Supplementary Material

Transcription

1 Evolution of substrate specificity in the Nucleobase-Ascorbate Transporter (NAT) protein family Anezia Kourkoulou, Alexandros A. Pittis & George Diallinas Supplementary Material

2 Supplementary Figure S1. Phylogeny of microbial NATs. The tree shown includes all functionally characterized NATs from bacteria and fungi and their closest homologues from other prokaryotic and eukaryotic microorganisms, obtained by blastp ( The AzgA sequences at the bottom of the tree are functionally characterized purine transporters of the so-called AzgA-like family, which includes proteins structurally similar with NATs, but show very little primary sequence similarity and lack NAT-specific functional motif (closest out-group). Multiple sequence alignments was built from the selected NAT sequences (accession number shown in the figure) using MUSCLE v7..26 ( MEGA was also used for testing the aligned sequences for optimal amino acid substitution model. According to the AIC, the LG+G+F model (Le SQ, Gascuel O. Mol Biol Evol. 28 Jul;25(7):137-2) was selected and a tree was created using a maximum likelihood (ML), and visualized by FigTree v1.4.3 ( Bootstrap values are shown in bold at the nodes of the clades. Evolutionary distances are also shown on the branches of the tree. The variable, four-amino acid, part of the NAT signature motif is shown at the right side of the tree (in bold the residues critical for specificity, see main text). Note that the microbial NAT sequences shown in this tree are collapsed and referred as UapA-like sequences in Figure 1 of the main text.

3 72.ACYPI858-PA {Acyrthosiphon pisum} T Y G E N I G V L A I T R T Y G E N I G V L A I T R Ganpr1_626 {Gonapodya prolifera} T Y A E N M G T M G A T R T Y A E N M G T M G A T R Ganpr1_1368 {Gonapodya prolifera} T Y A E N M G T M G A T R T Y A E N M G T M G A T R 68 8.MVEG_6T {Mortierella verticillata} 4468.Q54NF {Dictyostelium discoideum} 4468.Q54ZL {Dictyostelium discoideum} 368.Mucci2_147 {Mucor circinelloides} T Y A E N I G V L S V T Q T Y A E N I G V L S V T Q 47.Phybl2_13112 {Phycomyces blakesleeanus} T Y A E N I G V V A I T R T Y A E N I G V V A I T R 47.Phybl2_1117 {Phycomyces blakesleeanus} T Y S E N I G V M A V T K T Y S E N I G V M A V T K 644.RO3T_13 {Rhizopus oryzae} T Y S E N I G V M A V T Q T Y S E N I G V M A V T Q 368.Mucci2_12667 {Mucor circinelloides} T Y S E N I G V M A V T Q T Y S E N I G V M A V T Q 7175.CpipJ_CPIJ2253-RA {Culex pipiens} A F A Q N V G L V A V T G A F A Q N V G L V A V T G 72.ACYPI3-PA {Acyrthosiphon pisum} N N G V I Q L T G N N G V I Q L T G ATZT {Physcomitrella patens} T Y A Q N N G V L S L S R T Y A Q N N G V L S L S R AS4K1 {Physcomitrella patens} T Y A Q N N G V L S L S R T Y A Q N N G V L S L S R Cre1.g.t1.1 {Chlamydomonas reinhardtii} T F A Q N N G V I S L T N T F A Q N N G V I S L T N 355.Cre1.g48.t1.2 {Chlamydomonas reinhardtii} T F A Q N N G V I S L T N T F A Q N N G V I S L T N 355.Cre1.g46.t1.1 {Chlamydomonas reinhardtii} T F A Q N N G V I A L T N T F A Q N N G V I A L T N {Phaeodactylum tricornutum CCAP 155/1} T L S Q N N G I V A L T R T L S Q N N G I V A L T R {Fragilariopsis cylindrus} T L S Q N N G I L V D L E T L S Q N N G I L V D L E {Fragilariopsis cylindrus} T L S Q N N G I V A L T R T L S Q N N G I V A L T R B8CGC1 {Thalassiosira pseudonana} T F S Q N N G V I G I T K T F S Q N N G V I G I T K {Phaeodactylum tricornutum CCAP 155/1} T F S Q N N G V I A L T K T F S Q N N G V I A L T K {Fragilariopsis cylindrus} T Y S E N N G V V A L T K T Y S E N N G V V A L T K {Fragilariopsis cylindrus} T Y S E N N G V V A L T K T Y S E N N G V V A L T K Ganpr1_348 {Gonapodya prolifera} T F A Q N N G V I A L T K T F A Q N N G V I A L T K 6 8.MVEG_T {Mortierella verticillata} C Y A Q N N G I I S L T R C Y A Q N N G I I S L T R 8.MVEG_7135T {Mortierella verticillata} V F A Q N N G I I A L T R V F A Q N N G I I A L T R 47.Phybl2_1416 {Phycomyces blakesleeanus} T F A Q N N G I I A L T R T F A Q N N G I I A L T R 644.RO3T_32 {Rhizopus oryzae} T Y S N N N G V I A V T Q T Y S N N N G V I A V T Q 368.Mucci2_146 {Mucor circinelloides} T F S Q N N G I I A V T R T F S Q N N G I I A V T R 644.RO3T_3 {Rhizopus oryzae} T F S Q N N G V I A V T R T F S Q N N G V I A V T R 47.Phybl2_ {Phycomyces blakesleeanus} T F A Q N N G V V A L T R T F A Q N N G V V A L T R 644.RO3T_118 {Rhizopus oryzae} T F A Q N N G V V A M T R T F A Q N N G V V A M T R 368.Mucci2_ {Mucor circinelloides} T F A Q N N G V V A M T R T F A Q N N G V V A M T R 2.Rhoba1_1_172 {Rhodotorula graminis} I F A Q N N G V I S V T R I F A Q N N G V I S V T R 527.um.2 {Ustilago maydis} V F A Q N N G V I A I T K V F A Q N N G V I A I T K 536.Phchr11215 {Phanerochaete chrysosporium} I F A Q N N G V I A I T R I F A Q N N G V I A I T R 5346.CC1G_ {Coprinopsis cinerea} T F A Q N N G V I A I T R T F A Q N N G V I A I T R Ganpr1_157 {Gonapodya prolifera} T F S Q N T S V I A L S N T F S Q N T S V I A L S N 2.Rhoba1_1_561 {Rhodotorula graminis} T F S Q N V S V I A L S N T F S Q N V S V I A L S N 527.um36 {Ustilago maydis} T F S Q N S G V I S L T R T F S Q N S G V I S L T R 2146.Q5KHM2 {Cryptococcus neoformans var. neoformans JEC21} T F S Q N S G V I A L T R T F S Q N S G V I A L T R GSTUMT2261 {Tuber melanosporum Mel28} C F A Q N N G V I A L T G C F A Q N N G V I A L T G 516.CocheC5_13 {Bipolaris maydis} S Q A G N N G V I V L T G S Q A G N N G V I V L T G 5516.Botdo1_6 {Botryosphaeria dothidea} G Q A G N N G V I V L T G G Q A G N N G V I V L T G 561.A2QBM4 {Aspergillus niger} S Q A G N N G V I S L T G S Q A G N N G V I S L T G 441.PMAA_124.t1 {Talaromyces marneffei ATCC 124} S Q A G N N G V I S L T G S Q A G N N G V I S L T G 2812.SPAC13.1c.1 {Schizosaccharomyces pombe 2h-} 566.Saico1_634 {Saitoella complicata} C F A Q N N G V I A L T R C F A Q N N G V I A L T R 22.Lipst1_1_55 {Lipomyces starkeyi} 1165.Ascim {Ascobolus immersus RN} GSTUMT2651 {Tuber melanosporum Mel28} GSTUMT28181 {Tuber melanosporum Mel28} 2221.Q7 {Aspergillus nidulans FGSC A4} 557.Fusox1_5 {Fusarium oxysporum} V F A Q N N G V I V L T R V F A Q N N G V I V L T R 635.Triat153 {Trichoderma atroviride} 257.MGG_856T {Magnaporthe oryzae 7-15} PAAG_3721T {Paracoccidioides brasiliensis} 5141.Q7S3D5 {Neurospora crassa} 5145.Pa_5_818 {Podospora anserina} G1XCI1 {Arthrobotrys oligospora ATCC 247} 516.CocheC5_11127 {Bipolaris maydis} T F A Q N C G V V A L T R T F A Q N C G V V A L T R 1435.Aurpu_var_sub {Aureobasidium subglaciale EXF-2481} V F A Q N N G I I A L T R V F A Q N N G I I A L T R 5516.Botdo1_17 {Botryosphaeria dothidea} BC1T_11657 {Botrytis cinerea} 42.YALIE252g {Yarrowia lipolytica} T F A Q N N G V I A L T K T F A Q N N G V I A L T K 42.YALIE7g {Yarrowia lipolytica} 285.KLLAD4356g {Kluyveromyces lactis} T F A Q N N G V I S I T K T F A Q N N G V I S I T K 2811.AGOS_AFR622W {Eremothecium gossypii ATCC 18} T F A Q N N G V I A I T R T F A Q N N G V I A I T R 361.CLUG_16.1 {Clavispora lusitaniae} T F A Q N N G V I S I T K T F A Q N N G V I S I T K 54.C4_653C_A {Candida albicans} V F A Q N N G V I S I T K V F A Q N N G V I S I T K 537.HCAG_7147T {Histoplasma capsulatum} V F A Q N N G V I S L T R V F A Q N N G V I S L T R Oidma1_1 {Oidiodendron maius} 441.PMAA_41.t1 {Talaromyces marneffei ATCC 124} 557.A1CDS4 {Aspergillus clavatus} 2221.P4 {Aspergillus nidulans FGSC A4} V F A Q N N G V I A L T R V F A Q N N G V I A L T R 561.A2QMF8 {Aspergillus niger} 5.Pc2g1.t1 {Penicillium chrysogenum} 62.A8X8V6 {Caenorhabditis briggsae} T H T E N I G V I G V T R T H T E N I G V I G V T R 623.C51E3.6 {Caenorhabditis elegans} T H T E N I G V I G V T R T H T E N I G V I G V T R 623.Y5EAL.4.2 {Caenorhabditis elegans} T Y A E N I A L I H I T K T Y A E N I A L I H I T K 62.A8XX35 {Caenorhabditis briggsae} T Y A E N I A L I H I T K T Y A E N I A L I H I T K 623.T7G12.2 {Caenorhabditis elegans} T Y S E N I A I M Q V T K T Y S E N I A I M Q V T K 62.A8X6C5 {Caenorhabditis briggsae} T Y S E N I A I M Q V T K T Y S E N I A I M Q V T K 62.A8WQX8 {Caenorhabditis briggsae} 623.R11E3.2 {Caenorhabditis elegans} 62.A8X6E3 {Caenorhabditis briggsae} C Y A E N I A I M S V T K C Y A E N I A I M S V T K 623.T7G12.5a {Caenorhabditis elegans} 62.A8X6E {Caenorhabditis briggsae} ENSCSAVP12 {Ciona savignyi} S F S E N I G A I G I T R S F S E N I G A I G I T R 1.ENSCINP {Ciona intestinalis} S F S E N I G A I G I T R S F S E N I G A I G I T R ENSCSAVP132 {Ciona savignyi} S F S E N I A A I G V T R S F S E N I A A I G V T R 1.ENSCINP225 {Ciona intestinalis} 1.ENSCINP16143 {Ciona intestinalis} S F S Q N V A A I G I T R S F S Q N V A A I G I T R 1.ENSCINP11752 {Ciona intestinalis} S F S Q N V A A I G I T R S F S Q N V A A I G I T R 55.F1RM3 {Danio rerio} S Y S Q N I A A L G I T R S Y S Q N I A A L G I T R 71 8.ENSORLP246 {Oryzias latipes} 6.ENSGACP1234 {Gasterosteus aculeatus}.enstnip16716 {Tetraodon nigroviridis} 31.ENSTRUP13 {Takifugu rubripes} 55.B8JIT8 {Danio rerio} 6.ENSGACP16 {Gasterosteus aculeatus} 8.ENSORLP215 {Oryzias latipes}.enstnip11637 {Tetraodon nigroviridis} 31.ENSTRUP15116 {Takifugu rubripes} 8.ENSORLP124 {Oryzias latipes} S F S E N V A A L G I T K S F S E N V A A L G I T K 63 6.ENSGACP16471 {Gasterosteus aculeatus} S F S E N V A A L G I T K S F S E N V A A L G I T K.ENSTNIP671 {Tetraodon nigroviridis}.enstnip2317 {Tetraodon nigroviridis} S F S E N V A I L G I T K S F S E N V A I L G I T K 31.ENSTRUP16667 {Takifugu rubripes}.enstnip13643 {Tetraodon nigroviridis} 31.ENSTRUP17126 {Takifugu rubripes} 31.ENSTRUP4545 {Takifugu rubripes} 64.F6UZZ5 {Xenopus tropicalis} 2.ENSACAP14 {Anolis carolinensis} 31.GALT146 {Gallus gallus} 52.ENSTGUT58 {Taeniopygia guttata} ENSMODP11 {Monodelphis domestica}.ensecap1362 {Equus caballus} S F S Q N I A A L S I T K S F S Q N I A A L S I T K.ENSECAP15216 {Equus caballus} S Y S Q N I A A L S I T R S Y S Q N I A A L S I T R.ENSECAP155 {Equus caballus}.ensecap154 {Equus caballus} 563.ENSMLUP17132 {Myotis lucifugus} 85.ENSFCAP21447 {Felis catus} S S V P H V G A E G G H Q S S V P H V G A E G G H Q 23.ENSSSCP27 {Sus scrofa} S Y S E N V G A L S I T R S Y S E N V G A L S I T R 15.ENSCAFP {Canis lupus familiaris} 85.ENSLAFP1 {Loxodonta africana} S Y S E N V G V L G I T Q S Y S E N V G V L G I T Q ENSSSCP2655 {Sus scrofa}.ensbtap1 {Bos taurus}.ensbtap55 {Bos taurus}.ensbtap51345 {Bos taurus}.ensmusp41436 {Mus musculus} 1116.ENSRNOP31 {Rattus norvegicus} 1141.ENSCPOP15281 {Cavia porcellus} S Y S E N I G A L G I T R S Y S E N I G A L G I T R.ENSOCUP25 {Oryctolagus cuniculus} 55.F6NS11 {Danio rerio}.enstnip14 {Tetraodon nigroviridis} 31.ENSTRUP236 {Takifugu rubripes} 8.ENSORLP156 {Oryzias latipes} 6.ENSGACP258 {Gasterosteus aculeatus} F7DHZ {Xenopus tropicalis} 2.ENSACAP1616 {Anolis carolinensis} 52.ENSTGUT15224 {Taeniopygia guttata} 31.ENSGALT32 {Gallus gallus} ENSMODP148 {Monodelphis domestica} 15.ENSMEUP68 {Macropus eugenii} 65.ENSEEUP115 {Erinaceus europaeus} 23.ENSSSCP2185 {Sus scrofa} 35.ENSVPAP58 {Vicugna pacos} 15.ENSCAFP8511 {Canis lupus familiaris} 85.ENSFCAP247 {Felis catus} 85.ENSFCAP15 {Felis catus} 24 3.ENSTTRP351 {Tursiops truncatus}.ensbtap13 {Bos taurus} 1328.ENSPVAP14553 {Pteropus vampyrus}.ensecap184 {Equus caballus} 563.ENSMLUP25 {Myotis lucifugus} ENSPCAP655 {Procavia capensis} 85.ENSLAFP {Loxodonta africana} 347.ENSTBEP5 {Tupaia belangeri} 61.ENSDNOP4166 {Dasypus novemcinctus}.enscjat135 {Callithrix jacchus} 44.ENSMMUT1 {Macaca mulatta}.ensppyp1 {Pongo pygmaeus} 8.ENSPTRP46 {Pan troglodytes} 6.ENSP32851 {Homo sapiens}.ensggot16 {Gorilla gorilla gorilla} ENSETEP47 {Echinops telfairi} ENSOGAT1618 {Otolemur garnettii} 368.ENSMICT1246 {Microcebus murinus} 2.ENSDORP2131 {Dipodomys ordii}.ensmusp25212 {Mus musculus} 1116.ENSRNOP28 {Rattus norvegicus} ENSCPOP12685 {Cavia porcellus} 431.ENSSTOP13225 {Ictidomys tridecemlineatus}.ensocup5 {Oryctolagus cuniculus} 8.ENSOPRP23 {Ochotona princeps}.enstnip116 {Tetraodon nigroviridis} 31.ENSTRUP628 {Takifugu rubripes} 6.ENSGACP758 {Gasterosteus aculeatus} S S S P N I G V M G I T K S S S P N I G V M G I T K 8.ENSORLP361 {Oryzias latipes} 64.G1K3I1 {Xenopus tropicalis} 47 2.ENSACAP5 {Anolis carolinensis} 52.ENSTGUT63 {Taeniopygia guttata} 52.ENSTGUT1 {Taeniopygia guttata} 31.GALT261 {Gallus gallus} 15.ENSMEUP3547 {Macropus eugenii} 58.ENSOANP2183 {Ornithorhynchus anatinus} ENSMODP14568 {Monodelphis domestica} 85.ENSFCAP252 {Felis catus} 3.ENSTTRP51 {Tursiops truncatus} 23.ENSSSCP2 {Sus scrofa} 15.ENSCAFP3 {Canis lupus familiaris} 563.ENSMLUP31 {Myotis lucifugus} 1328.ENSPVAP5 {Pteropus vampyrus}.ensbtap351 {Bos taurus} 35.ENSVPAP135 {Vicugna pacos} S S C P N I G V L G I T K S S C P N I G V L G I T K 23.ENSSSCP262 {Sus scrofa} 8.ENSECAP121 {Equus caballus} 65.ENSEEUP12857 {Erinaceus europaeus} 254.ENSSARP36 {Sorex araneus} 431.ENSSTOP135 {Ictidomys tridecemlineatus} 1141.ENSCPOP {Cavia porcellus} 1116.ENSRNOP285 {Rattus norvegicus}.ensmusp215 {Mus musculus} 2.ENSDORP14356 {Dipodomys ordii} 37 8.ENSOPRP1567 {Ochotona princeps}.ensocup57 {Oryctolagus cuniculus} 61.ENSDNOP1718 {Dasypus novemcinctus} 347.ENSTBEP17 {Tupaia belangeri}.enstsyt1461 {Tarsius syrichta} 58.ENSCHOP18 {Choloepus hoffmanni} 13.ENSPCAP46 {Procavia capensis} 71.ENSETEP1314 {Echinops telfairi} 85.ENSLAFP117 {Loxodonta africana} 368.ENSMICT14355 {Microcebus murinus} 23.ENSSSCP2554 {Sus scrofa} 44.ENSMMUT44526 {Macaca mulatta} 6.ENSP {Homo sapiens}.ensggot {Gorilla gorilla gorilla}.ensptrp2266 {Pan troglodytes}.ensppyp1262 {Pongo pygmaeus} 3611.ENSOGAT2 {Otolemur garnettii}.enscjat54 {Callithrix jacchus} 72.ACYPI23-PA {Acyrthosiphon pisum} S S S E N V G N I G I T R S S S E N V G N I G I T R PHUM46-PA {Pediculus humanus corporis} S F S E N I G A I G V T R S F S E N I G A I G V T R EFX31 {Daphnia pulex} S Y S E N I G A I G V T K S Y S E N I G A I G V T K 666.EFX564 {Daphnia pulex} 666.EFX255 {Daphnia pulex} S Y S E N I G A I G V T K S Y S E N I G A I G V T K 666.EFX618 {Daphnia pulex} 72.ACYPI7-PA {Acyrthosiphon pisum} 72.ACYPI81-PA {Acyrthosiphon pisum} NV11256-PA {Nasonia vitripennis} T F G E N V G T I G V T K T F G E N V G T I G V T K 75 7.BGIBMGA3-TA {Bombyx mori} 7175.CpipJ_CPIJ32-RA {Culex pipiens} AGAP31-PA {Anopheles gambiae} 715.AAEL167-PA {Aedes aegypti} 7175.CpipJ_CPIJ4614-RA {Culex pipiens} 726.FBtr24.2.homologue {Drosophila willistoni} FBtr24.1.homologue {Drosophila persimilis} FBpp211 {Drosophila pseudoobscura pseudoobscura} 7222.FBtr24.1.homologue {Drosophila grimshawi} 7244.FBtr24.2.homologue {Drosophila virilis} 723.FBtr24.2.homologue {Drosophila mojavensis} 7217.FBtr24.1.homologue {Drosophila ananassae} FBtr24.1.homologue {Drosophila yakuba} FBpp1356 {Drosophila erecta} 724.FBpp2126 {Drosophila simulans} 72.FBpp252 {Drosophila sechellia} 7227.FBpp81718 {Drosophila melanogaster} 1.ENSCINP12358 {Ciona intestinalis} ENSCSAVP4 {Ciona savignyi} 1.ENSCINP1236 {Ciona intestinalis} A7RY {Nematostella vectensis} A7RXI6 {Nematostella vectensis} A7SXJ6 {Nematostella vectensis} A7RHH1 {Nematostella vectensis} A7RHH2 {Nematostella vectensis} A7T1W {Nematostella vectensis} A7RGN3 {Nematostella vectensis} S Y S E N I G A L G I T K S Y S E N I G A L G I T K A7RIC4 {Nematostella vectensis} S Y S Q N I G A L G I T K S Y S Q N I G A L G I T K A7RGN5 {Nematostella vectensis} S Y S E N I G A L G I T K S Y S E N I G A L G I T K A7SRV {Nematostella vectensis} S Y S Q N I G A I G I T K S Y S Q N I G A I G I T K A7RGN4 {Nematostella vectensis} S Y S Q N I G A I G I T K S Y S Q N I G A I G I T K 55.E7FEY {Danio rerio} S S A S N A C V I G L S Q S S A S N A C V I G L S Q 64.F6WZA7 {Xenopus tropicalis} S S I P N A G L A G L T Q S S I P N A G L A G L T Q 5 2.ENSACAP365 {Anolis carolinensis} S S M S N T C A A H L T K S S M S N T C A A H L T K 31.GALT1851 {Gallus gallus} A S I A N A C A G G L T Q A S I A N A C A G G L T Q 52.ENSTGUT57 {Taeniopygia guttata} S S I A N T C A T G F T Q S S I A N T C A T G F T Q 15.ENSMEUP256 {Macropus eugenii} ENSMODP148 {Monodelphis domestica} S S F P N V A T T S L T Q S S F P N V A T T S L T Q 85.ENSLAFP68 {Loxodonta africana} S S F P N V G T L S L T Q S S F P N V G T L S L T Q 3.ENSTTRP4 {Tursiops truncatus} 35.ENSVPAP663 {Vicugna pacos} 13.ENSPCAP348 {Procavia capensis} 71.ENSETEP87 {Echinops telfairi} L T P L T P 1328.ENSPVAP1163 {Pteropus vampyrus} 65.ENSEEUP75 {Erinaceus europaeus} 254.ENSSARP6352 {Sorex araneus} L I Q L I Q 3611.ENSOGAT1554 {Otolemur garnettii} 368.ENSMICT5 {Microcebus murinus} 47.ENSTSYT8134 {Tarsius syrichta} 431.ENSSTOP1 {Ictidomys tridecemlineatus} 2.ENSDORP {Dipodomys ordii} 62.ENSBTAP16751 {Bos taurus} S S F P N V G T V G L L Q S S F P N V G T V G L L Q 23.ENSSSCP21632 {Sus scrofa} S S F P N V G T M S L F Q S S F P N V G T M S L F Q 563.ENSMLUP14 {Myotis lucifugus} 28.ENSECAP53 {Equus caballus} S S F P N V G T V S L V Q S S F P N V G T V S L V Q 15.ENSCAFP22144 {Canis lupus familiaris} 85.ENSFCAP6 {Felis catus} 1141.ENSCPOP {Cavia porcellus}.ensmusp25 {Mus musculus} 1116.ENSRNOP24667 {Rattus norvegicus}.enscjat2232 {Callithrix jacchus}.ensppyp143 {Pongo pygmaeus} 44.ENSMMUT1137 {Macaca mulatta}.ensptrp2218 {Pan troglodytes}.ensggot1 {Gorilla gorilla gorilla} 6.ENSP46546 {Homo sapiens} 355.Cre17.g7168.t1.1 {Chlamydomonas reinhardtii} S Y A E N I G A I G L T G S Y A E N I G A I G L T G {Phaeodactylum tricornutum CCAP 155/1} S Y S E N I G A I S L T R S Y S E N I G A I S L T R BRADI1G271.1 {Brachypodium distachyon} T L T E N I H T L D I T K T L T E N I H T L D I T K 37.OS7T45-1 {Oryza sativa Japonica Group} T L T E N I H T L E N T K T L T E N I H T L E N T K 45.GRMZM2G171_P1 {Zea mays} Sb2g65.1 {Sorghum bicolor} 45.GRMZM5G1_P1 {Zea mays} 45.GRMZM2G1362_P1 {Zea mays} 45.GRMZM2G85_P3 {Zea mays} 26.VIT_s813g1.t1 {Vitis vinifera} T L T E N V H T I N I T K T L T E N V H T I N I T K 372.AT4G5.1 {Arabidopsis thaliana} T L T E N I H T I N I T K T L T E N I H T I N I T K {Arabidopsis lyrata} T L T E N I H T I N I T K T L T E N I H T I N I T K 5 36.POPTR_7s13.1 {Populus trichocarpa} T L T E N V H T V N I T K T L T E N V H T V N I T K VIT_s4g1.t1 {Vitis vinifera} 47.GLYMA8G21.1 {Glycine max} T L T E N V Q T I D T T K T L T E N V Q T I D T T K 47.GLYMA18G24.1 {Glycine max} T L T E N V H T I D T T K T L T E N V H T I D T T K 47.GLYMA1G2.1 {Glycine max} T L T E N M H T I D V T K T L T E N M H T I D V T K 47.GLYMAG22.1 {Glycine max} T L T E N T H T I D I T K T L T E N T H T I D I T K {Selaginella moellendorffii} T L T E N V H T I A V T R T L T E N V H T I A V T R {Selaginella moellendorffii} AS4J {Physcomitrella patens} ASZ {Physcomitrella patens} ARF1 {Physcomitrella patens} 37.OS1T {Oryza sativa Japonica Group} BRADI2G {Brachypodium distachyon} 45.GRMZM2G611_P3 {Zea mays} 4558.Sb3g43.1 {Sorghum bicolor} 372.AT2G21.1 {Arabidopsis thaliana} {Arabidopsis lyrata} 36.POPTR_s158.1 {Populus trichocarpa} 36.POPTR_4s1.1 {Populus trichocarpa} 26.VIT_6s61g131.t1 {Vitis vinifera} 47.GLYMA11G12.1 {Glycine max} 47.GLYMA12G6.1 {Glycine max} 47.GLYMA17G528.1 {Glycine max} 47.GLYMA12G367.1 {Glycine max} ASKP {Physcomitrella patens} T S A E T I G L I G L T K T S A E T I G L I G L T K ATGX2 {Physcomitrella patens} V A P E N A G L I G L T R V A P E N A G L I G L T R ATRH {Physcomitrella patens} AT18 {Physcomitrella patens} AT17 {Physcomitrella patens} AT2 {Physcomitrella patens} {Selaginella moellendorffii} A L V E N A G L I G L T R A L V E N A G L I G L T R {Selaginella moellendorffii} I S V E N A G L V G I T R I S V E N A G L V G I T R {Selaginella moellendorffii} I S V E N P G L V G T S Q I S V E N P G L V G T S Q {Selaginella moellendorffii} I S P E N A G L V G I T R I S P E N A G L V G I T R {Selaginella moellendorffii} V S V E N A G L V G L T R V S V E N A G L V G L T R {Selaginella moellendorffii} V S V E N A G L V G L T R V S V E N A G L V G L T R 37.OS12T5-2 {Oryza sativa Japonica Group} BRADI4G3.1 {Brachypodium distachyon} 4558.Sb8g18.2 {Sorghum bicolor} 45.GRMZM2G415_P1 {Zea mays} 372.AT2G {Arabidopsis thaliana} A S V E N V G L L G L T R A S V E N V G L L G L T R {Arabidopsis lyrata} A S V E N V G L L G L T R A S V E N V G L L G L T R 36.POPTR_2s138.1 {Populus trichocarpa} 36.POPTR_14s351.1 {Populus trichocarpa} VIT_18s1g1126.t1 {Vitis vinifera} 47.GLYMA14G.1 {Glycine max} V S V E N V G L L G L T H V S V E N V G L L G L T H 47.GLYMA17G {Glycine max} V S V E N V G L L G L T H V S V E N V G L L G L T H 36.POPTR_14s {Populus trichocarpa} 6 26.VIT_12s5g128.t1 {Vitis vinifera} 372.AT2G5.1 {Arabidopsis thaliana} V L V E N V G L L G L T R V L V E N V G L L G L T R {Arabidopsis lyrata} V L V E N V G L L G L T R V L V E N V G L L G L T R 47.GLYMA13G {Glycine max} 47.GLYMA2G {Glycine max} 47.GLYMA2G {Glycine max} 47.GLYMA14G522.1 {Glycine max} I S V E N V G L L G L T R I S V E N V G L L G L T R {Arabidopsis lyrata} 372.AT2G341.1 {Arabidopsis thaliana} 8 47.GLYMA8G {Glycine max} V S V E N V G L L G S N R V S V E N V G L L G S N R 47.GLYMA4G675.1 {Glycine max} 47.GLYMA6G6.1 {Glycine max} 26.VIT_1s3g146.t1 {Vitis vinifera} 36.POPTR_4s575.1 {Populus trichocarpa} 36.POPTR_11s717.1 {Populus trichocarpa} 37.OS1T75-1 {Oryza sativa Japonica Group} BRADI2G546.1 {Brachypodium distachyon} 4558.Sb3g351.1 {Sorghum bicolor} 45.GRMZM2G435_P4 {Zea mays} BRADI4G27.1 {Brachypodium distachyon} 4558.Sb2g {Sorghum bicolor} 45.GRMZM2G4_P1 {Zea mays} BRADI3G {Brachypodium distachyon} V S V E N V G F L G S T R V S V E N V G F L G S T R 4 37.OS8T36-1 {Oryza sativa Japonica Group} 4558.Sb8g165.1 {Sorghum bicolor} 4558.Sb7g14.1 {Sorghum bicolor} 45.GRMZM2G83_P1 {Zea mays} 45.GRMZM5G8517_P1 {Zea mays} A S V E N A G L L A V T R A S V E N A G L L A V T R 4558.Sb1g {Sorghum bicolor} A S V E N A G L L A V T R A S V E N A G L L A V T R BRADI1G11.1 {Brachypodium distachyon} A S V E N S G L L A I T R A S V E N S G L L A I T R 37.OS3T65-1 {Oryza sativa Japonica Group} A S V E N A G L L A L T R A S V E N A G L L A L T R 26.VIT_3sg15.t1 {Vitis vinifera} A S V E N T G L L G L T R A S V E N T G L L G L T R {Arabidopsis lyrata} A L V E N T G L L G L T K A L V E N T G L L G L T K 372.AT1G4.1 {Arabidopsis thaliana} A L V E N T G L L G L T K A L V E N T G L L G L T K 8 47.GLYMA1G424.2 {Glycine max} A S V E N A G L L G L K R A S V E N A G L L G L K R 47.GLYMA2G {Glycine max} A S V E N A G L L G L T R A S V E N A G L L G L T R 36.POPTR_1s2.1 {Populus trichocarpa} - - L E N S G L L E N S G L POPTR_s.1 {Populus trichocarpa} A S V E N A G L V G L T R A S V E N A G L V G L T R 26.VIT_18s1g8.t1 {Vitis vinifera} I S V E N V G L L A V T R I S V E N V G L L A V T R 47.GLYMA14G.1 {Glycine max} 47.GLYMA17G {Glycine max} 47.GLYMA4G48.1 {Glycine max} 47.GLYMA6G4.1 {Glycine max} POPTR_14s154.1 {Populus trichocarpa} 372.AT5G25.1 {Arabidopsis thaliana} K N V G L L A M T K K N V G L L A M T K {Arabidopsis lyrata} {Arabidopsis lyrata} 372.AT1G {Arabidopsis thaliana} 37.OST1-1 {Oryza sativa Japonica Group} V S V E N V G L L A L T H V S V E N V G L L A L T H BRADI4G24.1 {Brachypodium distachyon} V S V E N V G L L A V T H V S V E N V G L L A V T H 4558.Sb2g {Sorghum bicolor} V S V E N I G L L A L T R V S V E N I G L L A L T R 45.GRMZM2G414813_P2 {Zea mays} V S V E N I G L L A L T R V S V E N I G L L A L T R BRADI3G361.1 {Brachypodium distachyon} 37.OS8T6-1 {Oryza sativa Japonica Group} 4558.Sb7g251.1 {Sorghum bicolor} I C S E N A G L L A L T H I C S E N A G L L A L T H 45.GRMZM2G115635_P1 {Zea mays} 37.OS2T18-1 {Oryza sativa Japonica Group} BRADI3G5.1 {Brachypodium distachyon} V S I E N A G L L A L T R V S I E N A G L L A L T R 45.GRMZM5G812555_P1 {Zea mays} 4558.Sb4g2.1 {Sorghum bicolor} 37.OS3T-1 {Oryza sativa Japonica Group} BRADI1G348.1 {Brachypodium distachyon} 45.GRMZM2G451_P2 {Zea mays} 45.GRMZM2G3_P2 {Zea mays} 45.GRMZM2G8546_P1 {Zea mays} V H D R W K Y I W G S D K V H D R W K Y I W G S D K 4558.Sb1g2.1 {Sorghum bicolor} V S V E N A G L L G L S R V S V E N A G L L G L S R 58.5 {Arabidopsis lyrata} V S V E N A G L L A V T R V S V E N A G L L A V T R 372.AT1G154.1 {Arabidopsis thaliana} V S V E N A G L L A V T R V S V E N A G L L A V T R {Arabidopsis lyrata} 372.AT1G63.1 {Arabidopsis thaliana} 1 26.VIT_1s26g.t1 {Vitis vinifera} 36.POPTR_8s {Populus trichocarpa} 36.POPTR_1s155.1 {Populus trichocarpa} 47.GLYMA18G186.2 {Glycine max} 47.GLYMA8G4.2 {Glycine max} 47.GLYMA2G355.5 {Glycine max} V S V E N A G L L A L T Q V S V E N A G L L A L T Q 47.GLYMA1G416.2 {Glycine max} 47.GLYMA4G358.3 {Glycine max} 47.GLYMA6G16.4 {Glycine max} 47.GLYMA17G.1 {Glycine max} 47.GLYMA5G1.1 {Glycine max} 26.VIT_17sg5.t1 {Vitis vinifera} 36.POPTR_15s8.1 {Populus trichocarpa} 36.POPTR_12s.1 {Populus trichocarpa} {Arabidopsis lyrata} 372.AT5G628.1 {Arabidopsis thaliana} 372.AT5G4.1 {Arabidopsis thaliana} V S V E N A G L L A L T K V S V E N A G L L A L T K {Arabidopsis lyrata} V S V E N A G L L A L T K V S V E N A G L L A L T K.5

4 Supplementary Figure S2. Phylogenetic tree of the SVCT clade and the NAT signature motif The same ML phylogenetic tree as in Figure 1a is shown, here extended and with the sequence - followed by the species - names shown. The protein names correspond to the sequence ids as of Ensembl genomes, or the Uniprot name, for those cases that mapping was successful. Red spheres indicate duplication nodes (according to the species overlap algorithm), and the numbers next to the nodes correspond to alrt support values. On the right, the NAT signature motif is shown for each protein sequence. The ETE toolkit was used for tree visualization [66].

5 Supplementary Table S1. List of strain used in this study Strain Genotype wt uapa - uapaδ uapcδ::afpyrg azgaδ UapA UapA-Q48P UapA-A47S/Q48P UapA- F46S/A47S/Q48P UapA- A45S/F46S/A47S FurA uapa-gfp-argb uapaδ uapcδ::afpyrg azgaδ uapa-gfp -Q48P -argb uapaδ uapcδ::afpyrg azgaδ argb2 uapa-gfp -A47S/Q48P -argb uapaδ uapcδ::afpyrg azgaδ argb2 uapa-gfp -F46S/A47S/Q48P -argb uapaδ uapcδ::afpyrg azgaδ argb2 uapa-gfp -T45S/F46S/A47S -argb uapaδ uapcδ::afpyrg azgaδ argb2 gpdap-fura-gfp-panβ uapaδ uapcδ::pyrg azgaδ fcybδ::argb furdδ::ribob FurAΔ::riboBΔ cntaδ::ribob All strains shown possess the vea1 mutation which promotes conidiation. and argb2 are loss of function mutations leading to auxotrophic requirement of para-aminobenzoic acid and arginine respectively. AfpyrG is the Aspergillus fumigatus wild type pyrg gene(pyrimidine biosynthesis) used as a standard selection marker in A. nidulans transformation. panb and ribob are genes involved in pantothenic acid and riboflavin biosynthesis, used as selection markers for targeted gene knock out. All other genes, encoding transporters, are explained in the text. Supplementary Table S2. Oligonucleotides used for the construction of uapa mutants. Name Sequence 1 UapA A47S/Q48P F 5 -CCCCCATGACGACCTTTTCGCCGAACAACGGCGTG-3 2 UapA A47S/Q48P R 5 -CACGCCGTTGTTCGGCGAAAAGGTCGTCATGGGG-3 3 UapA F46S/A47S/Q48P F 5 -GACCCCCATGACGACCTCTTCGCCGAACAACGGCGTG-3 4 UapA F46S/A47S/Q48P R 5 -CACGCCGTTGTTCGGCGAAGAGGTCGTCATGGGGGTC-3 5 UapA T45S/F46S/A47S F 5 -CAATGACCCCCATGACGTCGTCGTCGCAGAACAACGGCGTG-3 6 UapA T45S/F46S/A47S R 5 -CACGCCGTTGTTCTGCGACGACGACGTCATGGGGGTCATTG-3