Supplementary Information. Mechanistic insights into a calcium-dependent family of α mannosidases in a human gut symbiont

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1 Supplementary Information Mechanistic insights into a calcium-dependent family of α mannosidases in a human gut symbiont Yanping Zhu 1,5* Michael DL Suits 2,*, Andrew J Thompson 2, Sambhaji Chavan 3, Zoran Dinev 4, Claire Dumon 1, Nicola Smith 1, Kelley Moremen 5, Yong Xiang 5, Aloysius Siriwardena 3, Spencer J Williams 4, Harry J Gilbert 1,5, and Gideon J Davies 2, 1 Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, U.K. 2 York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York, YO10 5YW, United Kingdom. 3 Université de Picardie Jules Vernes, Faculté des Sciences, Laboratoire des Glucides (CNRS-UMR 6219), 33, Rue Saint Leu, Amiens, France 4 School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia. 5 : Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia , USA Current address: INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F Toulouse; Université de Toulouse; INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F Toulouse; CNRS, UMR5504, F Toulouse, France Running title Human bowel α-mannosidase superfamily: function, structure and inhibition *these authors contributed equally Correspondence to either davies@ysbl.york.ac.uk or hgilbert@ccrc.uga.edu

2 % Intensity Mass (m/z) E+4 α-1, 2-Mannosidase Bt3990 Man Man9GlcNAc2 9 2 Man Man5GlcNAc2 5 GlcNAc 2 Supplemental Figure 1. Cleavage of Man 9 GlcNAc 2 by Bt2199 and Bt3990. The two enzymes were incubated with Man 9 GlcNAc 2 and the reaction products generated were then permethylated and subjected to MALDI-TOF MS analysis. The data show that Bt3990 is an exoacting α-1,2-mannosidases that targets all four α 1,2 mannosides in a Man 9 GlcNAc 2 glycan. Both Bt2199 and 3990 target the same substrate and generate the same end-product; data here are shown only for Bt3990

3 α 1,2 Mannobiose α 1,3 Mannobiose α 1,4 Mannobiose 0 min 0 min 0 min 1 min H 1β Man 1 (157) 1 min 1 min H 1α Man 1 (100) 3 min 3 min H 1β Man 1 (67) 6 min H 1α Man 1 (46) H 1β Man 1 H 1α Man 1 3 min H 1β Man 1 H 1α Man 1 (27) 6 min H 1β Man 1 (23) 20 min H 1α Man 1 H 1α Man 1 H 1β Man 1 10 min H 1β Man 1 H 1α Man 1 H 1β Man 1 12 min H 1α Man 1 H 1α Man 1 1 h H 1β Man 1 24 h H 1α Man 1 (100) H 1β Man 1 (51) H 1α Man 1 (100) 24 h H 1β Man 1 (55) H 1α Man 1 (100) 24 h H 1β Man 1 (59) Parts per million 1 H Parts per million 1 H Parts per million 1 H Supplemental Figure 2. Stereochemistry of mannobiose hydrolysis by representative B. thetaiotaomicron GH92 α-mannosidases. α-1,2-mannobiose, α-1,3-mannobiose and α-1,4-mannobiose were incubated with Bt3990, Bt3858 and Bt4073, respectively and the stereochemistry of the reaction was monitored by NMR (see experimental). Note that as 50 % of the mannose is derived from the reducing end of the disaccharide substrate, which will be a mixture of the two anomers in a ratio of 2:1 α:β, a significant amount of α-mannose is evident from even the very early phase of the reactions. However, it is also apparent that the ratio of α:β anomers of mannose (shown in parenthesis for selected spectra) in the initial phase of each reaction is significantly lower than when the monosaccharide has fully mutarotated after 24 h.

4 Supplemental Figure 3. ph profile for α mannosidase Bt3990 The buffers used were as follows: 100 mm Na citrate, ph , 100 mm MES ph , 100 mm MOPS ph 7.0, 100 mm HEPES ph 7.5, 100 mm Tris-HCl ph , 100 mm Bicine ph 9.0. The substrate deployed was 4NP-mannopyranoside 1 at a substrate concentration of 400 μμ (10-fold lower than the K M ) and the release of 4-nitrophenolate was as described in Methods.

5 Supplemental Figure 4. Alignment of the mature regions of B. thetaiotaomicron GH92 enzymes Bt GSSGSRWMIGPGPWMPFGMVKLMPDNEDAH WKAGYEYNVENI 321 Bt GTAHSRWMIAPGTWMPFSMVKLSPDNQDSG WQSGYDPSFESI 332 Bt GAVCPNGMMSVVPFNVMGSADNTYDKDARW WSTPYEHTNCFF 74 Bt GVYHGLGKTFPGATTPYGMVQVSPNTITGGD NSSGYSDEHKTI 65 Bt ADNGHTFPGACYPFGMIQTSPVT-GAVG WRYCSEYTYQDSLI 61 Bt GGHGHTFPGAVVPHGMIQPSPDT-RIDG WDACSGYYYDDNTI 63 Bt DFTGNTYPGAQAPFGMVQLSPDN-GLPG WDRISGYFYPDSTI 62 Bt IDGGLSGNNYPGATSPFGMIQLSPDTSEAPN WGDASGYDYNRNTI 62 Bt GFHGHVFVGTSVPYGMVQLGPNN-IHKG WDWCSGYHYSDSIL 61 Bt DGYGNVYPGAQIPFGGIQISPDTDSR FYDAASGYKYNHLTL 41 Bt NGMGHTFPGACTPFGWVQLSPDTDTIPHNVNGAYQKNAYEYCAGYQYRDKTI 65 Bt STFELSTGNTYPAIARPWGMNFWTPQTGKMGDG WQYTYTANKI 58 Bt SKHALSTGNTYPAIALPWGMNFWVPQTGKMGDG WAYTYDADKI 64 Bt SKFELSTGNTYPATALPWGMNFWTPQTGKMGDG WAYTYNADKI 62 Bt SKFELSTGNTYPATALPWGMNFWTPQTGKMGDG WAYTYDADKI 62 Bt SKISLSNGNTYPAIALPWGMNFWMPQTGKMGDG WAYTYASDKI 58 Bt STFAFSHGNTYPAVAVPWGMNFWSPQTGENGSG WMYTYTDSLM 63 Bt SNFGLSYGNTYPTTGMPYGMHMWSAQTGKNGDG FKYMYAVDKI 61 Bt2948 GVAVAGHEDPSGYTFPGVTEPFGMTEWTAHTLESKHAG TLHHRVPYWYKHNYI 79 Bt HLLVPTYPTVHLPNSMLRVYPERGD YTSDRV 54 Bt HLLVPTFPTIQLPNSMLRVYPERAD YTSELL 54 Bt TGLTSGYLYPGATYPYGMVQFTPSYFSKRS Bt DNGQVAPGAAAPFGMVCVCPDNDPRSHAG YDYAVTKV 57 Bt2105 MGFSHIHE----WTMTG--LLMMPTTGDLKIQPGTEK---QPDYGYRSRINKKTETARIG 372 Bt2111 GTFSHIHE----WTMAG---LGIMPANGPLKTEIGSQSSLVKDANSYRSAIDKTSEETKVG386 Bt1032 TGYSHVNLSGVGCP-ELGSLLLMPTTGELNVDYKEYG SKYKDEQASPG 121 Bt4073 EGFAFTQMSGVGWFGDLGNFLVMPTTGELQKIAGKED---GSIKGYRSSYDKATETAKAG 122 Bt1878 WGFTQTHLNGTGCM-DLGDILVMPVTG TRA---RAWDAYRSHFPKDKEAATPG 110 Bt3991 NGFSHTHVSGTGCC-DYGDVLLMPTVGKPQYLTTDPE---SQKLAYASAFSHENETAEPG 119 Bt3994 AGFSHTHLSGTGAG-DLYDISFMPVTLPYK-----EA---EAPLGIYSKFSHDEESAYAG 113 Bt3130 FGFSHTRLSGTGAS-DLIDITLMPTSS GRTSSAFTHDEEKARPG 105 Bt3773 IGFSHTHLSGTGCT-DLGDILIMPLNEIRTP-RGNQD---DIRDGYASKYSHDNEIARPE 116 Bt3965 MGFSLTHLSGTGIP-DLGDFLFIPGTGEMKLEPGTHE---DPDQGYRSRYSHDKEWASPN 97 Bt3963 VGFSHTHLSGTGHS-DLGDILLMPAVGDVKLNPGRAD---HPEEGYRSRFDHATEKATPG 121 Bt3990 RGFKQTHQPS-PWINDYGQFSIMPIVGQPVFD EEKRASWFAHKGEVATPY 107 Bt3527 RGFKQTHQPS-PWINDYGQFSIMPITGKAVFD QDQRASWFSHKAETATPY 113 Bt3784 RGFKQTHQPS-PWMNDYGQFSIMPITGGLVFD QDQRASWFSHKAEVAKPY 111 Bt2629 RGFKQTHQPS-PWMNDYGQFAIMPITGGLVFD QDRRASWFSHKAEVAKPY 111 Bt3962 RGFKQTHQPS-PWINDYGQFSIMPMTKQLKID QDSRASWFSHKAEKATPY 107 Bt2199 RGFRQTHQPS-PWINDYGTFSIMPLAGELKMS HKERLVPFSHQQEKATPY 112 Bt4092 RAFSQSHQCS-PWVSDYAVYSFMPMVGELVVN QDARATKFSHDNEIAKPH 110 Bt2948 SGFMGTHYPSGAVMFDYGAVELMPVVGQLKCR PEERSSSFTHITEKSKPH 129 Bt3858 NGLPVVVTSH----RGSSAFNLSPVQGEVSR PIVSYSYDLENITPY 96 Bt1769 KGLPLIVTNH----RERSAFNFSPYQGEKLR PVITYNYDNEHITPY 96 Bt4093 -GFVINQLSG-GGCEHMGNFPTFPVKGKLKMS PDNILNYRINVSEEKGHA 97 Bt3530 SGISVNRLSGVGCSGGGGNLRIRPVAPSQELH IKKSREKATPG 100 * *

6 Bt2105 YYSVNLTDYNIQAELTATTRSSLQRYTFNKAEQPRILVDFFFPAEYDWNLDDVYVKKVSD 432 Bt2111 YYKVDLTDYQIKAELTATSRCGFQRYTYPQDKDARVMIDLKIPSEYDYQIVEGSVKQTGA 446 Bt1032 YYSNYLTKYNIKTEVSATPRTGIARFTFPKGESHILLN----LGEGLTNESGAMLRRVSD 177 Bt4073 YYSVELTDYKIKVESSATPHCGILQFTFPSNEQSRIQIDLARRVGGTS--TSQYVKVLDD 180 Bt1878 YYTVELSDPQVKAELTASIHAALHRYTYHKADSASLLIDLQHGPAWREEQYHSQVNSCEV 170 Bt3991 YYSVFLDTYQVKAEITATKRGAIHRYTFPESTESGFIIDLDYSLQRQTN------YEMEI 173 Bt3994 YYQVRLKDYHINVELTATERCGIQRYTFPK-AEAAIFLNLKKAMN---WD---FTNDSHI 166 Bt3130 YYQVMLKDENINAELTTTQRNGIHRYQYPAGKDAEIILDMDHSADKGSWGR--RIINSQI 163 Bt3773 YYSLILDRYNIKAELTATDRVGFHRYTYPEGKPVSILIDLREGNGS NAYDSYI 169 Bt3965 YYAVELADYGVKAEMTSGVRSGMFRFTYPESDNAFIMIDMNHTLWQS CEWSNL 150 Bt3963 YYEVMLDDYGIKAQLTATQRTGVHKYTFPKGKDGHLILDLVHGIYNYDGK----VLWANL 177 Bt3990 YYKVYLAEHDIVTEMTPTERAVLFRFTFPENDHSYVVVDAFD KGSYIK 155 Bt3527 YYKVYLADHDVVTEIAPTERAAAFRFTFPENDHSYVVVDAFD NGSFVK 161 Bt3784 YYKVYLADHDVTTELVPTERAAMFRFTYPETKNAYVVIDAFD KGSYVK 159 Bt2629 YYKVYLADHDVTTELAPTERAVMFRFTYPETKNAYVIVDAFD KGSYVK 159 Bt3962 YYSVYLSEYNMTTEIAPTERCAYFRFTFPEASDAYVVVDAFD RGSYVK 155 Bt2199 NYSVTFNN-GLQTSLSATSRGAVFEVSFPEKEDQYVVVDAYN GGSSIT 159 Bt4092 YYKVTFDN-GITTEMAPTTRGVHLRFSYPTTGDAYLVLDGYT DMSEIK 157 Bt2948 FYEVMLDDYQVKAQLSATKTSAILQFTYPQSDSAYVVVDAMPSMFTAG------APGYIH 183 Bt3858 SYSVYLDEADIQVEYAPSHQAGIYHISFGTEGDNALVVNTKN GK 140 Bt1769 SFDVELDDNRMKAEYALSHQSAIYRITYEADKPAYLIVNSRN GS 140 Bt4093 GYYEAMVQEDIKAKLTVTERTGMASYEYPADQQYGTIIIGGGISATP IEQAAI 150 Bt3530 YYSTAFTN-GIKTELTATNAMAVERYKFPRSLSAALWIDFASTFED VATCHY 151 Bt2105 TEIEGWTLNDCRSTG-YHGVQR--YKLHFVMQFDKPFKTMNGWIRNK Bt2111 RRIEGFSKQLSKNVWSADADQN--YTIYFVIEFNKDIKKFGGWHDHT Bt1032 SEIEGMKLLG---TFCYNPQAV--FPIYFVMRVNKVPATTGYWKKQR Bt4073 YTIQGWMKCTPDGGGWGNGEGNSDYTVYYYAQFSKPLSNYGFWSADIPDEWVR Bt1878 NWEDAQTLAGHVNNTVWVDQDYF-FVMKFNRPVIDSLYL Bt3991 EVISDTEICGHKKTTYWAFDQYINFYAKFSKPFAYTLIT Bt3994 EVVDSVTIQGYRYSDGWARDQRIYFRTRFSKPFDKVELD Bt3130 RILNDHAVEGYRIITGWAKLRKIYFYMEFSSPILTSTLR Bt3773 RKVDDYTVEGYRYVRGWSPSRKVYFVLKSDQKIEKFTAY Bt3965 RMINDSTITGYKLVKGWGPERHVYFTATFSKKLTGLRFVQ Bt3963 RVENDTLLTGYRITNGWARTNYTYFAISLSQPIKDYGYK Bt3990 IIPEENKIIGYTTRNSGGVPEN--FKNYFIIEFDKPFTYKATVEN Bt3527 VIPSENKIIGYTTKNSGGVPAN--FKNYFVLEFDKPFTYTAAVAN Bt3784 VIPEENKIIGYSTKNSGGVPEN--FKNYFVIQFDKPFTFTSGVKE Bt2629 VIPEENKIIGYSTKNSGGVPEN--FKNYFVIQFDKPFTFVSTVFE Bt3962 VIPEENKIVGYTTRNSGGVPQN--FRNYFVIEFDKPFTFNKVWAD Bt2199 IEPEKRLVKGATRYNNGGVPDN--FANYFMMEFSHPVIEYGTYNG Bt4092 IDPAKRQISGWVN-NQRFVNDSKSFRNYFVVQFDKPFEDYGVWENQ Bt2948 IDPVRKEISGKSIQSARGYRET----GYFVVRFDKDFDSFGTFNLNNDYPEVIEEKYLFT 239 Bt3858 LVAEEKGVSGYQVIDNTP------TKIYLYLETSQLPLRKGVLADG Bt1769 IHANENFISGRQQLNDN TNVYVYIEAQEKPISAGILENG Bt4093 VITAPNKCEGYAEGGNFCGLRTP-YKVYFVAEFDTDAFETGTWKRE Bt3530 KRISETCIEGYVQAKNVCGHGC--YKLYFSLNTSQPFQLE

7 Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt2948 QKEGKWVNGLKGIYTQDSKGVGHLRSEKIDPVIDFDWDWYKPADDFSFNDYQVTWSGKLK 299 Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt Bt2948 APSTGEYTLGIQADDGARLYINGELLIDDWKSHSFSYQPTQKKISLEAGKMYDIKLEYYQ 359 Bt Bt Bt Bt

8 Bt VYSQIEQLHKSNMKSRQVFTVENNSQDKLDAGIFLDFNLNTGDDVMVRTGISLVSI 532 Bt LWETDTMTAHYPQR FGCYAEFDTTDHPEVMVRSGISYVDM 531 Bt PMTGVEAEWDRDQGKYKLYTRYGKEIAGDDVGAYFTFETEEGEQVEVQMGVSFVSI 275 Bt KRDEVVSIPYLTRISQAPVIKDKKELEGKHLGFFTEFPTKEGEQVEMKVGISFVDM 289 Bt PMGETEKG---KRI IATFDLKPGDELMMKVALSTTSV 242 Bt DSVTMDNG---KRLP VCKAVLHFNTKKDEEVLVKVGVSAVDI 251 Bt TTAIIKDK---QHIG--T AVIARFDFHTEEGEQILVNTAISGVSM 245 Bt DGGRVHEN---TAVINGT NLHGCFRFGQLNGKPLTCKVALSSVSM 244 Bt DDNTPKLW---DQLK-VE SVKSVLTFG--NVKEVKIKVAISSVSC 247 Bt DKKPVIYN--TSRFRSSY---EAWGKNLMACISFDTKAGEEVTVKTAISAVST 238 Bt DKEKVLYNGFWRRFKLEKNFPEITGRKIVAYFNFETAKDPELVVKVALSAVST 269 Bt GNLQENVAEQTTDHAGAIIGFKTRKGEQVNARIASSFISF 238 Bt GNIDTNKLEANDKHAGALIGFKTRKGEQVNVRVASSFISP 244 Bt NNILPNETEVQGNHTGAIIGFATQKGEIVHARVASSFISY 242 Bt NNILPNETEAKGNHTGAVIGFATKKGEIVHARVASSFISP 242 Bt YHLVETHLELQSNHVGAAIGFSTKKGEQVHAKVASSFISP 238 Bt DTLLHHQTDVAADYTCAYLKFDVPAGEKLTIRTASSFISP 242 Bt KDEVFPQKLDGAGKGYGAFIKFK--KGSKVQAKAASSYISA 241 Bt2948 HEWSSRIKLSWIRPDKKSSTSLLTGNRHLESSTKIGGYIRFKTGKNEVIKAIVGTSFISV 419 Bt KVDME--SKEGS---AIALYYGSEKNLNLRYGISFISA 213 Bt TIETSKDNAEGANACAAWRFADGTTTVNLRYGISFISE 217 Bt ELMPNTTFAEGEYSGVYFTFDVNKKKNIQYKIGVSYVSV 234 Bt EQKETTACLTFG-KKVRSVEVRIGLSALSS 218 * Bt2105 DNARLNLEEEIARPFGWNFDKVVTNQQDTWETLFQRVSITTDNYLLKQKFYTNLYRSISP 592 Bt2111 AGASNNLSNEITEPFGWNFEAVHKHQSDSWNNILNRVRIYSNDYREKVRFYTNLYRAFC- 590 Bt1032 ENARLNLDKEQS---GKNFEQVLSDARAQWNDDLSRITVEGGTDAQKTVFYTALYHLLIH 332 Bt4073 EGAANNFKQEIAS---KNFAQVKQEASDLWNKELSRIRISGGTDDEKTVFYTSLYHTMID 346 Bt1878 EGAKKNLQAEIPDW---NFDGVKLAAHDEWNSYLSRMDVEGTDDE-KTNFYTCFYHALIQ 298 Bt3991 AGARKNVESEIPEW---DFDKVRKDARTAWNNYLSKIDITTSDKEDKTIFYTALYHTAIS 308 Bt3994 EGAAKNLQAEVPEN---DFDKYLAETKANWNRQLGKIEVEGDNQDDKVNFYTALYHSMIA 302 Bt3130 ENARQNMEQEAPHW---DFDRYVAAADADWEKQLGKIEVKG-TEVQKEIFYTALYHTMIQ 300 Bt3773 DNAAMNLQAELSHW---DFDKVVKMSSDRWNKQLDKMTVESDDEAAKRVFYTAHYHTMIA 304 Bt3965 DGARNNMK-ELDGL---TFNELRAKGEALWEKELGKYTLTADRKT-KETFYTSAYHAALH 293 Bt3963 EGAVKNLRAEASGK---SFEQLAEAARTDWNNELDHFEIEGTPDQ-KAMFYTSLYHTMIN 325 Bt3990 EQAAANMN-ELGKD---NIEQLAQKGKDAWNQVLGKIEVEGGNLDQYRTFYSCLYRSLLF 294 Bt3527 EQAELNLK-ELGKD---NIDQIAAKGRKVWNDVLGRIEVEDDDIDHLRTFYSCLYRSVLF 300 Bt3784 EQAELNLK-ELGKD---SFDQLVTKGKDIWNREMSKVDVEDDNIDNLRTFYSCLYRSMLF 298 Bt2629 EQAELNLK-ELGKN---SFDQLVANGREIWNREMSKIEIEDDNIDNLRTFYSCLYRSMLF 298 Bt3962 EQAELNLK-EIGNK---TFEQTKEAGRKAWNDVLGRIKVEDDDENRMRTFYSCLYRSVLF 294 Bt2199 EQAAINFNREVADA---DVQLISGKAREQWNNYLGRVEAEGGTDEQLRTFYSCLYRTLLF 299 Bt4092 EQALITLNKELGKDK--NLEVTKARGQKTWNEVLNRIVVEGCTDEQMKTFYSCLFRANLF 299 Bt2948 EQARINLEREIGAK---SMETISAQTEALWNKELSVIDLPGATEQDKIVFYTALYHSFLL 476 Bt3858 EQAKKNLQRDITTY---DVKAVADAGRRIWNKTLGKIVIEGGSEDEKEIFYTSLYRTYER 270 Bt1769 EQAEKNLHRELKDY---NIKALAEAGRQIWNETLGRIQVEGGTEDDKTVFYSSFYRTFER 274 Bt4093 ENARENLKAENAEW---DFQKIQNQAEAKWNHYLGMIEVEGTNPDRTTQFYTHLYRSFIH 291 Bt3530 ELASWECARWEKMD----FEDVKSRTADQWEKQLSAIDVKGGKKDDRVIFYTSLYRTYLS 274 * * **

9 Bt2105 RTIWNDVNGEWIDMNGNKALIDKPGK---SVYGGDSAWGMHWTLGPFYNLLYPEYMSNWI 649 Bt2111 RNTFSDADRRWVDAAGNIQKLDDPDA---VALGCDAFWNTFWNLNQVWNLIAPEWSSRWV 647 Bt1032 PNVLQDVNGEYPAMESDQILTTKG-----TRYTVFSLWDTYRNVHQLLTLVYPERQMEMV 387 Bt4073 PRIYTDVDGRYIGGD-KKVHEQDGTF---TKRTIFSGWDVFRSQFPLQAMINPRLVSDAL 402 Bt1878 PNQISDVDGMYRNAADSIVK-AGTG----TFYSTFSLWDTYRAAHPFYTLMVPERVDDFV 353 Bt3991 PNLFTDADGRYLGMDLEVHQ-GDTIN---PLYTVFSLWDTFRALHPLMTIIDPNLNNQFI 364 Bt3994 PTIYSDVDGAYYGPDKKVHQ-SDG-W---VNYSTFSLWDTYRAAHPLFTYTEPERVNDMV 357 Bt3130 PNTMSDVNGEYMAADYTTRK-VANNE---THYTTFSLWDTFRASHPLYTLLEPERVTDFV 356 Bt3773 PTLYCDVNGEYRGMNDMIYT-DPK-K---ANYTTLSLWDTYRALNPLMTIIQPEMVDNVI 359 Bt3965 PFIFQDSDGQFRGLDKNIEK-AEG-F---TNYTVFSLWDTYRALHPWFNLVQQEVNADIA 348 Bt3963 PSVYMDVDGSYRGLDHNIHQ-AKG-F---TNYTIFSLWDTYRAEHPFLNLVKPERNADMV 380 Bt3990 PRKFYELDANGQPIHYSPYNGQVLPG---YMFTDTGFWDTFRCLFPLLNLMYPSVNKEMQ 351 Bt3527 PRSFYEIDAKGDIVHYSPYNGEVLPG---YMFTDTGFWDTFRCLFPFLNLMYPSMNMKMQ 357 Bt3784 PRSFYEIDAKGQVVHYSPYNGKVLPG---YMFTDTGFWDTFRCLFPFLNLMYPSMNQKMQ 355 Bt2629 PRSFYEIDAKGQVMHYSPYNGEVRPG---YMFTDTGFWDTFRCLFPFLNLMYPSMNQKMQ 355 Bt3962 PRMFHEVNAKGETVHYSPYNGEIRPG---YMFTDTGFWDTFRCLFPFVNLIYPSMGEKMQ 351 Bt2199 PREFYEFDSQGNPVYYSPYDGNVHDG---YMYTDNGFWDTFRAVHPLFTLLYPEVSERVT 356 Bt4092 SRKFYERKENGEPYYYSPYNGKIYDG---YMYTDNGFWDTFRSQFPLTNILHPTMQGRYM 356 Bt2948 PRSLSE-----DGKYRSPFDGKVHKG---ISFTDYSIWDTFRATHPLFVLLKPDFAGDLI 528 Bt3858 MINLSE-----DGKYYSAFDGKIHEDGGVPFYTDDWIWDTYRATHPLRILIEPQKELDMI 325 Bt1769 PICMSE-----GGRYFSAFDGKVHEDNGTPFYTDDWIWDTYRAAHPLRTLIDQQKEEDII 329 Bt4093 PNVCSDVNGEYMGADFRVHKSRSK------HYTSFSNWDTYRTQIQLLSMLDPEVASDIV 345 Bt3530 PADVSSPDGAYLGTDGKVYISEDFRY-----YSNWSLWDTFRTKFPLLVLTEPAKMRDMA 329 * Bt2105 YTYEQFYRRGG--WLPNGNPGMKYFRVMIGNPALPLIVSGYQHGIRDFDSQLMYRALIHQ 707 Bt2111 KSQLAMYDANG--WLAKGPSGMKYIPVMVGEHEIPLLVSTYQMGIRNYDAEKMFRAIVKM 705 Bt1032 RTMLDMYREHG--WFPKWELYGRETLTMEGDPSIPVIVDTWMKGLRDFDVDLAYEAMYKS 445 Bt4073 NSLITMADQSRREYYERWELLNSYSGCMIGNPALSVLADAYMKGIRTYDVEKAYQYAVNT 462 Bt1878 NSLIEQGEV--QGFLPIWALWGKENFCMIGNHGVSVIAEAYRKGFRGFDAERAFNMVKKT 411 Bt3991 NSLIRKHQE--GGIYPMWDLASNYTGTMIGYHAVPVIVDAYMKGYRNFDAHEAYKASLRA 422 Bt3994 KSFIAFFEQ--NGRLPVWNFYGSETDMMIGYHAVPVIVDAYLKGIGNFDAEKALAACVAT 415 Bt3130 KSMIRQYEY--YGYLPIWQLWGQDNYCMIGNHSIPVITDAILKGIPGIDMEKAYEAVYNS 414 Bt3773 NSMLSIYRQ--QDKLPIWPLMSGETNCMPGYSSVPVIADAYLKGFTGFDAEEALTAMKAT 417 Bt3965 NSMLAHYDKSVEKMLPIWSFYGNETWCMIGYHAVSVLADMIVKEVKGFDYERAYEAMKTT 408 Bt3963 ESMIKHEQQSVHGMLPIWSLMGNENWCMSGYHAVSVLADAITKGVFS-NVDEALSAMVST 439 Bt3990 EGLINTYLESG--FFPEWASP-GHRGCMVGNNSASILVDAYMKGVKVDDIKTLYEGLIHG 408 Bt3527 EGLVNTYKESG--FLPEWASP-GHRGCMVGNNSASVVADAYLKGLKGYDIETLWEAVKHG 414 Bt3784 EGLVNAYLESG--FLPEWASP-GHRDCMVGNNSASVVADAYIKGLRGYDIETLWEALKHD 412 Bt2629 EGLVNTYKESG--FLPEWASP-GHRDCMVGNNSASVVADAYIKGLRGYDIETLWEALKHG 412 Bt3962 EGLLNTYLESG--FFPEWASP-GHRGCMVGNNSASVVADAFMKNVTKADAEKMYEGLLKG 408 Bt2199 QSIINAYNESG--FMPEWASP-GHRGCMIGNNSVSLLVDAWMKGIQTVDAEKALEAMIHQ 413 Bt4092 NALLAAQEQCG--WLPSWSSP-GETGGMLGNHSISLLADAWAKGIRDFDPEKALKAYAHE 413 Bt2948 TGLLHAYDEGG--WLPKWPNP-GYTNCMMGTHSDAIIADAYVKGVRNFDVEKAKKAVLKN 585 Bt3858 RSYIRMAEQSDRRWMPTFPEVTGDSHRMNGNHAVAVIWDAYCKGLKDFDLEAAYEACKGA 385 Bt1769 ASYLRMAEQMGNMWMPTFPEVTGDTRRMNSNHAVATVADALAKGLK-VDTAKAYEACRKG 388 Bt4093 ISHQLFAEQSG-GSFPRWVMANIETGVMQGDPTPILIANAYAFGARNYDPKPIFKIMRKG 404 Bt3530 TSLIHLYATGK----KDWSTGFESTPTVRTEHAVILLLDAYRKGITNLDFRKGYAGMKQE 385

10 Bt2105 QTATMINYPGGG----QVGNESYPDYITKGYVPLYDDAWDWNS-----PHYQSYVSNTME 758 Bt2111 QTTPAQRVANG-----FAGNRDLETYLQHQYVPAD KGRFSNTLE 744 Bt1032 ATLPGAENLMR PDNDDYMSKGYVPLR EQYDNSVSHALE 483 Bt4073 SAKFGND SLGYTPEP LSISYTLE 485 Bt1878 QTVSHP LKSDWEVYT-KYGYFPTDLTKAES VSSTLE 446 Bt3991 AEYDTTGIKCPDLVLPHLMPKAKYYKNAIGYIPCDR-ENES VAKALE 468 Bt3994 ANLDNYRG IGLYKQLGYIPYNVTDHYNA ENWSLSKTLE 453 Bt3130 SVTSHPNSP FEVWEKYGFMPENIQTQS VSITLE 447 Bt3773 ATYEGQNG VPYVMAKGYIPADKIHEA TSIAME 449 Bt3965 AMNSNYDC LPEYREMGYVPFDKEAES VSKTLE 440 Bt3963 STVPYYEG VADYMKLGYIPLDKSGTA ASSTLE 471 Bt3990 TENVHPEVSSTGRLGYEY YNKLGYVPYD VKINENAARTLE 448 Bt3527 ANAVHPQVSSTGRLGYDY YNKLGYVPYN VGINENAARTLE 454 Bt3784 AN-AHLRGTASGRLAYDA YNKLGYVPNN IGIGQNVARTLE 451 Bt2629 AN-AHLRGTASGRLG YNQLGYVANN IGIGQNVARTLE 451 Bt3962 ANSVHPRVSTTGRRGYEY YNKLGYVPYD VKINENAARTLE 448 Bt2199 TQARHAEIASVGRDGFEY YDKLGYVPY PEVPEATAKTLE 452 Bt4092 AMNKGPWGGANGRGFWKE YFQLGYVPYP ESMGS-SAQTME 452 Bt2948 AYEKGNHIAWG-RLGIMD YERLGYVPV DKYGESVARTME 623 Bt3858 ITEKTLLPWLRCPLTELDK-----FYQEKGFFPALNPGEEETCKAVHSFERRQAVAVMLG 440 Bt1769 IEEKTLAPWSGAPAGWLDN-----FYRENGYIPALRVDEPENDPNVHPFEKRQPVAVTLG 443 Bt4093 AEEPGSKSQD VETRPGLKQYLDKGYYN ASIQLE 437 Bt3530 MERLPMRS PDQKME 399 Bt2105 YSYQDYCAAQYFDALNK-KDDFNTFMKRSDNWKNIFDPSTGYVRPRRPNGEWIENTNPYH 817 Bt2111 YSYDDWTVSQLAKALGK-EEYYRTFSNRGNWWKNAINPATGYAQMRYSNGEWEKNFDPFK 803 Bt1032 YYIADFALSRFADALGK-KKDAEMFYKRSLGYKHYYSKEFGTFRPILPDGTFYSPFNPRQ 542 Bt4073 YAYADWCVAQLAKALGK-EEDAKRFYEKGQAYRNMFDAEKGWFRPRNADGSWKAWPENAL 544 Bt1878 SVYDDYAAADMARRMGK-EEDAAYFAKRADYYKNLFDSQTNFMRPRKADGTWKSPFNPSD 505 Bt3991 YAYDDWCISIFAEAMND-FENKAKYERFAKAYEFYFDKSTRFMRGLDSNGEWRTPFNPRA 527 Bt3994 YAFDDYCIAEMANKMGK-KEIADEFYKRSQNYKNVYNPATSFMQPRDDKGNFIKDFKADE 512 Bt3130 QAFDDWCVAQLAAKLNK-DADYQRFHKRSEYYRNLFHPKTKFFQSKNDKGEWIEPFDPYQ 506 Bt3773 YAVDDWGIAAMAQKMGK-TADYETFSKRAHYYKNYFDSSIHFIRPKLEDGSWRTPYDPAR 508 Bt3965 YAYDDYCIAQAAKKLGK-EDDYHYFLNRALSYQTLIDPETKYMRGRDSKGDWRTPFTPVA 499 Bt3963 YAYDDWTIYQTALKAGN-KEIADTYRKRALNYRNIYDTSIGFARPRYSDGTFKKEFDVLQ 530 Bt3990 YAYDDWCIYRLAKELKRPKKEISLFAKRAMNYKNLFDKESKLMRGRNEDGTFQSP--FSP 506 Bt3527 YAYNDWCIYQLGKALNKPKKEIEIFAKRAMNYKNLYDPEHKLMRGKNEDGKFQSP--FNP 512 Bt3784 YAYNDWTIYTLGKKLGKPASEIDIFKQRALNYKNVYHPKRKLMVGKDDKGVFNPK--FDA 509 Bt2629 YAYNDWAIYTLGKKLGKPENEIDIYKKHALNYKNVYHPERKLMVGKDNKGVFNPN--FDA 509 Bt3962 YAYDDWCIYRMGEKLGRPAEELELYKSRSQNYRNLFDPETKLMRGKNADGTFQTP--FNP 506 Bt2199 YAYADWCIARFAESLGK-QDIADQYYQKAPNYRNLYYPEHGFMWTKDAKGNWRDR--FDA 509 Bt4092 YAYDDFCGYQLAKMVGN-KHYQEVFARQMYNYKNVFDKSIGFMRGKGVDGKWQEP--FDP 509 Bt2948 FAYDDYCLSRFFAEKGE-PDLSDKLGKRSKNFRNVLDKETKMVRARKADGSWSNPEDYDI 682 Bt3858 NCYDNWCLAQIARTLNK-TDDYKKFMRMSYTYRNVYNAETGFFHPKNKDGKFIEPFDYRY 499 Bt1769 TSYDQWCLSRIAQALNK-KEEAEYYLKCSYNYRNLYNKETAFFHPKDKEGQWIEPFDYRF 502 Bt4093 YTSADFAIGQFALHAVGDEFASWRYFHFARSWKNLYNSDTGWLQSRNPDGSWKSLG Bt3530 SAYDLWAMAKIAEIIGEKADSEQYRQRSVSLFEETWKKEFMNVTPA

11 Bt2105 APG------FCEGS---AWQFTWYVPHDVKSLINLIG-ERQFIDRLNAGFATSEKVSFNA 867 Bt2111 SGANHH---YVEGN---AWQLTFFVPQNVPALTEIIG-KELFIDRLSWGFKASEPWRYNA 856 Bt1032 GENFEPNPGFHEGNS---WNYTFYVPHDVYGLAKLMGGKKPFVNKLQMVFDEG Bt4073 TEEWYG---CIESN-AYQQGWFVPHDVPGMVELMGGKEEVIANLTNLFDHTP Bt1878 VGHAESTGGDYTEGN--AWQYTWHVQHDVPGLIALFGGEEPFLNKLDSLFTVK----LET 559 Bt3991 STHRS--DDYCEGT---AWQWTWFVPHDVEGLVKLMGGEDAFVEKLDSLFTVDSSLDGET 582 Bt3994 YTPH-----ICESN---GWQYFWSVQHDIDGLIDLTGGKNRFAEKLDSMFTYH-PAADEE 563 Bt3130 YGGNGGH-PFTEGN---AWQYFWYVPHNIQALMELTGGTKAFEQKLDTFFTST-YKS-EQ 560 Bt3773 SIHGVG--DFCEGN---GWQYTFFAPQDPYGLISLFGGDKPFTSKLDSFFTNN-DSMGEG 562 Bt3965 YQGPGSVHGWGDITEGFTMQYTWYVPQDVQGYINEAG-KELFRKRLDELFTVELPDDIPG 558 Bt3963 TYGEG FIEGNSWNFSFHVPHDVFGMIDLMGGEKTFVQKLDELFSMHLPEKYYE 583 Bt3990 LKWGDAFT---EGNS---WHYTWSVFHDPQGLIDLMGGKEMFVTMMDSVFAVPPIFDDSY 560 Bt3527 LKWGDAFT---EGNS---WHYTWSVFHDPQGLIDLMGGKDGFNQMMDSVFILPPVFDDSY 566 Bt3784 VDWSGEFC---EGNS---WHWSFCVFHDPQGLIDLMGGKKEFNNMMDSVFVIPGKQGMES 563 Bt2629 VDWSGEFC---EGNS---WHWSFCVFHDPQGLINLMGGKKEFNAMMDSVFVISGKLGMES 563 Bt3962 FKWGDAFT---EGNS---WHYTWSVFHDVQGLADLMGGRKMFVSMLDSVFNLPPIFDDSY 560 Bt2199 TEWGGPFT---EGSS---WHWTWSVFHDPEGLSELMGGHEPMIARLDSMFVAPNTYNYGT 563 Bt4092 LEWGGPFC---EGNA---WHYTWSVFHDVQGLIDLFGSDEKFTTKIDSVFTIPNIIKPGT 563 Bt2948 SVWSGFNP---KGVYNYKKNYTLFVPHDVPELIRFLGGTDSLAVFMDELFDK Bt3858 SGGQGARG---YYGENNGWIYRWDVQHNPADLIALMGGQASFIERLNQTFNEP----LGR 552 Bt1769 PGGMGARE---YYGENNGWVYRWDVPHNVADLISLMGGNEQFIANLDRTFTEP----LGR 555 Bt EDFRESTYKNYFWMVPYDIAGLVEIIGGKEKAEKRLDEFFTRLDAG Bt FEVMKNNGLYQGTRWQYRWAAPQYIDKMIEWVG-QDSLRLQLTYFFDHH * * Bt2105 LGDNMGAY PINHGNETNMQAAYLFSYTS-EPWHTQKWARAIQEKYYGMGPR 917 Bt2111 PGDQYWDF PVVQGNQQSMHFAFLFNWAG-QPWQTQRWSRSIIDRYYGFDTS 906 Bt LYDPANEPDIAYAHLFSYFKGEEWRTQKETQRLLDKYFTTKP- 634 Bt4073 -SDMLWND YYNHANEPVHFVPFLFNQLD-VPWYTQKWTRYICKNAY-ANKV 640 Bt1878 TQADVTGL IGQYAHGNEPSHHVTYLYALAG-RPERTQELIREIFDTQYKNKP- 610 Bt3991 TSADISGL IGQYAHGNEPSHHVIHLYNYVN-RPWRTQELVDSVYRSQYANNV- 633 Bt3994 LPIFSTGM IGQYAHGNEPSHHVIYLFNAVG-QQNLTQKYVAKVMNELYKNEP- 614 Bt3130 MNHNASGF VGQYAHGNEPSHHVAYLYNFAG-QPWKTQKYVSHILNTLYNNTS- 611 Bt3773 ASSDITGL IGQYAHGNEPSHHIAYLYTYAG-EQWKTAEKVRFIMSDFYTDQP- 613 Bt3965 -AHDIQGR IGAYWHGNEPCHHVAYLYNYLK-EPWKCQKWIRTIVDRFYGNTP- 608 Bt3963 HNEDITEECL-----VGGYVHGNEPSHHVPYLYAWTS-QPWKTQYWLREILNKMYKNDI- 636 Bt3990 YGQVIHEIREMTVMNMGNYAHGNQPIQHMIYLYDYAG-QPWKAQYWLRQVMDRMYTPGP- 618 Bt3527 YGGVIHEIREMQIMNMGNYAHGNQPIQHMLYMYNYSG-QPWKAQHWIREVMDKLYTPAP- 624 Bt3784 RG-MIHEMREMQVMNMGQYAHGNQPIQHMVYLYNYSG-EPWKAQHWVREIMDKLYTAGP- 620 Bt2629 RG-MIHEMREMQVMNMGQYAHGNQPIQHMVYLYNYSS-EPWKAQYWIREIMNKLYTAGP- 620 Bt3962 YGGVIHEIREMEIANMGNYAHGNQPIQHMIYLYNYAG-EPWKAQYWLREVMNRLYFATP- 618 Bt2199 YGFVIHEIAEMVALNMGQYAHGNQPVQHAIYLYDYIG-QPWKTQYHLRNVMDKLYNSGS- 621 Bt4092 YGGVIHEMKEMELANMGQYAHGNQPIQHMPYLYCYAG-QPWKTQYWVRQIVERLYNSTE- 621 Bt DIYYVGDEFVMHAPYMYNLCK-RPWMTQKRIYDIVNKYYLPTP- 773 Bt3858 SKFDFYHQLPDHTGNVGQFSMANEPCLHIPYLYNYAG-QPWMTQKRIRVLLNQWFRNDL- 610 Bt1769 SKYAFYAKLPDHTGNVGQFSMANEPSLHVPYLYNYAG-QPWKTQKRIRQMLKTWFRNDL- 613 Bt YNDAWFASGNEPSFHIPWIYNWIG-RPYKTQEIINRVLNEQYSSKI- 584 Bt LYNQGNEPDIHVPYLFNRLG-APEKTQQIVRSLMTEPMIHKYG 535

12 Bt DAYPGDEDQGQMSSWYILSSIGLFQMDGGCSKDPVWLLGS 957 Bt NAYLGDEDQGQMSAWFVMAALGLFQTDGGCNAEPVYELGS 946 Bt DGIPGNDDTGTMSAWAIFNMIGFYP---DCPGLPEYALTT 671 Bt EGIVGNEDVGQMSAWYILAASGIHP---SCPGNTRMEITS 677 Bt DGLCGNDDCGQMSAWYMFSAMGFYP---VDPVSGDYVFGA 647 Bt DGLSGNEDCGQMSAWYILNSMGFYQ---VCPGKPVYSIGR 670 Bt AGLCGNEDCGQMSAWYVFSAMGFYP---VNPVSGKYEIGT 651 Bt SGYAGNDDCGQMSAWYVFSAMGFYP---VNPADGRYIIGS 648 Bt DGIIGNEDCGQMSAWYLLSAMGFYQ---VNPSDGVFAFGS 650 Bt DALSGNDDCGQMSAWYMFNCIGFYP---VAPSSNIYNIGS 645 Bt NGLGGNDDCGQMSAWYLFSVMGFYP---VCPGTDQYVLGA 673 Bt DGYCGDEDNGQTSAWYVFSALGFYP---VCPGTDEYVMGT 655 Bt DGYCGDEDNGQTSAWYVFSAMGFYP---VCPGTDEYILGT 661 Bt DGYCGDEDNGQTSAWYVFSALGFYP---VCPGTDQYILGT 657 Bt DGYCGDEDNGQTSAWYVFSALGFYP---VCPGTDEYIIGT 657 Bt DGYCGDEDNGQTSAWYVFTALGFYP---VCPGSNEYVMGA 655 Bt KGYCGDEDNGQTSAWYVFSAMGFYP---VCPGMPEYAIGS 658 Bt KGYPGDEDQGGMSSWYILSSLGIYA---VCPGTDEYVIGS 658 Bt SGLPGNDDCGQLSSWYIFSAMGFYP---MCPASIEYQLGV 810 Bt MGVPGDEDGGGMTAFVVFSMMGFYP---VTPGSPTYNIGS 647 Bt MGIPGDEDGGGMTSFVVFSSLGFYP---VTPGLPAYTIGS 650 Bt DGLPGNDDLGTMGAWYVFACIGLYP---EIPGVGGFTVNT 621 Bt3530 GNSEFKTPYLGKAFKNAPEGYSPEMDEDDGTMSAWYVFGAMGFYP---LLVGDEYYDLTS 592 * * * Bt2105 PRFDRVEILLDNTYYSGKKLIIKAENVSKDNCYIQYVRFNNKRLSNNYIEWSKLKKGGTL 1017 Bt2111 PLFEKIKIDLGKHFGRGKQFIIKASNTSRENKYIQSAKLNGKKLNSFKFPAKELLKGGTL 1006 Bt1032 PVFNKVTIRLDPKWYKENELVIESNRTGSETLYINKVLLDGKKFNKYRITHDELVHGKRL 731 Bt4073 PVFDKVEFNLDSKYHQGKVFTIIAHNNNTNNLYIQKALLNGKEYNKCYLDFAEIAAGGTL 737 Bt1878 PQLPKIVLHLA----DGKTFTVIAENLSKEHKYVDSITLNGEPYTKNTISHEDILKGGTL 703 Bt3991 PAFDKAVINLP----GEKTFSIVAKNNSKSNKYIESVLLNGKPLDTPFFGHQDIVAGGVM 726 Bt3994 PLFPEMKLHLA----NGKTFTVLAPKVSKENIYIQSIKVDGQPYNKTYLTHEQIMSGTTV 707 Bt3130 PLLDECTLKLA----GNKEFRIRTIRKSPEDIYIQSVTLNGKKHKDFFITHQDIMNGGTM 704 Bt3773 PRFKKIEVKVR----GGKVFTVEAPNNSKDNIYIQKVYLNGKPYHKSYITYDDIINGSTL 706 Bt3965 PCAEAITVRMS----NGKNIEMTADNWSPKNLYVKELYVNGKKYDKSYLTYDDIRDGVKL 701 Bt3963 PYLPYLKLTLP----NGKTLEIKAPGVSDKKRYVQSLKLNGESYDKMYITHEDILKGGVL 729 Bt3990 PLFKKATLHFE----NGNSLVIDAPNNSTENFYIDSMSFNGADHTKNYLRHEDLFKGGTI 711 Bt3527 PYFKQMKLHLE----NGKTVTISAPNNGDDKRYISSMTLNGKDHTKNYVTHEDLMNGASI 717 Bt3784 PLFKSAKLHLE----NGKTVTIKASNNNTDNRYVKDMKVNGKAFTRNYLTHDQLLKGANI 713 Bt2629 PLFKSAKLHLE----NGKTITIKADNNQLDNRYIKEMKVNGKSQTRNFLTHDQLIKGANI 713 Bt3962 PYFKKATITLE----NGKKLEISAPKNSDANRYIRSLNYNGKNYTKNYLNHFDLLKGGRL 711 Bt2199 PLFKKVTLHLP----EGKNFVVSAADNAADRPYIRKALLNGQEFTRNYLTHDELKQGGEL 714 Bt4092 PLFKKATITME----NGNKFVIEAPENSKENLYIQSATLNGRLLDKNYIHYDDIAEGGVL 714 Bt2948 PCLPGFVLHLP----QNRTFTIKTKNFGKGNCYVRAVYLNGKPHRSSVITHSDIINGGEI 866 Bt3858 PVFQSAKMEVG----DGHYFEIIAENYAPDHKYIQSATLNGTPWNKPWFSHADIQNGGRL 703 Bt1769 PLFTDAKIRLS----NGAVFEIEAKNASTDNKYIQSATLNGKEWNKSWFSHDDLMSGGKL 706 Bt4093 PIFSSVKVHLK KGDIVIKGGSEKDIYIKSMKLNGKSTWIDWDQLNSGATI 674 Bt3530 PLFDRVLLRLT----NGNVLTIQTEGRKKKDAPIKSIHFNGKKIADYRISHNELIKGGEL 648 * *

13 Bt2105 HFIMGDRPNPN-AFK Bt2111 ELEMGDRPNKNWGIK Bt1032 IFDLK Bt4073 ELFMGDKPNTEWGVLSNI Bt1878 VYKMK Bt3991 EIRMTDHPTQWGVRQ Bt3994 EFEMGNTPLVEVEFEEQTQ Bt3130 VFKMGKKPSGWGK Bt3773 KFEMGKKPAKNFGKASANRPIVLNK Bt3965 RFVMSGKPNYKRAVSDEAVPPSISLPEKTMKYKSSIGF 739 Bt3963 EFKMSASPNKRRGVSVQDKPYSLTNGIN Bt3990 KVDMSNRPNLNRGTKEEDMPYSFSKE Bt3527 TFKMDSKPNEQRGTKETDFPYSFSNEFKKKK Bt3784 QYQMSPTPNKQRGTTEKDIPYSLSFE Bt2629 QFQMSPVPNKQRGTTEKDVPYSLSFE Bt3962 VFDMDNKPNKGRGINESDFPYSFSRDNK Bt2199 NLSMDSVPNQQRGTQPADFPYSYSK Bt4092 KFEMGSQPNKERCTSKYAAPFSLSKE Bt2948 LFELTDKPAYNWFQ Bt3858 VLQMGDKPNKKWGIASDAVPPSSESLPE Bt1769 VLVMGNKPNKTWASGAEDVPPSLEIK Bt4093 EYRTSGKPDMKWGAKVVPPSFP Bt3530 IYNYK Residues that bind calcium are highlighted in magenta, the putative catalytic residues in cyan and the motifs that are predicted to confer α-1,2 mannosidase activity in green. Residues conserved in all GH92 enzymes are indicated by an asterisk. The sequences were aligned using Clustal W.

14 1 C 4 3 H 2 1 C 4 O S 2 1 C 4 1 H 2 B 2,5 3,O B 3 H 4 1 S 5 O H 5 3 S 1 4 H 5 O H 1 1,4 B 4 C 1 B 1,4 4 H 3 2 H 1 1 S 3 2 H 3 5 S 1 1 H O B 3,O 2,5 B 5 H 4 1 C 4 2 S O 1 C 4 5 H O 1 C 4 Supplemental Figure 5. The map of paranoid ring interconversion in the formulation of Stoddart 1. The map is a planar projection of the spherical formulation of Pople and Cremer 2 more recently quantified by Bérces, Whitfield and Nukada 3. Potential pyranoside interconversion itineraries (passing through one four conformations, 3 H 4, 4 H 3, B 2,5 or 2,5 B close to the reaction transition-state), proposed by various groups (and partially reviewed in refs 4,5 ), are shown colored. In the case of glycoside hydrolase family GH92, the mannoimidazole 4 and kifunensine 5 are observed in approximate 1 S 5 / B 2,5 and 1,4 B / 1 S 5 conformations, respectively which in the absence of evidence to the contrary suggests catalysis around the B 2,5 putative near transition-state conformation (colored blue). 1. Stoddart, J.F. Stereochemistry of Carbohydrates, (Wiley Interscience, Toronto, 1971). 2. Cremer, D. & Pople, J.A. A general definition of ring puckering coordinates J Am Chem Soc 97, (1975). 3. Bérces, A., Whitfield, D.M. & Nukada, T. Quantitative description of six-membered ring conformations following the IUPAC conformational nomenclature Tetrahedron 57, (2001). 4. Davies, G.J., Ducros, V.M.-A., Varrot, A. & Zechel., D.L. Mapping the conformational itinerary of β-glycosidases by X-ray crystallography Biochem Soc Trans 31, (2003). 5. Vocadlo, D. & Davies, G.J. Mechanistic Insights into Glycosidase Chemistry Curr Opin Chem Biol 12, (2008).

15 Supplemental Figure 6. The Bt2199 α-mannosidase and its overlap with Bt3990 (a) Overlay of the Bt2199 swainsonine complex (yellow) with the Bt3990 swainsonine complex (green). Of particular note is the loop-turn-helix contributed by Bt2199 residues in pink which are rotated 180 degrees out of the active site relative to the equivalent residues ( ) in Bt3990 by virtue of crystal packing interactions. (b) Active site comparison of Bt3990 swainsonine complex (green) with the Bt2199 swainsonine complex (yellow) showing that upon ligand binding (in the absence of calcium) the positions of residues W88, D348, D642, D644 and E533 are restored. In Bt2199 the residue equivalent to Bt3990 residue N601 is oriented away from the active site and N602 displays high mobility. This suggests that the correct positioning of these residues demands appropriate Ca2+ coordination. (c) Comparison (divergent stereo) of the active centres of Bt2199 apo-enzyme (cyan/pink) with the Bt2199 swainsonine complex (yellow) highlighting the conformational changes that occur upon ligand binding (in the absence of calcium). In the absence of Ca2+ much of the catalytic centre remains mis-positioned for catalysis but upon ligand binding the loop (pink) moves out of the active centre to allow W94 (one of the α-1,2 mannosidase fingerprint motifs described in the text) to sit above the +1 subsite. Residues shift toward the -1 subsite, resulting in the reorientation of D646 and D648 (equivalent to Bt3990 residues D642 and D644; where 642 is the putative catalytic base).

16 Bt1769 Bt1769 Bt3858 Bt3965 Bt3963 Bt3963 Bt3773 Bt3773 Bt3994 Bt3130 Bt1878 Bt3991 Bt4073 Bt4073 Bt2629 Bt3784 Bt3527 Bt3527 Bt3990 Bt3990 Bt3530 Bt3530 Bt3962 Bt2105* Bt2105 Bt2199 Bt2199 Bt4092 Bt2948 Bt2948 Bt4093 Bt4093 Bt1032 Bt1032 Bt2111 Bt2111 Supplemental Figure 7. Phylogenetic tree of the GH92 enzymes of B. thetaiotaomicron. Enzymes displaying α1,2 (green), α1,3- (red), α1,4 (blue) and α1,6-mannosidase activities are shown. Enzymes thatt display low or no detectable t disaccharidase id activity it are in black. The asterisk indicates that the activity of Bt2105 was not determined as it was not expressed. The figure was constructed using Tree View. The bar shows the genetic distance of 0.1 substitutions per nucleotide. 0.1

17 Supplemental Table 1 Biochemical properties, expression profiles, cellular location and sequence conservation of GH92 α-mannosidases Enzyme Predicted cellular location a Bt1032 PP W Bt1769 OM W NA ++ No Bt1878 OM W No Bt2111 PP NE NA NA NA NA +++ No Bt2199 OM NE 100 NA 0.12 NA +++ Yes* Bt2629 PP YM, W, Mu NA NA NA NA ++ No Bt2948 PP NE NA NA NA NA +++ No* Bt3130 PP NE No Bt3527 PP S NA NA NA NA +++ Yes* Bt3530 CYT S NA NA +++ No* Bt3773 PP YM Yes* Bt3784 PP YM, W, Mu NA ++ No Bt3858 OM YM No Bt3962 PP NE 100 NA No** Bt3963 PP NE NA NA NA NA +++ No Bt3965 CYT NE No Bt3990 PP W NA +++ No Bt3991 OM W NA No Bt3994 OM NE NA NA +++ No** Bt4073 OM NE NA NA 100 NA ++ No Bt4092 PP S NA + No* Bt4093 PP S NA NA 100 NA ++ No a The predicted cellular location of the enzymes are the periplasm, PP; outer membrane, OM; cytoplasm, CYT. b The conditions in which the GH92s are expressed in B. thetaiotaomicron are as follows: W, weaning on diets containing plant polysaccharides; NE, no expression in the conditions tested; YM, yeast mannan; Mu, porcine mucein; S, suckling. c Relative enzyme activity was determined using substrate at a concentration of 1 mm d NA; no activity detected. e Expression was quantified by the amount of pure GH92 recovered after IMAC from cell-free extracts derived from one litre cultures where recombinant protein expression was induced with 200 μm IPTG for 20 h at 16 o C in E. coli strain TUNER. +++, >500 nm; nm; +, <50 nm f Proteins were dialyzed against 10 mm Tris/HCl buffer ph 8.0 or, if the protein precipitated in the no salt buffer, aggregation was assessed when the buffer was supplemented with 300 mm NaCl (*). GH92s that did not precipitate during dialysis but did so after storage at 4 o C for two weeks are indicated (**). Expression profile b Sequence conservation Relative mannosidase activity (%) c Recombinant expression in E. coli e His-Glu MOTIF Trp-MOTIF Precipitation during dialysis f α-1,2-man 2 α-1,3-man 2 α-1,4-man 2 α-1,6-m2 ++ No* NA d NA NA NA +++ No

18 Supplemental Table 2. The influence of metal ions on the activity of Bt3990 and Bt2199. Metal ions were stripped with either EDTA or EGTA and the activity after re introduction of different metals determined. Enzyme Metal added after EDTA/EGTA treatment % Activity a EDTA EGTA Bt2199 No metal 0 0 Bt2199 Ca Bt2199 Zn Bt2199 Mg Bt2199 Mn Bt2199 Co Bt2199 Ba Bt2199 Be Bt2199 Sr Bt2199 Sc Bt2199 Ti Bt2199 V Bt3990 No metal 0 0 Bt3990 Ca Bt3990 Zn Bt3990 Mg Bt3990 Mn Bt3990 Co Bt3990 Ba Bt3990 Be Bt3990 Sr Bt3990 Sc Bt3990 Ti Bt3990 V a The two enzymes were treated with 5 mm EDTA or EGTA in 100 mm MOPS ph 7.0 and assayed for enzyme activity using 1 mm 4 nitrophenyl α mannopyranoside. Metal ions were then added at a concentration of 7 mm and the recovery of enzyme activity was determined. Activity is expressed as a % of the activity recovered by the addition of calcium. The activity of untreated (no chelating agent or meal) Bt2199 and Bt3990 was 63 % and 65 % of the activity obtained after calcium addition.

19 Supplemental Table 3 Data collection and refinement statistics for GH92 α-1,2 mannosidases Bt3990. Bt2199 and their ligand complexes. Bt3990 Peak Bt3990 Mannoimidaz ole 6 Bt3990 Kifunensine 7 Bt3990 Swainsonine 5 Bt3990 Thiomannobi osiode 4 Bt3990 Thiomannobi osiode 4, plus Ca 2+ Bt2199 (apo) Bt2199 Swainsonine 5 Data collection Space group P2 1 P2 1 P P2 1 P2 1 P2 1 P P Cell dimensions a, b, c (Å) 64.3, 191.0, , 152.4, , 150.2, , 152.0, , 153.1, , 68.6, , 163.8, , 163.0, α, β, γ ( ) 90, 97.0, 90 90, 94.2, 90 90, 90, 90 90, 93.4, 90 90, 91.4, 90 90, 94.7, 90 90, 90, 90 90, 90, 90 Resolution (Å) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) R merge 0.10 (0.48) 0.13 (0.47) 0.13 (0.49) 0.12 (0.49) 0.15 (0.47) 0.05 (0.36) 0.13 (0.49) 0.09 (0.51) I / σi 23.3 (3.2) 7.6 (2.1) 6.2 (2.1) 9.7 (2.9) 7.1 (3.0) 15.4 (2.0) 11.8 (3.9) 11.2 (4.1) Completeness (%) 97.6 (96.6) 100 (100) 88.4 (89.3) 98.0 (95.2) 100 (99.9) 97.7 (95.4) 100 (100) 99.9 (99.9) Redundancy 7.5 (7.2) 3.8 (3.8) 3.5 (3.5) 4.0 (3.9) 4.2 (3.9) 3.2 (2.6) 6.9 (7.0) 6.2 (6.1) Refinement Resolution (Å) No. reflections R work / R free 0.16/ / / / / / / /0.19 No. atoms Protein Ligand/ion 0/4 112/8 64/4 96/8 96/4 144/6 0/0 12/0 Water B-factors Protein Ligand/ion 0/24 5/5 34/34 25/30 15/0 31/29 0/0 34/0 Water R.m.s deviations Bond lengths (Å) Bond angles ( )

20 Supplemental Table 4 Catalytic activity of wild type and variants of Bt3990 and Bt2199 Enzyme PNP-α-Mannopyranoside α-1,2-mannobiose DNP-α-Mannopyranoside Bt3990 k cat (min -1 ) K M (mm) k cat / K M Relative to k cat (min -1 ) K M (mm) k cat / K M Relative to k cat / K M Relative to (min -1 wild type (min -1 mm -1 ) wild type (min -1 mm -1 ) wild mm -1 ) Wild-type 5.6± ± x 10 3 ±3 x ± x E533A 0.2± ± ± ± x x E585A NA a NA ± ± x x Q602A 0.14± ± x 10 2 ±47 16± x x 10-2 D642A NA NA NA ± ± x x 10-4 E643A NA NA NA 0 NA NA x x 10-4 D644A NA NA NA ± ± x x 10-5 S86A 0.4± ± x x 10 3 ±1.2 x ± x x x 10-2 E533Q 8.8±1.8 10± ± ± x x N601A 0.033± ± x ± ± x x 10-3 D644N NA NA NA ± ± x x 10-4 Bt2199 Wild type 6.3± ± x 10 3 ±1.4 x ± x b - E537A NA NA ± ± x E537Q 3.6± ± ± ± x D648A NA NA NA ± ± x 10-6 D648N NA NA NA ± ± x 10-6 a NA; no activity detected b -; not determined.

21 Supplemental Table 5 Inhibition of B. thetaiotaomicron GH92 α-mannosidases by mannoimidazole (6) and other inhibitors. BT GH92s Inhibitor K i (μm) 1032 Mannoimidazole (6) Mannoimidazole Mannoimidazole Mannoimidazole Mannoimidazole Mannoimidazole Mannoimidazole Mannoimidazole Mannoimidazole Mannoimidazole Mannoimidazole Mannoimidazole Mannoimidazole Mannoimidazole Mannoimidazole Mannoimidazole Swainsonine (5) Deoxymannojirimycin 13 x 10 3 (10) 2199 Kifunensine (7) Swainsonine (5) Deoxymannojirimycin 12 x 10 3 (10) 3990 Kifunensine (7) 140 The K i values were determined using 4NP-mannopyranoside 1 as the substrate and thus only those enzymes that hydrolyzed this aryl-glycoside were evaluated.

22 Supplemental Table 6 Primers deployed to clone the BT92 genes and introduce specific mutations Primers used to clone the GH92 genes BT GH92s Primers sequences 5 to 3 BT1032 Forward 5 CTC CAG CCA TGG GTG GAG AGG GTA AGG CCC CC Reverse 5 CTC CAG CTC GAG TTT CAA ATC AAA TAT CAA GCG TTT ACC BT1769 Forward 5 CTC CAG CCA TGG GTA ACG GTA GCC TGC AAA CAG Reverse 5 CTC CAG CTC GAG CTT GAT TTC TAA TGA TGG AGG BT1878 Forward 5 CT CCA GCC ATG GCA GAA ACG AAA GAT TAC ACC CAG TAT G Reverse 5 CAC GTC CTC GAG TTT CAT CTT ATA TAC GAG TGT ACC ACC BT2105 Forward 5 CTC CAG GCT AGC AAA CGA ACT ATT GTA CAA ATT ATC TTT TTC NheI Reverse 5 CTC CAG CTC GAG TTT AAA TGC ATT TGG ATT TGG TCG BT2111 Forward 5 GGA ATT CCA TAT GAC CAA GAC TGT CTG GCA AAT CGG NdeI Reverse 5 CTC CAG CTC GAG TTT GAT TCC CCA ATT TTT ATT CGG AC BT2199 Forward 5 CTC CAG CCA TGG GTC AGC AGT CAA AGA AGA CGG TAG Reverse 5 CTC CAG CTC GAG CTT GAT TTC TAA TGA TGG AGG BT2629 Forward 5 CTC CAG CCA TGG GTG TTA AGA ATC CTG TAG ACT ATG Reverse 5 CTC CAG CTC GAG TTC AAA CGA AAG AGA GTA AG BT2948 Forward 5 CTC CAG CCA TGG GTC AAG AGG AAA AAG CCT CAT GG Reverse 5 CTC CAG CTC GAG TTG AAA CCA ATT ATA TGC AGG C BT3130 Forward 5 CTC CAG CCA TGG GCC AGG CAG GGG AAA TCA CTA AAT ATG Reverse 5 CTC CAG CTC GAG CCA TCC GGA TGG TTT CTT ACC BT3527 Forward 5 CTC CAG CCA TGG GTA TGG ATG CTA AAA CGA TTA CC Reverse 5 CTC CAG CTC GAG CTT TTT CTT TTT AAA TTC GTT GG BT3530 Forward 5 CTC CAG CCA TGG GTA CAC CGT CAG TTG CAC AAA ATA C

23 BT3773 BT3784 BT3858 BT3962 BT3963 BT3965 BT3990 BT3991 BT3994 BT4073 BT4092 Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse 5 CTC CAG CTC GAG TTT ATA ATT ATA AAT CAA TTC TCC GC 5 CT CCA ACC ATG GCA AAA CAG GAC TAT TCA AAG AGT GAG 5 CTG GGT CTC GAG TTT ATT CAA AAC GAT CGG CCT ATT GG 5 CTC CAG CCA TGG GTG TCA AGA ATC CGG TAG ACT ATG 5 CTC CAG CTC GAG TTC AAA TGA AAG GGA ATA GGG 5 CTC CAG CCA TGG GTG ATC AGA CGC CCA CGA AAG 5 CTC CAG CTC GAG TTC CGG CAA ACT CTC TGA AG 5 CTCGTGCC ATG GCA CAA AAG CAG CCG GTG GAT TAC G 5 CAA GTG CTC GAG CTT GTT ATC ACG AGA GAA GGA ATA GG 5 CTC CAG CCA TGG GTC AGC AAC CGG TAG ATT ACG T 5 CTC CAG CTC GAG ATT GAT TCC ATT AGT TAA TGA ATA A 5 CTC CAG CCA TGG GTG CTC AAA CTG AAA AGC TGA C 5 CTC CAG CTC GAG AAA CCC AAT TGA TGA TTT ATA C 5 CTC CAG CCA TGG GTA AAG ACT GGA CGC AGT ACG 5 CTC CAG CTC GAG TTC TTT GGA AAA AGA ATA AGG C 5 CTC CAG CCA TGG GTC AGC AAC CGG TAG ATT ACG T 5 CTC CAG CTC GAG ATT GAT TCC ATT AGT TAA TGA ATA A 5 CT CCA GCC ATG GCA GAA ATT GAT TAC ACA TCG TAT GTA AAT CC 5 TGG TCC TCG AGC TGG GTC TGT TCT TCA AAT TCG 5 CTC CAG CCA TGG GTA CAG AAG ATT TAA TTC CGA CTG 5 CTC CAG CTC GAG AAT GTT AGA TAA AAC ACC CC 5 CTC CAG CCA TGG GTC AGC TAA AAG ATC TGG TAC AGT ATG 5 CTC CAG CTC GAG TTC TTT AGA CAA AGA GAA AGG CG Primers used in site-directed mutagenesis Primer Name Bt3990 D644A Primer Sequence (5' to 3') TTACTGCGGAGACGAAGCCAACGGACAAACTTCTG

24 Bt3990 D644A_antisense CAGAAGTTTGTCCGTTGGCTTCGTCTCCGCAGTAA Bt3990 D415A Bt A_antisense Bt3990 E643A TGAAAGGAGTAAAAGTCGATGCTATCAAGACGTTGTATGAAGG CCTTCATACAACGTCTTGATAGCATCGACTTTTACTCCTTTCA' GGTTACTGCGGAGACGCAGACAACGGACAAACT Bt3990 E643A_antisense AGTTTGTCCGTTGTCTGCGTCTCCGCAGTAACC' Bt3990 E533A GGAGATGCCTTTACGGCAGGAAACAGCTGGCAT' Bt3990 E533A_antisense ATGCCAGCTGTTTCCTGCCGTAAAGGCATCTCC Bt3990 E585A TACGGACAAGTGATTCACGCAATTCGTGAAATGACAGTG Bt3990 E585A_antisense CACTGTCATTTCACGAATTGCGTGAATCACTTGTCCGTA Bt3990 Q602A TGGGCAACTATGCACATGGTAATGCGCCGATCCAGCA Bt3990 Q602A_antisense TGCTGGATCGGCGCATTACCATGTGCATAGTTGCCCA Bt3990 D642A CGGTTACTGCGGAGCCGAAGACAACGGAC Bt3990 D642A_antisense GTCCGTTGTCTTCGGCTCCGCAGTAACCG Bt3990 S86A Bt3990 S86A_antisense Bt3990 E533Q CAAACAGACACACCAGCCCGCTCCGTGGATCAATGATTAT ATAATCATTGATCCACGGAGCGGGCTGGTGTGTCTGTTTG GGGGAGATGCCTTTACGCAGGGAAACAGCTGGCATTA Bt3990 E533Q_antisense TAATGCCAGCTGTTTCCCTGCGTAAAGGCATCTCCCC Bt3990 N601A TGAATATGGGCAACTATGCACATGGTGCTCAGCCGATCCAGC Bt3990 N601A_antisense GCTGGATCGGCTGAGCACCATGTGCATAGTTGCCCATATTCA Bt3990 D644N GGACGGTTACTGCGGAGACGAAAACAACGGACAAA Bt3990 D644N_antisense TTTGTCCGTTGTTTTCGTCTCCGCAGTAACCGTCC Bt2199-E537A GCGGTCCTTTCACAGCAGGAAGTTCCTGGCA Bt2199-E537A_antisense TGCCAGGAACTTCCTGCTGTGAAAGGACCGC Bt2199-D648A GTGGCGACGAGGCCAACGGGCAGAC Bt2199-D648A_antisense GTCTGCCCGTTGGCCTCGTCGCCAC Bt2199-E537Q TGGGGCGGTCCTTTCACACAGGGAAGTTCCTGG

25 Bt2199-E537Q_antisense CCAGGAACTTCCCTGTGTGAAAGGACCGCCCCA Bt2199-D648N CTGTGGCGACGAGAACAACGGGCAGAC Bt2199-D648N_antisense GTCTGCCCGTTGTTCTCGTCGCCACAG