page S1

Crystal structure of the prolyl-acyl carrier protein oxidase involved in the

biosynthesis of the cyanotoxin anatoxin-a

Karine Moncoqab*, Leslie Regadcb, Stéphane Mannde, Annick Méjeandeb, and Olivier Plouxde*

aCNRS, UMR 7099, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005

Paris, France

bUniversité Paris Diderot-Paris 7, 75013 Paris, France

cINSERM, UMR-S973, MTi, 35 rue Hélène Brion, 75205 Paris Cedex 13, France

dChimie ParisTech, ENSCP, Laboratoire Charles Friedel, 11 rue Pierre et Marie Curie, 75231 Paris

Cedex 05, France

eCNRS, UMR 7223, 75005 Paris, France

*Corresponding authors: O.P.: Phone: +(33) 1 44 27 67 01; E-mail: olivier-ploux@chimie-

paristech.fr. K.M.: Phone: +(33) 1 58 41 51 04; E-mail: moncoq@ibpc.fr.

Supporting Information. Figures S1 to S10.

page S2

Figure S1. Sequence of wild-type AnaB and of the recombinant histidine-tagged AnaB and their

calculated average molecular weight. The N-terminal sequence that has been added after cloning

into the pET-28 vector is in bold type.

>AnaBwt MDFAWNSQQIQFRKKVIQFAQQSLISDLIKNDKEEIFNRDAWQKCSEFGV 50 HGWPIPARYGGQELDILTTAYALQGLGYGCKDNGLIFAMNAHIWACEMPL 100 LTFGTEEQKEKYLPLLCRGGWIASHAATEPQAGSDIYSLKTTAQKDGDKY 150 ILNGYKHYVTNGTIADLFIIFATIDPSLGKEGLTTFMIEKDTPGLILSKP 200 ISKMGMRTAEVPELRLENCEVSAANRLGEEGTGLAIFNHSMEWERGFILA 250 AAVGTMERLLEQSIRYARSHKQFGQAIGKFQLVANKLVEMKLRLENAKAY 300 LYKVAWMKENKQMALLEASMANLYISEAWVQSCLEAIEIHGAYGYLTNTE 350 LERELRDAIASKFYSGTSEIQRVVIAKFLGL 381 Mw (average mass): 42 859.30 Da >H6AnaB MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFDFAWNSQQIQFRKK 50 VIQFAQQSLISDLIKNDKEEIFNRDAWQKCSEFGVHGWPIPARYGGQELD 100 ILTTAYALQGLGYGCKDNGLIFAMNAHIWACEMPLLTFGTEEQKEKYLPL 150 LCRGGWIASHAATEPQAGSDIYSLKTTAQKDGDKYILNGYKHYVTNGTIA 200 DLFIIFATIDPSLGKEGLTTFMIEKDTPGLILSKPISKMGMRTAEVPELR 250 LENCEVSAANRLGEEGTGLAIFNHSMEWERGFILAAAVGTMERLLEQSIR 300 YARSHKQFGQAIGKFQLVANKLVEMKLRLENAKAYLYKVAWMKENKQMAL 350 LEASMANLYISEAWVQSCLEAIEIHGAYGYLTNTELERELRDAIASKFYS 400 GTSEIQRVVIAKFLGL 416 Mw (average mass): 46 417.10 Da

page S3

Figure S2. AnaB elution on a calibrated gel filtration column. A: AnaB was first purified on a

nickel affinity column, concentrated by ultrafiltration and loaded (2 mg) on a Superdex S200

column (see the Materials and Methods section for details). B: the column was calibrated with

commercial molecular weight markers and the logarithm of the molecular weight was plotted

against the constant Kav. C: the Stokes radius was plotted against the constant Kav. The apparent

molecular weight of AnaB was 151 kDa and its apparent Stokes radius was 4.67 nm. The molecular

weight markers were the followings:

Protein Mw (kDa) RS (nm)a Ferritin 440 6.10 Aldolase 158 4.81 Conalbumin 75 3.64 Ovalbumin 43 3.05 Carbonic anhydrase 29 2.30 Ribonuclease 13.7 1.64

aData taken from: Erickson, H. P. (2009) Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy. Biol. Proced. Online 11, 32-51.

A

 

B

 C

 

 

 

0

500

1000

1500

2000

2500

0 5 10 15 20 25

Abs

orba

nce

(mA

U)

Elution volume Ve (mL)

3.5

4.0

4.5

5.0

5.5

6.0

0.2 0.3 0.5 0.6 0.8

log

Mw

Kav

1.0

2.0

3.0

4.0

5.0

6.0

7.0

0.2 0.3 0.5 0.6 0.8

Rs (n

m)

Kav

page S4

Figure S3. Multiple alignment (CLUSTAL X)1 of the sequence of AnaB with that of its homologs

for which a three-dimensional structure has been published (the sequences were downloaded from

the PDB website). A star indicates identity, a colon strong similarity, and a dot weak similarity. The

corresponding dendogram is shown (drawn using NJPlot).2 The enzyme names and sources are

collected in a Table, below.

3D9G --------------------------------------------------VDFKLSPSQL 10 3MKH ------------------------------------------------MAIDFHLSASQK 12 3D6B -----------------------------------MAAATFH--WDDPLLLDQQLADDER 23 1SIQ ---------------------------------------EFD--WQDPLVLEEQLTTDEI 19 2IX6 MAVLSSADRASNEKKVKSSYFDLPPMEMSVAFPQATPASTFPPCTSDYYHFNDLLTPEEQ 60 1IVH ------------------------------------------HSLLPVDDAINGLSEEQR 18 AnaB -----------------------------------------------MDFAWN---SQQI 10 1RX0 ------------------------------------MVQTGHRSLTSCIDPSMGLNEEQK 24 3MDD -------------------------------------------------GFSFELTEQQK 11 1JQI ---------------------------------------------LHSVYQSVELPETHQ 15 1BUC --------------------------------------------------MDFNLTDIQQ 10 3B96 -----------------------------ESKSFAVGMFKGQLTTDQVFPYPSVLNEEQT 31 3MPI --------------------------------------------------MDFNLSKELQ 10 1W07 ------------------------------------------------MEGIDHLADERN 12 2FON -----------------------------MRGSHHHHHHGSACELVREMEGVDYLADERK 31 1R2J ----------------------------------------------------------MP 2 3DJL -----------MHWQTHTVFNQPIPLNNSNLYLSDGALCEAVTREGAGWDSDFLASIGQQ 49 3D9G EARRHAQAFAN--TVLTKASAEYSTQK---DQLSRFQATRPFYREAVRHGLIKAQVPIPL 65 3MKH GTYQAARSLAR--NLLMPARQTYLQHPP--NSPLRFQSTQPTYAAAVSAGILKGQISPAH 68 3D6B MVRDAAHAYAQ--GKLAPRVTEAFRHE-------TTD--AAIFREMGEIGLLGPTIPEQY 72 1SIQ LIRDTFRTYCQ--ERLMPRILLANRNE-------VFH--REIISEMGELGVLGPTIKG-Y 67 2IX6 AIRKKVRECME--KEVAPIMTEYWEKA-------EFP--FHITPKLGAMGVAGGSIKG-Y 108 1IVH QLRQTMAKFLQ--EHLAPKAQEIDRSN-------EFKNLREFWKQLGNLGVLGITAPVQY 69 AnaB QFRKKVIQFAQ--QSLISDLIKNDKEE-------IFN--RDAWQKCSEFGVHGWPIPARY 59 1RX0 EFQKVAFDFAA--REMAPNMAEWDQKE-------LFP--VDVMRKAAQLGFGGVYIQTDV 73 3MDD EFQATARKFAR--EEIIPVAAEYDRTG-------EYP--VPLLKRAWELGLMNTHIPESF 60 1JQI MLRQTCRDFAE--KELVPIAAQLDKEH-------LFP--TSQVKKMGELGLLAMDVPEEL 64 1BUC DFLKLAHDFGE--KKLAPTVTERDHKG-------IYD--KELIDELLSLGITGAYFEEKY 59 3B96 QFLKELVEPVSRFFEEVNDPAKNDALE-------MVE--ETTWQGLKELGAFGLQVPSEL 82 3MPI MLQKEVRNFVN--KKIVPFADQWDNEN-------HFP-YEEAVRPMGELGFFGTVIPEEY 60 1W07 KAEFDVEDMKIVWAGSRHAFEVSDRIAR------LVASDPVFEKSNRARLSRKELFKSTL 66 2FON KAGFDVDEMKIVWAGSRHDFELTDRISK------LVASDPGFSKEGRTMLPRKELFKNTL 85 1R2J ERDALLTDLVG------DRAAEWDTSG-------ELP--RDLLVRLGADGLLCAEVAAEH 47 3DJL LGTAESLELGRLANVNPPELLRYDAQGRRLDDVRFHPAWHLLMQALCTNRVHNLAWEEDA 109 3D9G GGTM---ESLVHESIILEELFAVEPATSITIV---ATALGLMPVILCDSPSLQEKFLKPF 119 3MKH GGTG---GTLIESAILVEECYSVEPSAALTIF---ATGLGLTPINLAAGP-QHAEFLAPF 121 3D6B GGPG---LDYVSYGLIAREVERVDSGYRSMMS--VQSSLVMVPIFEFGSDAQKEKYLPKL 127 1SIQ GCAG---VSSVAYGLLARELERVDSGYRSAMS--VQSSLVMHPIYAYGSEEQRQKYLPQL 122 2IX6 GCPG---LSITANAIATAEIARVDASCSTFIL--VHSSLGMLTIALCGSEAQKEKYLPSL 163 1IVH GGSG---LGYLEHVLVMEEISRASGAVGLSYG--AHSNLCINQLVRNGNEAQKEKYLPKL 124 AnaB GGQE---LDILTTAYALQGLGYGCKDNGLIFAMNAHIWACEMPLLTFGTEEQKEKYLPLL 116 1RX0 GGSG---LSRLDTSVIFEALATGCTSTTAYIS--IHN-MCAWMIDSFGNEEQRHKFCPPL 127 3MDD GGLG---LGIIDSCLITEELAYGCTGVQTAIE--ANT-LGQVPLIIGGNYQQQKKYLGRM 114 1JQI SGAG---LDYLAYSIALEEISRGCASTGVIMS--VNNSLYLGPILKFGSSQQKQQWITPF 119 1BUC GGSGDDGGDVLSYILAVEELAKYDAGVAITLS--ATVSLCANPIWQFGTEAQKEKFLVPL 117 3B96 GGVG---LCNTQYARLVEIVGMHDLGVGITLG--AHQSIGFKGILLFGTKAQKEKYLPKL 137 3MPI GGEG-MDQGWLAAMIVTEEIARGSSALRVQLN--MEVLGCAYTILTYGSEALKKKYVPKL 117 1W07 RKCAHAFKRIIELRLNEEEAGRLRHFIDQPAYVDLHWGMFVPAIKGQGTEEQQKKWLSLA 126 2FON RKAAYAWKRIIELRLSQEEATMLRRYVDEPAFTDLHWGMFIPAIKGQGTDKQQEKWLPLA 145 1R2J GGLG---LGSRENGEFTAHVGSLCSSLRSVMT---SQGMAAWTVQRLGDAGQRATFLKEL 101 3DJL RSGAFVARAARFMLHAQVEAGSLCPITMTFAATPLLLQMLPAPFQDWTTPLLSDRYDSHL 169 . :

page S5

3D9G ISGEGE--PLASLMHSEPNGTANWLQKGGPGLQTTARKVGNEWVISG-----EKLWPSNS 172 3MKH LSGEGS--PLASLVFSEPGGVANALEKGAPGFQTTARLEGDEWVING-----EKMWATNC 174 3D6B ATGE----WIGCFGLTEPNHGS---DPGSMVTRARKVPG--GYSLSG-----SKMWITNS 173 1SIQ AKGE----LLGCFGLTEPNSGS---DPSSMETRAHYNSSNKSYTLNG-----TKTWITNS 170 2IX6 AQLN----TVACWALTEPDNGS---DASGLGTTATKVEG--GWKING-----QKRWIGNS 209 1IVH ISGE----YIGALAMSEPNAGS---DVVSMKLKAE--KKGNHYILNG-----NKFWITNG 170 AnaB CRGG----WIASHAATEPQAGS---DIYSLKTTAQ--KDGDKYILNG-----YKHYVTNG 162 1RX0 CTME----KFASYCLTEPGSGS---DAASLLTSAK-KQGD-HYILNG-----SKAFISGA 173 3MDD TEEP----LMCAYCVTEPGAGS---DVAGIKTKAE-KKGD-EYIING-----QKMWITNG 160 1JQI TNGD----KIGCFALSEPGNGS---DAGAASTTAR-EEGD-SWVLNG-----TKAWITNS 165 1BUC VEGT----KLGAFGLTEPNAGT---DASGQQTIAT-KNDDGTYTLNG-----SKIFITNG 164 3B96 ASGE----TVAAFCLTEPSSGS---DAASIRTSAVPSPCGKYYTLNG-----SKLWISNG 185 3MPI SSAE----FLGGFGITEPDAGS---DVMAMSSTAE--DKGDHWLLNG-----SKTWISNA 163 1W07 NKMQ----IIGCYAQTELGHGS---NVQGLETTATLDPKTDEFVIHTPTQTASKWWPGGL 179 2FON YKMQ----IIGCYAQTELGHGS---NVQGLETTATFDPQTDEFVIHSPTLTSSKWWPGGL 198 1R2J TSGK-----LAAVGFSERQAGS---DLSAMRTRVR--LDGDTAVVDG-----HKVWTTAA 146 3DJL LPGGQKRGLLIGMGMTEKQGGS---DVMSNTTRAERLEDG-SYRLVG------HKWFFSV 219 . :* : : . . : : : 3D9G GGWDYKGADLACVVCRVSDDPSKPQDPNVDPATQIAVLLVTRETIANNKKDA-YQILGEP 231 3MKH AGWDFKGCDLACVVCR---DATTPLEEGQDPENKVMIILVTRADLDRNGEGS-FEVLRHV 230 3D6B P-----IADVFVVWAKLD----------EDGRDEIRGFILEKGCKG-------LSAPAIH 211 1SIQ P-----MADLFVVWARC-----------EDG--CIRGFLLEKGMRG-------LSAPRIQ 205 2IX6 T-----FADLLIIFARNT----------TTN--QINGFIVKKDAPG-------LKATKIP 245 1IVH P-----DADVLIVYAKTDL---A----AVPASRGITAFIVEKGMPG-------FSTSKKL 211 AnaB T-----IADLFIIFATIDP---S----LG--KEGLTTFMIEKDTPG-------LILSKPI 201 1RX0 G-----ESDIYVVMCRTG----------GPGPKGISCIVVEKGTPG-------LSFGKKE 211 3MDD G-----KANWYFLLARSDPDP------KAPASKAFTGFIVEADTPG-------VQIGRKE 202 1JQI W-----EASATVVFASTD---------RSRQNKGISAFLVPMPTPG-------LTLGKKE 204 1BUC G-----AADIYIVFAMTD---------KSKGNHGITAFILEDGTPG-------FTYGKKE 203 3B96 G-----LADIFTVFAKTPVTDPA----TGAVKEKITAFVVERGFGG-------ITHGPPE 229 3MPI A-----QADVLIYYAYTDK---------AAGSRGLSAFVIEPRNFP-------GIKTSNL 202 1W07 G----KVSTHAVVYARLIT---------NGKDYGIHGFIVQLRSLEDHSPLPNITVGDIG 226 2FON G----KVSTHAVVYARLIT---------DGKDYGVNGFIVQLRSLEDHKPLPGVTVGDIG 245 1R2J A-----YADHLVVFGLQE-------------DGSGAVVVVPADTPG-------VRVERVP 181 3DJL P-----QSDAHLVLAQTAG--------------GLSCFFVPRFLPD--GQRNAIRLERLK 258 . ..: 3D9G ELAG---HITTSGPHTRFTEFHVPHENLLCTPGLKAQGLVETA-----------FAMSAA 277 3MKH ATPG---HTSVSGPHVRYTNVRVPTKNVLCPAGQGAK-VAFGA-----------FDGSAV 275 3D6B GKVG---LRASITGEIVLDEAFVPEENILPHVKG-LRGPFTC------------LNSARY 255 1SIQ GKFS---LRASATGMIIMDGVEVPEENVLPGASS-LGGPFGC------------LNNARY 249 2IX6 NKIG---LRMVQNGDILLQNVFVPDEDRLPGVNS-FQDTSKV------------LAVSRV 289 1IVH DKLG---MRGSNTCELIFEDCKIPAANILGHENKGVYVLMSG------------LDLERL 256 AnaB SKMG---MRTAEVPELRLENCEVSAANRLGEEGTGLAIFNHS------------MEWERG 246 1RX0 KKVG---WNSQPTRAVIFEDCAVPVANRIGSEGQGFLIAVRG------------LNGGRI 256 3MDD INMG---QRCSDTRGIVFEDVRVPKENVLTGEGAGFKIAMGT------------FDKTRP 247 1JQI DKLG---IRASSTANLIFEDCRIPKENLLGEPGMGFKIAMQT------------LDMGRI 249 1BUC DKMG---IHTSQTMELVFQDVKVPAENMLGEEGKGFKIAMMT------------LDGGRI 248 3B96 KKMG---IKASNTAEVFFDGVRVPSENVLGEVGSGFKVAMHI------------LNNGRF 274 3MPI EKLG---SHASPTGELFLDNVKVPKENILGKPGDGARIVFGS------------LNHTRL 247 1W07 TKMGNGAYNSMDNGFLMFDHVRIPRDQMLMRLSKVTREGEYVPSDVPKQLVYGTMVYVRQ 286 2FON MKFGNGAYNSMDNGVLSFDHVRIPRDQMLMRVSQVTKEGKYVQSDIPRQLLYGTMVYVRQ 305 1R2J KPSG---CRAAGHADLHLDQVRVPAGAVLAGSGASLPMLVAAS-----------LAYGRK 227 3DJL DKLG---NRSNASCEVEFQDAIG---WLLGLEGEGIRLILKMG------------GMTRF 300 . : 3D9G LVGAMAIGTARAAFEEALVFAKSDTRGGS------KHIIEHQSVADKLIDCKIRLETSRL 331 3MKH LVGAMGVGLMRAAFDAALKFAKEDNRGGA------VPLLERQAFADLLSGVKIQTEAARA 329 3D6B GIAWGALGAAESCWHIARQYVLDRKQFG-------RPLAANQLIQKKLADMQTEITLGLQ 308 1SIQ GIAWGVLGASEFCLHTARQYALDRMQFG-------VPLARNQLIQKKLADMLTEITLGLH 302 2IX6 MVAWQPIGISMGIYDMCHRYLKERKQFG-------APLAAFQLNQQKLVQMLGNVQAMFL 342 1IVH VLAGGPLGLMQAVLDHTIPYLHVREAFG-------QKIGHFQLMQGKMADMYTRLMACRQ 309 AnaB FILAAAVGTMERLLEQSIRYARSHKQFG-------QAIGKFQLVANKLVEMKLRLENAKA 299 1RX0 NIASCSLGAAHASVILTRDHLNVRKQFG-------EPLASNQYLQFTLADMATRLVAARL 309 3MDD PVAAGAVGLAQRALDEATKYALERKTFG-------KLLAEHQGISFLLADMAMKVELARL 300 1JQI GIASQALGIAQASLDCAVKYAENRHAFG-------APLTKLQNIQFKLADMALALESARL 302 1BUC GVAAQALGIAEAALADAVEYSKQRVQFG-------KPLCKFQSISFKLADMKMQIEAARN 301 3B96 GMAAALAGTMRGIIAKAVDHATNRTQFG-------EKIHNFGLIQEKLARMVMLQYVTES 327 3MPI SAAAGGVGLAQACLDAAIKYCNERRQFG-------KPIGDFQMNQDMIAQMAVEVEAARL 300 1W07 TIVADASNALSRAVCIATRYSAVRRQFGAHNGGIETQVIDYKTQQNRLFPLLASAYAFRF 346 2FON SIVADASLAMSRAVCIATRYSAVRRQFGSQNGGQETQVIDYKTQQNRLFPLLASAYAFRF 365 1R2J SVAWGCVGILRACRTAAVAHARTREQFG-------RPLGDHQLVAGHIADLWTAEQIAAR 280 3DJL DCALGSHAMMRRAFSLAIYHAHQRHVFG-------NPLIQQPLMRHVLSRMALQLEGQTA 353 . * : :

page S6

3D9G LVWKAVTTLEDEAL--------EWKVKLEMAMQTKIYTTDVAVECVIDAMKAVGMKSYAK 383 3MKH LTWKAAHAMENGPG--------DYDARRELALAAKVFCSEAAVKACTDVINAVGISAYDL 381 3D6B GVLRLGRMKD------------EGTAAVEITSIMKRNSCGKALDIARLARDMLGGNGISD 356 1SIQ ACLQLGRLKD------------QDKAAPEMVSLLKRNNCGKALDIARQARDMLGGNGISD 350 2IX6 MGWRLCKLYE------------TGQMTPGQASLGKAWISSKARETASLGRELLGGNGILA 390 1IVH YVYNVAKACD------------EGHCTAKDCAGVILYSAECATQVALDGIQCFGGNGYIN 357 AnaB YLYKVAWMKE------------NKQMALLEASMANLYISEAWVQSCLEAIEIHGAYGYLT 347 1RX0 MVRNAAVALQE-----------ERKDAVALCSMAKLFATDECFAICNQALQMHGGYGYLK 358 3MDD SYQRAAWEIDS-----------GRR-NTYYASIAKAYAADIANQLATDAVQVFGGNGFNT 348 1JQI LTWRAAMLKDN-----------KKP-FTKESAMAKLAASEAATAISHQAIQILGGMGYVT 350 1BUC LVYKAACKKQE-----------GKP-FTVDAAIAKRVASDVAMRVTTEAVQIFGGYGYSE 349 3B96 MAYMVSANMDQ-----------GATDFQIEAAISKIFGSEAAWKVTDECIQIMGGMGFMK 376 3MPI LAYKAAAAKDE-----------GRLNNGLDVAMAKYAAGEAVSKCANYAMRILGAYGYST 349 1W07 VGEWLKWLYTDVTERLAASDFATLPEAHACTAGLKSLTTTATADGIEECRKLCGGHGYLW 406 2FON VGEWLKWLYTDVTQRLAANDFSTLPEAHACTAGLKSLTTSATADGIEECRKLCGGHGYLC 425 1R2J VCEYASDHWDE-----------GSPEMVPATILAKHVAAERAAAGAATAAQVLASAGARE 329 3DJL LLFRLARAWDRR------ADAKEALWARLFTPAAKFVICKRGMPFVAEAMEVLGGIGYCE 407 . . 3D9G DMSFPRLLNEVMCYPLFDGGNIGLRRRQMQRVMALEDYEPWAATYGSSKVDKSRL----- 438 3MKH QRPFSDLLNTAVVLPIFDGGNVGIRRRHLQQLMLKPTYDAWSSTYGSFPGSHHHHHH--- 438 3D6B EFGVARHLVNLEVVNTYEGTHDIHALILGRAQTGIQAFF--------------------- 395 1SIQ EYHVIRHAMNLEAVNTYEGTHDIHALILGRAITGIQAFTASK------------------ 392 2IX6 DFLVAKAFCDLEPIYTYEGTYDINTLVTGREVTGIASFKPATRSRLDDDGKLEHHHHHH- 449 1IVH DFPMGRFLRDAKLYEIGAGTSEVRRLVIGRAFNADFH----------------------- 394 AnaB NTELERELRDAIASKFYSGTSEIQRVVIAKFLGL-------------------------- 381 1RX0 DYAVQQYVRDSRVHQILEGSNEVMRILISRSLLQE------------------------- 393 3MDD EYPVEKLMRDAKIYQIYEGTAQIQRIIIAREHIGRYK----------------------- 385 1JQI EMPAERYYRDARITEIYEGTSEIQRLVIAGHLLRSYRS---------------------- 388 1BUC EYPVARHMRDAKITQIYEGTNEVQLMVTGGALLR-------------------------- 383 3B96 EPGVERVLRDLRIFRIFEGTNDILRLFVALQGCMDKGKELSGLGSALKNPFGNAGLLLGE 436 3MPI EYPVARFYRDAPTYYMVEGSANICKMIIALDQLGVRKANRKGHHHHHH------------ 397 1W07 CSGLPELFAVYVPACTYEGDNVVLQLQVARFLMKTVAQLGSGKVPVGTTAYMGRAAHLLQ 466 2FON SSGLPELFAVYVPACTYEGDNVVLQLQVARFLMKTISQLGTGKKPVGTVSYMGRIEHLMQ 485 1R2J GHVVERAYRDAKLMEIIEGSSEMCRVMLAQHALALPA----------------------- 366 3DJL ESELPRLYREMPVNSIWEGSGNIMCLDVLRVLNKQAGVYDLLSEAFVEVKGQDRYFDRAV 467 * 3D9G ------------------------------------------------------------ 3MKH ------------------------------------------------------------ 3D6B ------------------------------------------------------------ 1SIQ ------------------------------------------------------------ 2IX6 ------------------------------------------------------------ 1IVH ------------------------------------------------------------ AnaB ------------------------------------------------------------ 1RX0 ------------------------------------------------------------ 3MDD ------------------------------------------------------------ 1JQI ------------------------------------------------------------ 1BUC ------------------------------------------------------------ 3B96 AGKQLRRRAGLGSGLSLSGLVHPELSRSGELAVRALEQFATVVEAKLIKHKKGIVNEQFL 496 3MPI ------------------------------------------------------------ 1W07 CRSGVQKAEDWLNPDVVLEAFEARALRMAVTCAKNLSKFENQEQGFQELLADLVEAAIAH 526 2FON CRSDVKQAEDWLKPSAVLEAFEARSARMSVACAKNLSKFENQEEGFAELAADLVEAAVAH 545 1R2J ------------------------------------------------------------ 3DJL RRLQQQLRKPAEELGREITHQLFLLGCGAQMLKYASPPMAQAWCQVMLDTRGGVRLSEQI 527 3D9G ------------------------------------------------------------ 3MKH ------------------------------------------------------------ 3D6B ------------------------------------------------------------ 1SIQ ------------------------------------------------------------ 2IX6 ------------------------------------------------------------ 1IVH ------------------------------------------------------------ AnaB ------------------------------------------------------------ 1RX0 ------------------------------------------------------------ 3MDD ------------------------------------------------------------ 1JQI ------------------------------------------------------------ 1BUC ------------------------------------------------------------ 3B96 LQRLADGAIDLYAMVVVLSRASRSLSEGHPTAQHEKMLCDTWCIEAAARIREGMAALQSD 556 3MPI ------------------------------------------------------------ 1W07 CQLIVVSKFIAKLEQDIGGKGVKKQLNNLCYIYALYLLHKHLGDFLSTNCITPKQASLAN 586 2FON CQLIVVSKYIEKLQQNIPGKGVKQQLEVLCGIYSLFILHKHQGDFLGTGYITSKQGSLAN 605 1R2J ------------------------------------------------------------ 3DJL QNDLLLRATGGVCV---------------------------------------------- 541

page S7

3D9G ------------------------------------------------------------ 3MKH ------------------------------------------------------------ 3D6B ------------------------------------------------------------ 1SIQ ------------------------------------------------------------ 2IX6 ------------------------------------------------------------ 1IVH ------------------------------------------------------------ AnaB ------------------------------------------------------------ 1RX0 ------------------------------------------------------------ 3MDD ------------------------------------------------------------ 1JQI ------------------------------------------------------------ 1BUC ------------------------------------------------------------ 3B96 PWQQELYRNFKSISKALVERGGVVTSNPLGF----------------------------- 587 3MPI ------------------------------------------------------------ 1W07 DQLRSLYTQVRPNAVALVDAFNYTDHYLNSVLGRYDGNVYPKLFEEALKDPLNDSVVPDG 646 2FON DQLRALYSQLRPNAVSLVDAFNYTDHYLGSILGRYDGNVYPKLYEAAWKDPLNKSDIADG 665 1R2J ------------------------------------------------------------ 3DJL ------------------------------------------------------------ 3D9G ------------------ 3MKH ------------------ 3D6B ------------------ 1SIQ ------------------ 2IX6 ------------------ 1IVH ------------------ AnaB ------------------ 1RX0 ------------------ 3MDD ------------------ 1JQI ------------------ 1BUC ------------------ 3B96 ------------------ 3MPI ------------------ 1W07 YQEYLRPVLQQQL----- 659 2FON FHEYIRPLLKQQLRTAKL 683 1R2J ------------------ 3DJL ------------------

1 IVH

2 IX6

3D6B

1SIQ1

R2J

3DJL

3D9G

3MKH

1W07

2FON

3MPI

3B96

1JQI 1

BUC

1RX0

3MDD

AnaB

0 . 1

page S8

PDB

accession

Enzyme Enzyme

class

Source

3D9G nitroalkane oxidase NAOX Fusarium oxysporum

3MKH nitroalkane oxidase NAOX Podospora anserina

3D6B glutaryl-CoA dehydrogenase GCD Burkholderia pseudomallei

1SIQ glutaryl-CoA dehydrogenase GCD Homo sapiens

2IX6 short-chain acyl-CoA oxidase ACOX4 Arabidopsis thaliana

1IVH isovaleryl-CoA dehydrogenase IVD Homo sapiens

4IRN prolyl-acyl carrier protein oxidase AnaB Oscillatoria sp. PCC 6506

1RX0 isobutyryl-CoA dehydrogenase IBD Homo sapiens

3MDD medium-chain acyl-CoA dehydrogenase MCAD Sus scrofa

1JQI short-chain acyl-CoA dehydrogenase SCAD Rattus norvegicus

1BUC butyryl-CoA dehydrogenase BCAD Megasphaera elsdenii

3B96 very long-chain acyl-CoA dehydrogenase VLCAD Homo sapiens

3MPI glutaryl-CoA dehydrogenase GCD Desulfococcus multivorans

1W07 acyl-CoA oxidase 1 ACOX1 Arabidopsis thaliana

2FON acyl-CoA oxidase 1 ACOX1 Solanum lycopersicum

1R2J acyl-ACP dehydrogenase, FkbI ACAD Streptomyces hygroscopicus

3DJL alkylation response protein, AidB ACAD Escherichia coli

(1) Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) CLUSTAL W: improving the

sensitivity of progressive multiple sequence alignment through sequence weighting, position-

specific gap penalties and weight matrix choice, Nucleic Acids Res. 22, 4673-4680.

(2) Perrière, G., and Gouy, M. (1996) WWW-query: An on-line retrieval system for biological

sequence banks, Biochimie 78, 364-369.

page S9

Figure S4. An artist view of the topology of AnaB secondary structure. α-Helices are represented

by cylinders, and β-strands by arrows, at approximate scale. The secondary structure elements are

labeled using the nomenclature proposed for MCAD (PDB accession: 3MDD).1

 

     (1) Kim, J. J., Wang, M., and Paschke, R. (1993) Crystal structures of medium-chain acyl-CoA

dehydrogenase from pig liver mitochondria with and without substrate, Proc. Natl. Acad. Sci. U. S.

A. 90, 7523-7527.  

A

H

B

C

D

E

F

G

I

J

1

2

3

4 5

6

7 7

N

C K

page S10

Figure S5. Stereo view of Figure 1D. Superposition of AnaB and human IVD monomers. AnaB is

colored as in Figure 1A, IVD chain in grey, and the FAD as in Figure 1A. Helices A, B, D, E and G

where the main differences between AnaB and IVD fold are visible, are highlighted while the rest

of the chains are shown in transparent cartoons.

αB αA

αE

αG αD

β4-β5 loop

αB αA

αE

αG αD

β4-β5 loop

page S11

Figure S6. Stereo view of the superposition of the α-E α-G region in the structure of AnaB with

that of MCAD. A: Helices E and G (with the catalytic base E244) of AnaB are represented in blue

and yellow, respectively, while helices J and K with their connecting loop are colored in orange.

The superimposed structure of MCAD is displayed in grey (with the catalytic base E376). The

residues responsible for making the substrate pocket shallower in AnaB and the corresponding

residues in MCAD are labeled and shown in sticks. The distance between the Cα atoms of I248 and

N90 in AnaB is 5.4 Å, and the corresponding distance in MACD (between and V259 and E99) is

8.0 Å. For clarity the ligands are not represented in A. B: The cofactor FAD of AnaB and the

octanoyl-CoA substrate of MCAD are displayed in magenta and grey lines, respectively.

A

B

αE

αG W94

E244 E376 E99

 V259

T96

αJ

αK  I248

 F87

 N90

αE

αG W94

E244 E376 E99

 V259

αJ

αK  I248

 F87

 N90

αE

αG W94

E244

 E376 E99

 V259

αJ

αK  I248

 F87

 N90

FAD

C8-CoA

T96

αE

αG W94

E244

 E376 E99

 V259

αJ

αK  I248

 F87

 N90

FAD

C8-CoA

T96

page S12

Figure S7. Schematic representation of the interactions between FAD and the polypeptide chain of

AnaB (chain A). The figure was generated using the LIGPLOT software1 available on line at the

PDBsum website2.

(1) Wallace, A. C., Laskowski, R. A., and Thornton, J. M. (1996) LIGPLOT: a program to

generate schematic diagrams of protein-ligand interactions, Protein Eng. 8, 127-134.

(2) Laskowski, R. A. (2009) PDBsum new things, Nucleic Acids Res. 37, D355-D359.

2.59

2.98

2.81

3.01

2.66

2.93

2.78

2.94

2.53

2.732.46

3.08

2.74

P

O3P O2P

O1P

O5*

C5*

C4*

O4*

C3*

O3*

C2*

O2*

C1*

N10

C10

C9A

C9

C8

C8M

C7

C7M

C6

C5A

N5

C4A

C4

O4

N3

C2 O2

N1

AN3 AC4

AC2

AN1 AC6

AN6

AC5 AN7

AC8

AN9

AC1*

AC2*AO2*

AC3*

AO3*

AO4*

AC4*

AC5*

AO5*

AP

AO2 AO1

C O

CB CG2

OG1

CA

N

C

O

CB

CG2

OG1

CA N

C O

CB

OG

CA N

C

O

CE2

CD2

CZ

CE1

CD1

CG

CB

CA

N

C

O

CD

OE2

OE1

CG

CB CA

N

C O

NE2 CD2

CE1

ND1

CG CB

CA

N

C

O CA

CB

N

C

O

CD NE2

OE1

CG

CB

CA

N

C

O

CA CB

N

C

O

NE2

CD2

CE1

ND1

CG

CB

CA

N

C

O

CD OE2

OE1

CG CB

CA

N

Phe 280(B)

Tyr 158(A)

Gly 133(A)

Phe 273(B)

Ile 277(B)

Val 283(B)

Gly 341(B)

Val 159(A)

Ile 339(B)

Phe 87(A)

Thr 367(A)

Ile 370(A)

Val 373(A)

Fad 400(A)

Thr 128(A)

Thr 160(A)

Ser 134(A)Phe 363(A)

Glu 369(A)

His 125(A)

Ala 342(B)

Gln 281(D)

Ala 127(A)

His 270(B)

Glu 338(B)

page S13

Figure S8. Stereo view of the superposition of AnaB (yellow) and IVD (cyan) monomers in the

region surrounding the adenosine moiety of the CoA persulfide ligand (magenta) of IVD. FAD,

CoASSH, and important residues are displayed as sticks. Hydrogen bonds are shown as dotted

lines. For clarity, the full-length proteins are shown as transparent cartoons. There are two residues

of IVD, Ser190 and Arg191 that form hydrogen bonds with the ribose 3'-phosphate of the CoASSH

ligand. The corresponding residues in AnaB, Lys180 and Glu181, are strikingly different, and, in

the overlaid structure, seem to make steric or charge repulsions, with the adenosine part of

CoASSH. In addition, the presence of the bulky residue Tyr137 unique to AnaB when compared to

all others ACADs, seems to be incompatible with the binding of the adenosine CoA moiety.

Y137

V145 FAD

K180

S190

R191

CoA-SSH

E181

Y137

V145 FAD

K180

S190

R191

CoA-SSH

E181

page S14

Figure S9. Comparison of the location of His residues near the flavin N5-C4a locus, in AnaB

(yellow) and in glucose oxidase (grey, pdb 1GAL). Stereo view.

His559

His516

His125

His559

His516

His125

page S15

Figure S10. Ramachandran plot for AnaB.

MolProbity Ramachandran analysis

http://kinemage.biochem.duke.edu Lovell, Davis, et al. Proteins 50:437 (2003)

General case

-180

1800-180

180

0

Phi

Psi

Pre-proline

Trans proline

Glycine

Isoleucine and valine

Cis proline

-180

1800-180

180

0

Phi

Psi

-180

1800-180

180

0

Phi

Psi

-180

1800-180

180

0

Phi

Psi

-180

1800-180

180

0

Phi

Psi

-180

1800-180

180

0

Phi

Psi

A 158 TYRB 158 TYRD 158 TYRF 158 TYRG 158 TYRH 158 TYR

95.6% (2877/3008) of all residues were in favored (98%) regions.99.8% (3002/3008) of all residues were in allowed (>99.8%) regions.

There were 6 outliers (phi, psi): A 158 TYR (55.0, 87.9) B 158 TYR (54.8, 87.9) D 158 TYR (55.0, 88.0) F 158 TYR (55.1, 87.4) G 158 TYR (54.8, 87.0) H 158 TYR (54.7, 88.0)

rcsb077122_pdbv3.pdb, model 1