (Fig. 5) showed that plant AtPAO2,3,4-like PAOs are widespread across main lineages of land plants including bryophytes, lycophytes, gymnosperms and angiosperms. Te phylogenetic tree further shows that angiosperm isoforms of Amborella, monocots and eudicots belong to two sister clades (BS = 100, node a; Fig. 5), one including AtPAO2,3-like PAOs (BS = 75, node c), and the other including AtPAO4-like PAOs (BS = 100, node b). Terefore, in keeping with previous studies­ 54, AtPAO2,3-like and AtPAO4-like PAOs arose through a gene duplication before the origin of angiosperms (Fig. 5, box a). Furthermore, within the AtPAO4-like clade, all Poales monocots have two PAO copies clustered into two sister clades, suggesting that an additional duplication event took place in the AtPAO4-like PAO of this lineage of monocots (Fig. 5, box a). AtPAO2,3,4-like PAO genes of land plants have highly conserved intron positions and upstream untranslated open reading frames­ 64 (uORFs). Of note, the AtPAO2 and AtPAO3 uORFs are more conserved with each other than with that of AtPAO464, which is consistent with phylogenetic results. All plant PAO proteins of Clade IV have a type I peroxisomal targeting signal (PTS1) at the carboxyl terminal and indeed peroxisomal localization has been shown for AtPAO2, AtPAO3, ­AtPAO428,29, as well as for OsPAO3, OsPAO4, and ­OsPAO531. Interest- ingly, AtPAO4-like PAOs of one of the two monocot groups bear a non-canonical, but functional, peroxisomal targeting signal at the carboxyl terminal (CRT)31. Despite shared subcellular localization AtPAO4-like PAOs and AtPAO2,3-like PAOs difer in catalytic properties. Indeed, while AtPAO2 is equally active with either Spm or Spd, AtPAO3 has greater activity with Spd and AtPAO4 with Spm­ 30. In a similar way, while OsPAO3 of the AtPAO2,3 group is active mainly with Spd, OsPAO4 and OsPAO5 of the AtPAO4 group is mainly active with Spm and T-Spm31. Whether these diferences refect distinct physiological roles is still unknown. Within Clade IV, the AtPAO2,3,4-like clade of land plant PAOs is closely related to the PAOs of green algae (chlorophytes and trebouxiophyceae). Te PAOs of green algae V. carteri and C. reinhardtii have a similar gene structure to each other sharing some intron positions, but diferent from those of the PAO genes of plants and of the other green algae (trebouxiophyceae) for which genomic sequences are available (e.g., Ct-Cv1 of Chlo- rella variabilis). Furthermore, C. reinhardtii PAO, but not the V. carteri PAO, has a PTS1 signal for peroxisomal

Scientifc Reports | (2020) 10:17858 | https://doi.org/10.1038/s41598-020-74708-3 8 Vol:.(1234567890) www.nature.com/scientificreports/

Pd-Rc5 77 Pd-Pt6 Pd-Jc6 100 Pd-Cts2 Pd-Cc5 100 Pd-Md7 100 Pd-Md6 Pd-Pr1 Pd-Tc3 Pd-Vv7 Pd-Eg4 100 Pd-Va2 100 Pd-Pv4 88 Pd-Gm9 98 100 Pd-Mt4 Pd-Ca1 Pd-Gm8 99 Pd-St9 100 Pd-Sl2 Pd-Cpa4 75 Pd-Nt4 97 Pd-St7 100 Pd-Nt5 92 100 Pd-St6 Eudicots Pd-Sl4 100 Pd-St8 100 Pd-Sl3 100 Pd-Nt6 94 Pd-Ns2 71 Pd-Cfc2 100 Pd-Sei6 Pd-Sei5 Pd-Cs1 Pd-Mn2 88 Pd-Cms8 100 Pd-Cms7 100 Pd-Cms9 Pd-Cr1 Pd-At5 Pd-Es5 100 100 Pd-Br7 71 100 Pd-Bj1 100 Pd-Rs5 100 Pd-Rs4 Pd-Br8 Pd-Th7 100 100 Pd-Th6 Pa-Amt6 Amborellales Pm-Hv5 Pm-Os1 100 Pm-Bd2 87 Pm-Zm4 Monocots 100 Pm-Sb5 Pm-Si5 Pm-Ma7 100 Pl-Sm6 100 Pl-Sm5 Lycopodiophyta 100 Pl-Sl1 Ph-Mp5 Bryophyta

0.4

Figure 4. Maximum likelihood phylogeny of plant PAOs of the AtPAO5-like clade (see Fig. 2) based on 59 amino acidic sequences. Bootstrap values of support (BS) are reported along the branches (BS > 70).

localization, though peroxisomal localization has still to be demonstrated. Te PAO gene of C. reinhardtii has also an uORF which however does not exhibit similarity to the uORF of the AtPAO2,3,4 plant PAO genes. Te close relationships of PAOs of many protists (amoebozoans, cryptista, haptista, rhizarians, and stramenopiles) with land plant PAOs of the AtPAO2,3,4-like clade allows identifcation of their common ancestors along the early diversifcation of eukaryotes, thus much earlier than during the diversifcation of streptophytes as suggested in previous studies­ 54.

Active site analysis of PAOs. Te amino acid residues Glu62, Glu170, Tyr298, Lys300, Phe403 and Tyr439 of ZmPAO1 (Clade I) are key amino acids of the catalytic site as shown by resolution of the crystal structure­ 58,65, molecular modelling studies and site-directed mutagenesis­ 36,66. Glu62 and Glu170 are located close to the cofac- tor FAD and residue Tyr298 is found in close proximity to Lys300, the ε-amino group of which is H-bonded through a water molecule with the N5 atom of FAD, an atom which participates in the catalytic ­mechanism58. Moreover, Phe403 and Tyr439 fank the catalytic tunnel on opposite sides and form a kind of ‘aromatic sand- wich’58. Sequence alignments of PAOs of Clades I to IV, as well as of Archaea, Diatom and Stramenopile clades showed that Lys300 residue of ZmPAO1 is strictly conserved in all PAOs (Table 1). Tis residue is also present in RePuO and Mm-MAOs (Table 1). Furthermore, except for some fungal PAOs, all PAOs analysed (and also RePuO, Mm-MmMAO-A and Mm-MmMAO-A) have an aromatic amino acid (either Phe or Tyr) at position Phe403 of ZmPAO1 (Table 1). Tyr439 is highly conserved in Clade I (including fungal PAOs) and is also pre- sent in the Archaea Clade, while it is substituted mostly by Tr or Ser residues in the PAOs of the other clades, with the exception of the oomycete proteins in which it is substituted by Ala. Tyr439 is also conserved in Mm- MmMAO-A and Mm MmMAO-B and substituted by His in RePuO (Table 1). Tese observations suggest that the ‘aromatic sandwich’ Phe403/Tyr439 is a particular characteristic of Clade I PAOs, as well as of the archaea PAO/MAO-like sequences. Glu62 is highly conserved in the ZmPAO1-like PAOs of monocots, Amborella (Pa-

Scientifc Reports | (2020) 10:17858 | https://doi.org/10.1038/s41598-020-74708-3 9 Vol.:(0123456789) www.nature.com/scientificreports/

a) Eudicots GD d Monocots b AtPAO4-like Monocots 100 Pd-Gm6 GD a Amborella Amt-4 100 Pd-Gm7 100 Pd-Pv3 Eudicots 100 c Pd-Mt3 Monocots AtPAO2,3-like Pd-Ca4 100 Amborella Amt-5 Pd-Md5 100 Pd-Md4 Pd-Pr3 94 Pd-Jc4 Gymnosperm 98 Pd-Rc3 Lycopodiophyta Pd-Pt5 Pd-Cc4 Bryophyta Pd-Vv6 100 Pd-St5 88 Pd-Sl6 Eudicots 100 Pd-Nt3 99 Pd-St4 100 Pd-Sl5 Pd-Cpa3 Pd-Sei4 Pd-Gh4 Pd-Eg3 100 100 Pd-Cms6 72 Pd-Cms5 Pd-At4 100 Pd-Es4 100 98 Pd-Rs3 Pd-Br6 Pd-Th5 100 Pm-Sb3 100 Pm-Zm2 Pm-Si7 100 Pm-Os5 Pm-Bd4 92 d 100 b 95 Pm-Zm3 Monocots 96 Pm-Sb4 100 99 Pm-Si6 Pm-Os4 100 Pm-Ma6 Pm-Ma5 Pa-Amt4 Amborellales 87 Pd-At2 97 Pd-Cms4 Pd-Es2 Pd-Br2 100 Pd-Br3 Pd-Rs1 98 Pd-Br4 100 Pd-Rs2 78 Pd-Br5 100 100 Pd-At3 Pd-Es3 99 Pd-Th3 Pd-Th4 97 Pd-Pt4 94 Pd-Pt3 Pd-Jc5 Pd-Rc2 100 Pd-Gm3 100 Pd-Gm5 a 93 Pd-Pv2 100 100 86 Pd-Mt2 Pd-Ca3 Eudicots 100 Pd-Gm4 Pd-Ca2 Pd-Cs4 100 Pd-Md2 100 Pd-Md3 Pd-Pr4 100 Pd-Cm2 Pd-Cs3 100 Pd-Gh3 Pd-Tc2 100 Pd-Vv5 Pd-Cc3 100 Pd-St3 100 Pd-Sl8 100 Pd-Nt2 100 Pd-St2 100 Pd-Sl7 85 98 Pd-Cpa2 Pd-Sei3 Pd-Sei2 77 Pm-Bd3 98 Pm-Hv4 c 100 Pm-Os3 99 Pm-Si8 75 100 Pm-Sb2 Monocots 100 Pm-Ma3 100 Pm-Ma2 Pm-Ma4 Pa-Amt5 Amborellales 86 Pp-Ac1 73 Pp-Pp1 Pinophyta Pp-Wn1 100 74 Pl-Sm3 Lycopodiophyta 86 Pl-Sm4 100 Pb-Pp4 91 Pb-Pp1 Bryophyta Ph-Mp4

0.2

Figure 5. Maximum likelihood phylogeny of plant PAOs of the AtPAO2,3,4-like clade (see Fig. 2) based on 100 amino acidic sequences. Main clades are labelled from a to c; bootstrap values of support (BS) are reported along the branches (BS > 70). Box (a) illustrates the evolutionary model for the AtPAO2,3,4-like proteins with two gene duplications (GD) in correspondence of node a (giving rise to the AtPAO2,3 and AtAPO4 subclades) and d.

Scientifc Reports | (2020) 10:17858 | https://doi.org/10.1038/s41598-020-74708-3 10 Vol:.(1234567890) www.nature.com/scientificreports/

Amt2 and Pa-Amt3), and the dicot Vitis vinifera (Pd-Vv1, Pd-Vv2, Pd-Vv3), but highly varies (Asn, Ile; Phe, His) in the extracellular PAOs present in some other dicots (node d, Fig. 3). It is also present in several fungal PAOs of Clade I, while it is substituted by an Ala residue in all AtPAO1-like PAOs and by a His residue in most of the other PAOs clades (Clades II, III, and IV, and stramenopile clades). Glu170 is also well conserved in PAO-like sequences of the various clades (including the bacterial and archaeal proteins of Clade IV, and of those of the stramenopile clades), with the exception of the land plant AtPAO5-like PAOs in which it is substituted by Gln. Furthermore, in some PAO-like sequences ZmPAO1 Glu170 residue is not well-defned (Table 1, ND) due to the presence of gaps and/or regions of low sequence homology. Ser402, which in the Mm-MmMAO-A and Mm MmMAO-B is substituted by a Cys residue involved in covalent binding to the isoalloxazine ring of the FAD, is also highly conserved (Table 1). Only the AtPAO4-like PAOs of Clade IV have a Cys residues at this position which, however, is not involved in covalent binding of the FAD­ 30. Unlike the other residues of the ZmPAO1 catalytic site, Tyr298 highly varies across the PAOs. In particular, while an aromatic residue is present at this position in the PAOs of Clade I, it is substituted by a Tr residue in vertebrate SMOXs, by a Val residue in the plant and algal PAOs of Clade II and some invertebrate PAOs (such as insect PAOs and the two PAOs from the amphioxus Branchiostoma foridae), and by a Asn residue in all vertebrate PAOXs­ 67. Further studies are neces- sary to understand whether these variations in amino acid residues of the PAO catalytic sites correlate to varia- tions in substrate specifcity. Conclusions Te tree of life of polyamine oxidases suggests a common origin for archaeal PAO-like proteins and eukary- otic PAOs, which probably also involved the evolution of monoamine oxidases. Within eukaryotes, four main clades of PAOs were identifed, likely originated from an ancestral eukaryotic PAO before the split of the main supergroups and followed by specifc gene losses in each supergroup. As a result, while some eukaryotes present a high diversity of PAO isoforms belonging to multiple clades (e.g. land plants and stramenopiles), some others have PAOs belonging to one (animals) or two clades (e.g. fungi and green algae). Within each of these clades, phylogenetic patterns revealed that PAOs have undergone several diversifcation events. Evolution of Clade I and Clade IV is shaped by multiple gene duplications. Conversely, only a few gene duplication events occurred within Clade II and Clade III. Clade I PAOs have additionally experienced peptide deletion leading to functional changes and diversifcation in subcellular localization. Te latter has been a pervasive process along eukaryotic PAO evolution, most organisms having PAOs in two or three diferent subcellular compartments (extracellular space, cytosol and peroxisomes), which suggests diferent physiological roles. Te large variety of PAOs analysed in the present study may facilitate structure–function studies. Methods Protein sequence homology search and retrieval. Te amino acid sequence of PAOs were retrieved by sequence similarity searches using BLASTP­ 68 (NCBI, Uniprot and Phytozome databases) and TBLASTN (NCBI TSA database). As query sequences the amino acid sequence of the following PAOs, for which enzymatic activity had been previously verifed, were used: Arabidopsis thaliana AtPAO1, AtPAO2, AtPAO3, AtPAO4, ­AtPAO59,27,30, Zea mays ­ZmPAO135, Pseudomonas aeruginosa ­SpdH40, Saccharomyces cerevisiae ­FMS125, Usti- lago maydis UmPAO­ 2, Mus musculus ­SMOX24 and M. musculus PAOX­ 23. Following an initial search on the entire databases, several lineages were not represented in the dataset. To further assess the presence of PAO-like proteins in these lineages, we repeated the search using the same query sequences and specifcally targeting, for each eukaryotic super-group, those species for which genomic resources were available (Supple- mentary Table S1). Among retrieved sequences, we selected those having a sequence identity with the query sequence ≥ 20%, a coverage ≥ 60% and an E-value ≤ 1e−6. Selected sequences were further validated based on sequence length (selecting sequences in the range of 400–650 amino acids), annotation of protein function, and the presence of particular domains. FAD-dependent PAO-like sequences with SWIRM domains, which are involved in histone oxidative ­demethylation69 rather than in polyamine metabolism, were excluded.

Sequence Analysis. Subcellular localization was inferred based on amino acid sequences using PSORT and SignalP. Genomic exon–intron structure comparison was performed by means of alignment between genomic and cDNA sequences. Amino acid residues of catalytic site were retrieved by multiple sequence alignments per- formed using Clustal Omega 1.2.170 and based on ZmPAO1 and Fms1 crystal ­structure26,58,65,66.

Phylogenetic analysis. Multiple amino acid sequence alignments were performed using Clustal Omega 1.2.1. On large data sets, Clustal Omega outperforms other packages in terms of execution time and ­quality70. Multiple sequence alignments were not trimmed. Phylogenetic analyses of the amino acid sequences were performed using the Maximum Likelihood (ML) method on fve distinct datasets for a total of 428 PAO-like sequences (see Supplementary Table S1). Multiple sequence alignments and phylogenetic trees are provided in Supplementary Data. Te frst dataset included 300 sequences of Bacteria (37), Archaea (16), and Eukaryotes (247). Subsequently, ML analyses were performed on the monophyletic group including all eukaryotic PAOs and a few prokaryotic PAOs (253 sequences) based on a new alignment. Additionally, to increase the taxonomic representation, we built, through additional similarity searches, three extended datasets of land plant PAOs for each of the AtPAO1-like (Clade I), AtPAO5-like (Clade II), and AtPAO2,3,4-like (Clade IV) clades, and we made new alignments for each of these clades. Phylogenetic trees were rooted using the midpoint method, which is a valuable method when a proper outgroup is not available or difcult to ­identify71. Additionally, we tested the root position using the Minimal Ancestor Deviation (MAD) method, that has been shown to outperform

Scientifc Reports | (2020) 10:17858 | https://doi.org/10.1038/s41598-020-74708-3 11 Vol.:(0123456789) www.nature.com/scientificreports/

existing ­methods72. Details on numbers of taxa, sites and informative sites are reported for each alignment in Supplementary Table S3. For each dataset the best-ft model of amino acid replacement was selected by ModelTest-NG 0.1.573, using an optimize Maximum-Likelihood topology and branch lengths for each model (-t ml) and the Akaike Information Criterion (AIC). Te WAG model­ 74 with gamma distributed rates across site (+ G) and a proportion of invari- ant sites (+ I) was selected for the eukaryote dataset, and the JTT model­ 75 with gamma distributed rates and a proportion of invariant sites (+ I) was selected for each plant PAO dataset. ML tree searches were performed with IQ-tree76 (for dataset larger than 200 sequences) and PhyML 3.077 (for dataset smaller than 100 sequences) using the best-ft model and 100 random starting trees. Node support for the resulting phylogenetic tree was evalu- ated by 1000 bootstrap replicates in IQ-tree (using both the ultrafast bootstrap approximation and the SH-like approximate likelihood ratio test) and by 100 bootstrap replicates in PhyML. Phylogenetic analyses were carried out on the T-REX ­webserver78 and the CIPRES Science Gateway 3.379 (at https​://www.phylo​.org/). Code availability GenBank accession numbers of all sequences used in this study are reported in Supplementary Table S1.

Received: 6 February 2020; Accepted: 29 September 2020

References 1. Hamana, K. & Matsuzaki, S. Widespread occurrence of norspermidine and norspermine in eukaryotic algae. J. Biochem. 91, 1321–1328 (1982). 2. Valdés-Santiago, L., Guzmán-de-Peña, D. & Ruiz-Herrera, J. Life without putrescine: disruption of the gene-encoding polyamine oxidase in Ustilago maydis odc mutants. FEMS Yeast Res. 10, 928–940 (2010). 3. Tavladoraki, P. et al. Polyamine catabolism: target for antiproliferative therapies in animals and stress tolerance strategies in plants. Amino Acids 42, 411–426 (2012). 4. Hamana, K., Niitsu, M., Samejima, K. & Matsuzaki, S. Linear and branched pentaamines, hexaamines and heptaamines in seeds of Vicia sativa. Phytochemistry 30, 3319–3322 (1991). 5. Casero, R. A. Jr. & Pegg, A. E. Spermidine/spermine N1-acetyltransferase-the turning point in polyamine metabolism. FASEB J. 7, 653–661 (1993). 6. Grienenberger, E. et al. A BAHD acyltransferase is expressed in the tapetum of Arabidopsis anthers and is involved in the synthesis of hydroxycinnamoyl spermidines. Plant J. 58, 246–259 (2009). 7. Morimoto, N. et al. Dual biosynthesis pathway for longer-chain polyamines in the hyperthermophilic archaeon Termococcus kodakarensis. J. Bacteriol. 192, 4991–5001 (2010). 8. Michael, A. J. Molecular machines encoded by bacterially-derived multi-domain gene fusions that potentially synthesize, N-meth- ylate and transfer long chain polyamines in diatoms. FEBS Lett. 585, 2627–2634 (2011). 9. Ahou, A. et al. A plant spermine oxidase/dehydrogenase regulated by the proteasome and polyamines. J. Exp. Bot. 65, 1585–1603 (2014). 10. Illingworth, C. et al. Te diverse bacterial origins of the Arabidopsis polyamine biosynthetic pathway. FEBS Lett. 549, 26–30 (2003). 11. Ober, D. & Hartmann, T. , the frst pathway-specifc enzyme of pyrrolizidine alkaloid biosynthesis, evolved from . Proc. Natl. Acad. Sci. USA 96, 14777–14782 (1999). 12. Ohnuma, M. et al. N1-aminopropylagmatine, a new polyamine produced as a key intermediate in polyamine biosynthesis of an extreme thermophile, Termus thermophilus. J. Biol. Chem. 280, 30073–30082 (2005). 13. Cacciapuoti, G. et al. Te frst agmatine/cadaverine aminopropyl : biochemical and structural characterization of an enzyme involved in polyamine biosynthesis in the hyperthermophilic archaeon Pyrococcus furiosus. J. Bacteriol. 189, 6057–6067 (2007). 14. Knott, J. M. Biosynthesis of long-chain polyamines by crenarchaeal polyamine synthases from Hyperthermus butylicus and Pyro- baculum aerophilum. FEBS Lett. 583, 3519–3524 (2009). 15. Okada, K. et al. Identifcation of a novel aminopropyltransferase involved in the synthesis of branched-chain polyamines in hyperthermophiles. J. Bacteriol. 196, 1866–1876 (2014). 16. Minguet, E. G. et al. Evolutionary diversifcation in polyamine biosynthesis. Mol. Biol. Evol. 25, 2119–2128 (2008). 17. Fuell, C., Elliott, K. A., Hanfrey, C. C., Franceschetti, M. & Michael, A. J. Polyamine biosynthetic diversity in plants and algae. Plant Physiol. Biochem. 48, 513–520 (2010). 18. Solé-Gil, A., Hernández-García, J., López-Gresa, M. P., Blázquez, M. A. & Agustí, J. Conservation of thermospermine synthase activity in vascular and non-vascular plants. Front. Plant Sci. 10, 663. https​://doi.org/10.3389/fpls.2019.00663​ (2019). 19. Wang, Y. et al. Cloning and characterization of a human polyamine oxidase that is inducible by polyamine analogue exposure. Cancer Res. 61, 5370–5373 (2001). 20. Wu, T., Yankovskaya, V. & McIntire, W. S. Cloning, sequencing, and heterologous expression of the murine peroxisomal favopro- tein, ­N1-acetylated polyamine oxidase. J. Biol. Chem. 278, 20514–20525 (2003). 21. Cona, A., Rea, G., Angelini, R., Federico, R. & Tavladoraki, P. Functions of amine oxidases in plant development and defence. Trends Plant Sci. 11, 80–88 (2006). 22. Vujcic, S., Liang, P., Diegelman, P., Kramer, D. L. & Porter, C. W. Genomic identifcation and biochemical characterization of the mammalian polyamine oxidase involved in polyamine back-conversion. Biochem. J. 370, 19–28 (2003). 23. Vujcic, S., Diegelman, P., Bacchi, C. J., Kramer, D. L. & Porter, C. W. Identifcation and characterization of a novel favin-containing spermine oxidase of mammalian cell origin. Biochem. J. 367, 665–675 (2002). 24. Cervelli, M., Polticelli, F., Federico, R. & Mariottini, P. Heterologous expression and characterization of mouse spermine oxidase. J. Biol. Chem. 278, 5271–5276 (2003). 25. Landry, J. & Sternglanz, R. Yeast Fms1 is a FAD-utilizing polyamine oxidase. Biochem. Biophys. Res. Commun. 303, 771–776 (2003). 26. Huang, Q., Liu, Q. & Hao, Q. Crystal structures of Fms1 and its complex with spermine reveal substrate specifcity. J. Mol. Biol. 348, 951–959 (2005). 27. Tavladoraki, P. et al. Heterologous expression and biochemical characterization of a polyamine oxidase from Arabidopsis involved in polyamine back conversion. Plant Physiol. 141, 1519–1532 (2006). 28. Moschou, P. N. et al. Bridging the gap between plant and mammalian polyamine catabolism: a novel peroxisomal polyamine oxidase responsible for a full back-conversion pathway in Arabidopsis. Plant Physiol. 147, 1845–1857 (2008). 29. Kamada-Nobusada, T., Hayashi, M., Fukazawa, M., Sakakibara, H. & Nishimura, M. A putative peroxisomal polyamine oxidase, AtPAO4, is involved in polyamine catabolism in Arabidopsis thaliana. Plant Cell Physiol. 49, 1272–1282 (2008). 30. Fincato, P. et al. Functional diversity inside the Arabidopsis polyamine oxidase gene family. J. Exp. Bot. 62, 1155–1168 (2011).

Scientifc Reports | (2020) 10:17858 | https://doi.org/10.1038/s41598-020-74708-3 12 Vol:.(1234567890) www.nature.com/scientificreports/

31. Ono, Y. et al. Constitutively and highly expressed Oryza sativa polyamine oxidases localize in peroxisomes and catalyze polyamine back conversion. Amino Acids 42, 867–876 (2012). 32. Tavladoraki, P., Cona, A. & Angelini, R. Copper-containing amine oxidases and FAD-dependent polyamine oxidases are key players in plant tissue diferentiation and organ development. Front. Plant Sci. 7, 824. https​://doi.org/10.3389/fpls.2016.00824​ (2016). 33. Kim, D. W. et al. Polyamine oxidase 5 regulates Arabidopsis thaliana growth through a thermospermine oxidase activity. Plant Physiol. 165, 1575–1590 (2014). 34. Liu, T., Kim, D. W., Niitsu, M., Berberich, T. & Kusano, T. Oryza sativa polyamine oxidase 1 back-converts tetraamines, spermine and thermospermine, to spermidine. Plant Cell Rep. 33, 143–151 (2014). 35. Tavladoraki, P. et al. Maize polyamine oxidase: primary structure from protein and cDNA sequencing. FEBS Lett. 426, 62–66 (1998). 36. Polticelli, F. et al. Lys300 plays a major role in the catalytic mechanism of maize polyamine oxidase. 44, 16108–16120 (2005). 37. Liu, T. et al. Polyamine oxidase 7 is a terminal catabolism-type enzyme in Oryza sativa and is specifcally expressed in anthers. Plant Cell Physiol. 55, 1110–1122 (2014). 38. Tabor, C. W. & Kellogg, P. D. Identifcation of favin dinucleotide and heme in a homogeneous spermidine dehydrogenase from Serratia marcescens. J. Biol. Chem. 245, 5424–5433 (1970). 39. Hisano, T. et al. Characterization of membrane-bound spermidine dehydrogenase of Citrobacter freundii. Biosci. Biotechnol. Biochem. 56, 1916–1920 (1992). 40. Dasu, V. V., Nakada, Y., Ohnishi-Kameyama, M., Kimura, K. & Itoh, Y. Characterization and a role of Pseudomonas aeruginosa spermidine dehydrogenase in polyamine catabolism. 152, 2265–2272 (2006). 41. Okada, M., Kawashima, S. & Imahori, K. Substrate binding characteristics of the of spermidine dehydrogenase from Serratia marcescens. J. Biochem. 85, 1235–1243 (1979). 42. van Hellemond, E. W., van Dijk, M., Heuts, D. P., Janssen, D. B. & Fraaije, M. W. Discovery and characterization of a putrescine oxidase from Rhodococcus erythropolis NCIMB 11540. Appl. Microbiol. Biotechnol. 78, 455–463 (2008). 43. Kopacz, M. M., Rovida, S., van Duijn, E., Fraaije, M. W. & Mattevi, A. Structure-based redesign of binding in putrescine oxidase. Biochemistry 50, 4209–4217 (2011). 44. Foster, A., Barnes, N., Speight, R. & Keane, M. A. Genomic organisation, activity and distribution analysis of the microbial putres- cine oxidase degradation pathway. Syst. Appl. Microbiol. 36, 457–466 (2013). 45. Kopacz, M. M., Heuts, D. P. & Fraaije, M. W. Kinetic mechanism of putrescine oxidase from Rhodococcus erythropolis. FEBS J. 281, 4384–4393 (2014). 46. Michael, A. J. Polyamines in eukaryotes, bacteria, and archaea. J. Biol. Chem. 291, 14896–14903 (2016). 47. Michael, A. J. Polyamine function in archaea and bacteria. J. Biol. Chem. 293, 18693–18701 (2018). 48. Shaw, F. L. et al. Evolution and multifarious horizontal transfer of an alternative biosynthetic pathway for the alternative polyamine sym-homospermidine. J. Biol. Chem. 285, 14711–14723 (2010). 49. Kitashiba, H., Honda, C. & Moriguchi, T. Identifcation of polyamine oxidase genes from apple and expression analysis during fruit development and cell growth. Plant Biotechnol. 23, 425–429 (2006). 50. Takahashi, Y. et al. Characterization of fve polyamine oxidase isoforms in Arabidopsis thaliana. Plant Cell Rep. 29, 955–965 (2010). 51. Wang, W. & Liu, J. Genome-wide identifcation and expression analysis of the polyamine oxidase gene family in sweet orange (Citrus sinensis). Gene 555, 421–429 (2015). 52. Polticelli, F. et al. Molecular evolution of the polyamine oxidase gene family in Metazoa. BMC Evol. Biol. 12, 90. https​://doi. org/10.1186/1471-2148-12-90 (2012). 53. Cervelli, M., Salvi, D., Polticelli, F., Amendola, R. & Mariottini, P. Structure–function relationships in the evolutionary framework of spermine oxidase. J. Mol. Evol. 76, 365–370 (2013). 54. Bordenave, C. D., Granados Mendoza, C., Jiménez Bremont, J. F., Gárriz, A. & Rodríguez, A. A. Defning novel plant polyamine oxidase subfamilies through molecular modeling and sequence analysis. BMC Evol. Biol. 19, 28. https​://doi.org/10.1186/s1286​ 2-019-1361-z (2019). 55. Samasil, K., Lopes de Carvalho, L., Mäenpää, P., Salminen, T. A. & Incharoensakdi, A. Biochemical characterization and homology modelling of polyamine oxidase from cyanobacterium Synechocystis sp. PCC 6803. Plant Physiol. Biochem. 119, 159–169 (2017). 56. Edmondson, D. E., Binda, C., Wang, J., Upadhyay, A. K. & Mattevi, A. Molecular and mechanistic properties of the membrane- bound mitochondrial monoamine oxidases. Biochemistry 48, 4220–4230 (2009). 57. Gribaldo, S., Poole, A. M., Daubin, V., Forterre, P. & Brochier-Armanet, C. Te origin of eukaryotes and their relationship with the Archaea: are we at a phylogenomic impasse?. Nat. Rev. Microbiol. 8, 743–752 (2010). 58. Binda, C. et al. A 30-angstrom-long U-shaped catalytic tunnel in the crystal structure of polyamine oxidase. Structure 7, 265–276 (1999). 59. Binda, C., Mattevi, A. & Edmondson, D. E. Structure-function relationships in favoenzyme-dependent amine oxidations: a comparison of polyamine oxidase and monoamine oxidase. J. Biol. Chem. 277, 23973–23976 (2002). 60. Ochman, H., Lawrence, J. G. & Groisman, E. A. Lateral gene transfer and the nature of bacterial innovation. Nature 405, 299–304 (2000). 61. Wang, H., Liu, B., Li, H. & Zhang, S. Identifcation and biochemical characterization of polyamine oxidases in amphioxus: Implica- tions for emergence of vertebrate-specifc spermine and acetylpolyamine oxidases. Gene 575, 429–437 (2016). 62. Liu, T., Wook Kim, D., Niitsu, M., Berberich, T. & Kusano, T. Polyamine oxidase 1 from rice (Oryza sativa) is a functional ortholog of Arabidopsis Polyamine oxidase 5. Plant Signal. Behav. 9, 29773 (2014). 63. Sagor, G. H. et al. Te polyamine oxidase from lycophyte Selaginella lepidophylla (SelPAO5), unlike that of angiosperms, back- converts thermospermine to norspermidine. FEBS Lett. 589, 3071–3078 (2015). 64. Guerrero-González, M. L., Rodríguez-Kessler, M. & Jiménez-Bremont, J. F. uORF, a regulatory mechanism of the Arabidopsis polyamine oxidase 2. Mol. Biol. Rep. 41, 2427–2443 (2014). 65. Binda, C., Angelini, R., Federico, R., Ascenzi, P. & Mattevi, A. Structural bases for inhibitor binding and in polyamine oxidase. Biochemistry 40, 2766–2776 (2001). 66. Fiorillo, A. et al. Te structure of maize polyamine oxidase K300M mutant in complex with the natural substrates provides a snapshot of the catalytic mechanism of polyamine oxidation. FEBS J. 278, 809–821 (2011). 67. Sjögren, T. et al. Te structure of murine N1-acetylspermine oxidase reveals molecular details of vertebrate polyamine catabolism. Biochemistry 56, 458–467 (2017). 68. Altschul, S. F. et al. Gapped BLAST and PSI–BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997). 69. Spedaletti, V. et al. Characterization of a lysine-specifc histone demethylase from Arabidopsis thaliana. Biochemistry 47, 4936–4947 (2008). 70. Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539. https​://doi.org/10.1038/msb.2011.75 (2011). 71. Hess, P. N. & De Moraes Russo, C. A. An empirical test of the midpoint rooting method. Biol. J. Linn. Soc. 92, 669–674 (2007). 72. Tria, F., Landan, G. & Dagan, T. Phylogenetic rooting using minimal ancestor deviation. Nat. Ecol. Evol. 1, 0193 (2017).

Scientifc Reports | (2020) 10:17858 | https://doi.org/10.1038/s41598-020-74708-3 13 Vol.:(0123456789) www.nature.com/scientificreports/

73. Darriba, D. et al. ModelTest-NG: a new and scalable tool for the selection of DNA and protein evolutionary models. bioRxiv 612903;https​://doi.org/10.1101/61290​3 (2019). 74. Whelan, S. & Goldman, N. A. General empirical model of protein evolution derived from multiple protein families using a Maximum-Likelihood approach. Mol. Biol. Evol. 18, 691–699 (2001). 75. Jones, D. T., Taylor, W. R. & Tornton, J. M. Te rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8, 275–282 (1992). 76. Nguyen, L.-T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: A fast and efective stochastic algorithm for estimating maximum likelihood phylogenies. Mol. Biol. Evol. 32, 268–327 (2015). 77. Guindon, S. & Gascuel, O. A. simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704 (2003). 78. Boc, A., Diallo, A. B. & Makarenkov, V. T-REX: a web server for inferring, validating and visualizing phylogenetic trees and net- works. Nucleic Acids Res. 40(W1), W573–W579 (2012). 79. Miller, M. A., Pfeifer, W. & Schwartz, T. Creating the CIPRES Science Gateway for Inference of Large Phylogenetic Trees. In: SC10 Workshop on Gateway Computing Environments (GCE10) (2010). Acknowledgements We thank Anthony Michael (UT Southwestern) for useful suggestions. Tis work was supported by the Ital- ian Ministry of Education, University and Research (Grant to Department of Science, University ‘Roma Tre’-‘Dipartimenti di Eccellenza’, ARTICOLO 1, COMMI 314–337. LEGGE 423 232/2016; PRIN 2017– CUP F84I19000730005), and University ‘Roma Tre’. Author contributions D.S. and P.T. jointly conceived the study, performed the analyses, and wrote the manuscript.

Competing interests Te authors declare no competing interests. Additional information Supplementary information is available for this paper at https​://doi.org/10.1038/s4159​8-020-74708​-3. Correspondence and requests for materials should be addressed to P.T. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afliations. Open Access Tis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. Te images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/.

© Te Author(s) 2020

Scientifc Reports | (2020) 10:17858 | https://doi.org/10.1038/s41598-020-74708-3 14 Vol:.(1234567890)