Characterization of a Tryptophan 6-Halogenase from Streptomyces Toxytricini

SUPPORTING INFOMATION

Characterization of a tryptophan 6-halogenase from Streptomyces toxytricini

Jia Zeng• Jixun Zhan

Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT 84322-4105, USA

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Supplementary Fig. 1Amino acid sequence alignment of STTH and nine other flavin-dependent halogenases

STTH ------MNTRNPDKVV 10

KtzR ------VVTAKTPDRVV 11

PyrH ------MIRSVV 6

RebH ------MSG-KIDKIL 9

Thal ------MDN-RIKTVV 9

KtzQ ------MDDNRIRSIL 10

PrnA ------MNK-PIKNIV 9

MibH MARSEESNTLARLFDVLGDDAAAAREWVTEPHRLIASNERLGTAPEAPADDDPEAIRTVG 60

Rdc2 ------MSVPKSCTIL 10

ChlA ------MDTNIINHYDII 12

STTH IVGGGTAGWMTASYLKKAFGERVSVTLVESGTIGT------VGVGEATFSDIR-HFFEF 62

KtzR IVGGGTAGWMTAAYLKTAFGDRLSITVVESSRIGT------IGVGEATFSDIQ-HFFQF 63

PyrH IVGGGTAGWMTASYLKAAFDDRIDVTLVESGNVRR------IGVGEATFSTVR-HFFDY 58

RebH IVGGGTAGWMAASYLGKALQGTADITLLQAPDIPT------LGVGEATIPNLQTAFFDF 62

Thal ILGGGTAGWMTAAYLGKALQNTVKIVVLEAPTIPR------IGVGEATVPNLQRAFFDY 62

KtzQ VLGGGTAGWMSACYLSKALGPGVEVTVLEAPSISR------IRVGEATIPNLHKVFFDF 63

PrnA IVGGGTAGWMAASYLVRALQQQANITLIESAAIPR------IGVGEATIPSLQKVFFDF 62

MibH VIGGGTAGYLTALALKAKR-PWLDVALVESADIPI------IGVGEATVSYMVMFLHHY 112

Rdc2 VAGGGPAGSYAAAALAREG---NDVVLLEADQHPR------YHIGESMLPSLR-PLLRF 59

ChlA IIGGGIAGLSATRHLLLKMPELSGRIAVIEPRSERRDNLDEDYKVGESTVEVSAMFFAKE 72

STTH LDLRE---EEWMPACNATYKLAVRFQDWQRPG------HHFYHPFEQMRSVD 105

KtzR LNLRE---QDWMPACNATYKLGIRFENWRHVG------HHFYQPFEQIRPVY 106

PyrH LGLDE---REWLPRCAGGYKLGIRFENWSEPG------EYFYHPFERLRVVD 101

RebH LGIPE---DEWMRECNASYKVAIKFINWRTAGEGTSEARELDGG-PDHFYHSFGLLKYHE 118

Thal LGIPE---EEWMRECNASYKMAVKFINWRTPGEGSPDPRTLDDGHTDTFHHPFGLLPSAD 119

KtzQ LGIAE---DEWMRECNASYKAAVRFVNWRTPGDGQATPRRRPDGRPDHFDHLFGQLPEHE 120

PrnA LGIPE---REWMPQVNGAFKAAIKFVNWRKSPDPSRD------DHFYHLFGNVPNCD 110

MibH LGIDP---AEFYQHVRPTWKLGIRFEWGSRPE------GFVAPFDWGTGSV 154

Rdc2 IDLED---KFDAHGFQKKLGAAFKLTSKREGY------TDFIAAHGPRGYSW 102

ChlA LELQDYLIENHPPKFSLQFHWPRELSKTDTIEDYYSTWAVKNPDIQAFQLNRCKIERDLL 132

STTH GFPLTDWWL---QNGP-TDRFDRDCFVMASLCDAGRSPRYLNGSLLQQEFDERAEEPAGL 161

KtzR GFPLTDWWL---HDAP-TDRFDTDCFVMPNLCEAGRSPRHLDGTLADEDFVEEGDELANR 162

PyrH GFNMAEWWL---AVGDRRTSFSEACYLTHRLCEAKRAPRMLDGSLFASQVDES---LGRS 155

RebH QIPLSHYWFDRSYRGKTVEPFDYACYKEPVILDANRSPRRLDGSKVTN------166

Thal QIPLSHYWAAKRLQGETDENFDEACFADTAIMNAKKAPRFLDMRRATN------167

KtzQ NLPLSQYWAHRRLNGLTDEPFDRSCYVQPELLDRKLSPRLMDGTKLAS------168

PrnA GVPLTHYWLRKREQG-FQQPMEYACYPQPGALDGKLAPCLSDGTRQMS------157

MibH GLVG------SLRETGNVNEATLQAMLMTEDRVPVYRGEGGHVSLMKYLP------198

Rdc2 NVVRSESDEILFNHARESGAQAFQGIKINAIEFEPYEEEYPNGEKVANPGKPTS------156

ChlA KMVIAQGAVYYHGRVRNVDNLDNDDMKSIDVEILSEVESGADFKLQQSIERITLT----- 187

STTH TMSEHQGKTQFPYAYHFEAALLAEFLSGYSKDR-GVKHVVDEVLEVKLDDRGWISHVVTK 220

KtzR TMSEHQGKSQFPYAYHFEAALLAKFLTGYAVDR-GVEHVVDDVLDVRLDQRGWIEHVVTA 221

PyrH TLAEQR--AQFPYAYHFDADEVARYLSEYAIAR-GVRHVVDDVQHVGQDERGWISGVHTK 212

RebH ------YAWHFDAHLVADFLRRFATEKLGVRHVEDRVEHVQRDANGNIESVRTA 214

Thal ------YAWHFDASKVAAFLRNFAVTKQAVEHVEDEMTEVLTDERGFITALRTK 215

KtzQ ------YAWHFDADLVADFLCRFAVQKLNVTHVQDVFTHADLDQRGHITAVNTE 216

PrnA ------HAWHFDAHLVADFLKRWAVER-GVNRVVDEVVDVRLNNRGYISNLLTK 204

MibH ------FAYHMDNARLVRYLTELATRR-GVHHVDATVAEVRLDGPDHVGDLITT 245

Rdc2 ------AKWSSKDGSSGDIAFKYLVDATGRIGIMSTKYLKNRHYNEGLKNLAIW 204

ChlA -----TDYIVDASGRNFTVGSRTDNILKDPKHLFGLDNASTWVRVKNTERSLFDFKSQDV 242

STTH EHGDIGGDLFVDCTGFRGVLLNQALGVP----FVSYQDTLPNDS--AVALQVPLDMEARG 274

KtzR EHGEIHGDLFVDCTGFRGLLLNKALGVP----FVSYQDTLPNDS--AVALQVPLDMQRRG 275

PyrH QHGEISGDLFVDCTGFRGLLINQTLGGR----FQSFSDVLPNNR--AVALRVPRENDE-D 265

RebH TGRVFDADLFVDCSGFRGLLINKAMEEP----FLDMSDHLLNDS--AVATQVPHDDDANG 268

Thal SGRILQGDLFVDCSGFRGLLINKAMEEP----FIDMSDHLLCNS--AVATAVPHDDEKNG 269

KtzQ SGRTLAADLFIDCSGFRSVLMGKVMQEP----FLDMSKHLLNDR--AVALMLPHDDEKVG 270

PrnA EGRTLEADLFIDCSGMRGLLINQALKEP----FIDMSDYLLCDS--AVASAVPNDDARDG 258

MibH DGRRLHYDFYVDCTGFRSLLLEKALGIP----FESYASSLFTDA--AITGTLA---HGGH 296

Rdc2 GYYKNNIPWAQGTPRENQPFFEGMRDGAGWCWTIPLHNGTVSVG--AVMRKDLFFEKKKS 262

ChlA TCSWTYDTNHFFGPGYWIWMIPLERGSRDYSIGVSYHRDKIQPSQLNSLDKFMSFLEKNQ 302

STTH IPPYTRATAKEAGWIWTIPLIGRIGTGYVYAKDYCSPEEAERTLREFVGPEAADVEAN-- 332

KtzR IVPNTTATAREAGWIWTIPLFGRVGTGYVYAKDYLSPEEAERTLREFVGPAAADVEAN-- 333

PyrH MRPYTTATAMSAGWMWTIPLFKRDGNGYVYSDEFISPEEAERELRSTVAPGRDDLEAN-- 323

RebH VEPFTSAIAMKSGWTWKIPMLGRFGTGYVYSSRFATEDEAVREFCEMWHLDPETQPLN-- 326

Thal VEPYTSSIAMEAGWTWKIPMLGRFGSGHVYSDHFATQDEATLAFSKLWGLDPDNTEFN-- 327

KtzQ IEPYTSSLAMRSGWSWKIPLLGRFGSGYVYSSQFTSQDEAAEELCRMWDVDPAEQTFN-- 328

PrnA VEPYTSSIAMNSGWTWKIPMLGRFGSGYVFSSHFTSRDQATADFLKLWGLS-DNQPLN-- 315

MibH LKPYTTATTMNAGWCWTIPTPESDHLGYVFSSAAIDPDDAAAEMAR---RFPGVTREA-- 351

Rdc2 LGENATNTQIMAECMKLCPTIG------ELLAPAELVSDIKQATDYSYSATAYA-- 310

ChlA KLLYNLIKSGEIVDFHRWPKLAHTSKTFFSKNNWCVIGDAAAIFDPFYSTGMVMIAMEIE 362

STTH ----HIRMRIGRSEQSWKNNCVAIG-----LSSGFVEPLESTGIFFIHHAIEQLVKHFPA 383

KtzR ----HIRMRIGRSQESWRNNCVAIG-----LSSGFVEPLESTGIFFIHHAIEQLVKHFPA 384

PyrH ----HIQMRIGRNERTWINNCVAVG-----LSAAFVEPLESTGIFFIQHAIEQLVKHFPG 374

RebH ----RIRFRVGRNRRAWVGNCVSIG-----TSSCFVEPLESTGIYFVYAALYQLVKHFPD 377

Thal ----HVRFRVGRNRRAWVRNCVSVG-----LASCFVEPLESSGIYFIYAAIHMLAKHFPD 378

KtzQ ----NVRFRVGRSRRAWVRNCVAIG-----VSAMFVEPLESTGLYFSYASLYQLVKHFPD 379

PrnA ----QIKFRVGRNKRAWVNNCVSIG-----LSSCFLEPLESTGIYFIYAALYQLVKHFPD 366

MibH ----LVRFRSGRHREAWRGNVIAVG-----NSYAFVEPLESSGLLMIATAVQILVSLLPS 402

Rdc2 ----GPNFRIVGDAGCFIDPFFSSG-----HHLAVAGALAAA--VSINASIKGDCTEYEA 359

ChlA CLTEMLKFKLSNAANDYHRRVEAFDKLIRCVTQINNHLIKDHSNHLGNASIMSWRIYFES 422

STTH GDWHPQLRAG------YNSAVANVMDGVREFLVLHYLGAARNDTRYWKDT-KTRAVPDA 435

KtzR ADWNPKSRDM------YNSAVAHVMDGIREFLVIHYRGAARADNQYWRDT-KTRPLPDG 436

PyrH ERWDPVLISA------YNERMAHMVDGVKEFLVLHYKGAQREDTPYWKAA-KTRAMPDG 426

RebH KSLNPVLTAR------FNREIETMFDDTRDFIQAHFYFSPRTDTPFWRAN-KELRLADG 429

Thal KTFDKVLVDR------FNREIEEMFDDTRDFLQAHYYFSPRVDTPFWRAN-KELKLADS 430

KtzQ KRFRPILADR------FNREVATMYDDTRDFLQAHFSLSPRDDSEFWRAC-KELPFADG 431

PrnA TSFDPRLSDA------FNAEIVHMFDDCRDFVQAHYFTTSRDDTPFWLANRHDLRLSDA 419

MibH SRRDPLPSNV------ANQALAHRWDAIRWFLSIHYRFNGRLDTPFWKEARAETDISG- 454

Rdc2 SRWHAKKVDEG------YTLFLLVVMAALKQIRMQENPVLSDVDEDGFDRAFQFLRPVIQ 413

ChlA STYFSILLPAYIGKYHLCPIFSDHFTTDHENGLALRNQLLATLDYANENSINIGFMDNHR 482

STTH LAERIERWKV----QLPDSENVFPYYHGLP------PYSYMAILLGTGAIGLRPSPAL 483

KtzR LAERIECWQT----QLPDTETIYPYYHGLP------PYSYMCILMGGGAIRTPASAAL 484

PyrH LARKLELSAS----HLLDEQTIYPYYHGFE------TYSWITMNLGLGIVPERPRPAL 474

RebH MQEKIDMYRAGMAINAPASDDAQLYYGNFEEEFRNFWNNSNYYCVLAGLGLVPDAPSPRL 489

Thal IKDKVETYRAGLPVNLPVTDEG-TYYGNFEAEFRNFWTNGSYYCIFAGLGLMPRNPLPAL 489

KtzQ FAEKVEMYRAGLPVELPVTIDDGHYYGNFEAEFRNFWTNSNYYCIFAGLGFLPEHPLPVL 491

PrnA IKEKVQRYKAGLPLTTTSFDDS-TYYETFDYEFKNFWLNGNYYCIFAGLGMLPDRSLPLL 478

MibH IEPLLRLFSAGAPLTGRDSFARYLADGAAPLFYGLEGVDTLLLGQEVPARLLPPRESPEQ 514

Rdc2 GSGSSEAVKKFTKEDVAQTIDFAVHALNNMAELDMDIPEHMINGDKEGENGVTNGNNGAA 473

ChlA GGQLLGDWSPTSSWDYDHALSLAKYGHKRLNLPKCLSWSNFYLSLIICKLYYRVYGIGAL 542

STTH ALADP--AAAEKEFTAIRDRARFLVDTLPSQYEYFAAM-GQRV------523

KtzR ALTDQ--GAAQKEFAAVRDRAAQLRDTLPSHYEYLARMRGLDV------525

PyrH LHMDP--APALAEFERLRREGDELIAALPSCYEYLASIQ------511

RebH AHMPQATESVDEVFGAVKDRQRNLLETLPSLHEFLRQQHGR------530

Thal AYKPQSIAEAELLFADVKRKGDTLVESLPSTYDLLRQLHGAS------531

KtzQ EFRPEAVDRAEPVFAAVRRRTEELVATAPTMQAYLRRLHQGT------533

PrnA QHRPESIEKAEAMFASIRREAERLRTSLPTNYDYLRSLRDGDAGLSRGQRGPKLAAQESL 538

MibH WRARAAAARSLASRGLRQSEALDAYAADPCLNAELLSDSDSWAGERVAVRAGLR------568

Rdc2 KTAGLASNMEKLTNDEEKVLNGLRILGKAAPGGTLADFEGTAIDGLMPRLEHGKLGLNKV 533

ChlA WNSSFFKSLSNTTYRFSKFYFLSKLHSFNMIGTPNNDYYDKIQKDFKSYNYNQNNIVDWK 602

STTH -

KtzR -

PyrH -

RebH -

Thal -

KtzQ -

PrnA -

MibH -

Rdc2 -

ChlA Y 603

Supplementary Fig. 2Bathochromic shift of the UV absorbance of 6-chloro-L-Trp

Supplementary Fig. 3ESI-MS (+) spectra ofL-Trp, 6-chloro-L-Trp and 6-bromo-L-Trp.