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SupportinginformationStructuralcharacterizationofEasH(Aspergillusjaponicus)–anoxidaseinvolvedincycloclavinebiosynthesisDorotaJakubczyk,LorenzoCaputi,ClareE.M.Stevenson,DavidM.LawsonandSarahE.O’Connor*

Tableofcontents

ContentsTableofcontents....................................................................................................................1

SI-1Supportingmethods........................................................................................................2

SI-1.1Generalmaterialsandmethods...................................................................................2

SI-1.2Cloning,overexpression,purificationofAj_EasH.........................................................2

SI-1.3InvitroassaysforthewildtypeAj_EasHandalloftheAj_EasHmutants...................2

SI-1.4Massspectrometry(invitroassays).............................................................................2

SI-1.5Mutagenesis.................................................................................................................3

SI-1.6Crystallizationandstructuredetermination................................................................3

SI-1.7Crystallographicmethods.............................................................................................3

SI-1.8Dockingparameters.....................................................................................................4

SI-2Supportingfigures...........................................................................................................5

SI-2.1ReactionscatalysedbyAj_EasHhomologues..............................................................5

SI-2.2SequencealignmentofAj_EasH,Cp_EasHandAsqJ....................................................6

SI-2.3SDS-PAGEanalysisoftheAj_EasHfractionscollectedduringgelfiltrationchromatography.....................................................................................................................7

SI-2.4Co-elutionofcycloclavineauthenticstandardwithanAj_EasHassayproduct...........7

SI-2.5InhibitionofEasHbydivalentmetalcations................................................................8

SI-2.6B-factorsofthe“lid”region(residues52-72)..............................................................8

SI-2.7ComparisonofmonomersofAj_EasHhomologues.....................................................9

SI-3Supportingtables..........................................................................................................10

TableS1.Primersusedinthisstudy.....................................................................................10

TableS2.Crystallographystatistics......................................................................................11

Supportingreferences..........................................................................................................12

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2016

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SI-1Supportingmethods

SI-1.1GeneralmaterialsandmethodsChanoclavine-Ialdehyde1,waspreparedaspreviouslyreported.1AcetonitrilewasofLC-MSgradeandwas purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and solvents were ofanalytical grade and used as purchased. GST-tagged HRV-3C Protease was purchased from AcroBiosystems. All buffers and solutions were prepared in Milli-Q water. Primers for cloning weresynthesizedbyIntegratedDNATechnologies(USA)andsequencingoftheconstructswasperformedbySourcebioscience(UK).CloneAmpHiFipolymerase(ClontechTakara)wasusedforPCRamplification.AVarianCary50BioScanningSpectrometerwasusedtoacquireUV-Visspectra.

SI-1.2Cloning,overexpression,purificationofAj_EasHFor heterologous protein expression, easH was amplified from the previously published vectorpEVE19142 and cloned into theE. coli expression vectors pOPINJ and pOPINF using the In-FusionHDcloning kit (Clontech Takara)3 by using the following primers: Fwd 5’AAGTTCTGTTTCAGGGCCCGACCATTACCGAAACTTCTAAGC, Rev 5’ATGGTCTAGAAAGCTTTAGGCCTTCTTAGCAACTTGATTAG.Expression of Aj_EasH was carried out in 2YT medium supplemented by carbenicillin (100 µg/mL).EscherichiacoliBl21(DE3)cellsweregrowntoanOD600of0.5priorto inductionwithIPTG(100µM)andgrownfor16hoursat18°Cpriortoharvesting.Cellswerere-suspendedinbuffer(50mMK2HPO4,100mMNaCl,10%(v/v)glycerol,pH=7.0)andlysedbycelldisruptor.Cellulardebriswaspelletedbycentrifugation(23000xgfor0.5hour).EasHwaspurifiedbyaffinitychromatographyona5mLGSTrapHP column (GE Healthcare). The GST tag was then removed by treatment with 3C protease beforefurtherpurificationof theproteinbygel filtrationonaTRICORNSuperdex20010/300GLcolumn(GEHealthcare).ThegelfiltrationchromatographywasperformedonanAktapurifier(GEHealthcare)at0.6mL/minusing50mMphosphatebufferpH7.0containing150mMNaClasmobilephase.Elutionwasfollowedat280nmand0.5mLfractionswerecollected.TheyieldofEasHwasestimatedtobe2mgperlitre of culture. Fractions containing EasH, as demonstrated by SDS-PAGE, were collected andconcentratedusingAmicon10kDaMWCOcentrifugalfilters.

SI-1.3InvitroassaysforthewildtypeAj_EasHandalloftheAj_EasHmutantsInvitro assayscontained0.5μMAj_EasA,0.5μMAj_EasG,5μMchanoclavine-Ialdehyde,100μMα-ketoglutaricacid,100μML-ascorbicacid,500μMofNADP+and10μMFeSO4heptahydratein100mMK2HPO4 buffer (pH = 7.5). Aj_EasH or itsmutants were added at a concentration of 0.5 μM and thereactions were incubated at room temperature for 30 minutes. Aliquots (10 μL) were quenched bydilutionin40μLmixtureof20μLof0.1%formicacid(inwater)and20μLofmethanolandanalyzedbyUPLC-MS/MSasdescribedinsectionSI-1.4.

SI-1.4Massspectrometry(invitroassays)UltraperformanceliquidchromatographywasperformedonaWatersAcquityUPLCsystemconsistingofabinarypump,anonlinevacuumdegasser,anautosampler,andacolumncompartment.Separationof

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theanalyteswasachievedonaWatersAcquityUltraPerformanceBEHC18columnwith1.7µmparticlesize,2.1x50mm,keptat30 °C.MobilephaseAwaswatercontaining0.1%(v/v) formicacid;mobilephaseBwasacetonitrilecontaining0.1%(v/v)formicacid.Thegradientprofilewas0min,5%B;from0to5min,lineargradientto30%B;from5to7min,backtoinitialconditionsof5%B.Flowratewas0.6mL/minandtheinjectionvolumeofboththestandardsandthesampleswas2μL.Aftereachinjection,the needle was rinsed with 600 μL of weak wash solution (water/acetonitrile, 90:10) and 200 μL ofstrongwashsolution(acetonitrile/water,90:10).Sampleswerekeptat10°Cduringtheanalysis.Mass spectrometry detection was performed on a Waters Xevo TQ-S instrument equipped with anelectrospray(ESI)sourceoperatedinpositivemode.Capillaryvoltagewas3.8kV;thesourcewaskeptat150°C;desolvationtemperaturewas200°C;conegasflow,150L/h;anddesolvationgasflow,800L/h.Unitresolutionwasappliedtoeachquadrupole.Forqualitativestudies,theacquisitionwasperformedin full scan and SIR (single ion recording), while for cycloclavine 4 quantification a MRM (multiplereaction monitoring) method was used. Based on collision induced dissociation studies the firstquadrupolewassetontheparention[M+H]+=239.2whilstthethirdquadrupolewassettom/z144.1(conevoltage36V;collisionenergy24V).

SI-1.5MutagenesisMutants were generated by overlap extension PCR. The primers used to generate the mutants arereported in Supplementary Table 1. Full-length PCR products were gel purified, ligated into pPOINFandpOPINJ expression vectors and transformed into competent E. coli Stellar strain cells (ClontechTakara).3Mutantconstructsweresequencedtoverifythemutantgenesequenceandcorrectinsertion.

SI-1.6CrystallizationandstructuredeterminationForcrystallization,theproteinwasconcentratedto6.6mg/mLandfilteredthrough0.1µmPVDFfilter(Durapore). α-Keto-glutarate was added to the protein solution to a concentration of 1 mM beforesetting up crystallization trials. PACT Premier (Molecular Dimensions) and PEGs suite (Qiagen)crystallization screenswere conducted by sitting-drop vapour diffusion inMRC 96-well crystallizationplates (Molecular Dimensions) with a mixture of 0.3 µL well solution and 0.3 µL protein solution.Solutions were dispensed by an OryxNano robot (Douglas Instruments). The initial screens providedseveral hits after four weeks incubation at 20°C. The best crystals for the complex Aj_EasH/α-keto-glutaratewere obtained from condition C12 from the PACT Premier (Molecular Dimensions): 0.01MZnCl2 buffer; pH 7.0 and 20% (w/v) PEG 6000. Crystalswere harvested from themother liquor usingLithoLoops(MolecularDimensions)andcryoprotectedwithwellsolutionsupplementedwith20%(v/v)ethyleneglycol.

SI-1.7CrystallographicmethodsX-ray diffraction images were recorded using a Pilatus 6M detector (Dectris) on beamline I02 at theDiamondLightSource(Oxfordshire,UK),atawavelengthof0.9795Å,fromasinglecrystalmaintainedat100KbyaCryojetcryocooler(OxfordInstruments).TheresultantdatawereintegratedusingXDS4andscaled and merged using AIMLESS.5 A summary of the data collection and processing statistics ispresented in Supplementary Table 2. An initial model of the EasH/α-ketoglutarate complex wasobtainedbymolecularreplacementusingPHASER6withthestructureofCp_EasH(PDBcode4NAO)asthe search model. Two copies of the subunit were located in the asymmetric unit, which formed a

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biologically relevant homodimer giving an estimated solvent content of 47%. Themodel was refinedwithREFMAC57andmanual correctionsweremade inCOOT.8Aswellas the ironandaKGpresent ineach copy of the active site, therewere four zinc ions bound at crystal contacts. Additional electrondensityatoneofthesecontactswas interpretedasathirdmoleculeofaKG.ThestatisticsofthefinalmodelareshowninSupplementaryTable2;thiswasdepositedintheProteinDataBankwithaccessioncode5M0T.

SI-1.8DockingparametersEnamine5wasdockedintotheAj_EasHcrystalstructureusingAutodock4.2.9

Theligand(enamine)waspreparedwithoutanyactivetorsions.Themoleculewastreatedasrigidandthereceptorconsistedofthedesolvatedhigh-resolutioncrystalstructure.Thesearchspacewasdefinedbya61x61x61Åboxwitha0.375Ågridspacingandencompassedtheentireactivesitecavity.SearcheswereperformedusingtheLamarckianGeneticAlgorithm,consistingof100runswithapopulationsizeof150and2,500,000energyevaluations.Atotalof4,574generationswereanalysedandclusteredwithanRMStoleranceof2Åpercluster.Thisresultedinfourdistinctclusters,whichconstituted70%,9%,15%and6%oftheresultantconformations,respectively.9

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SI-2Supportingfigures

SI-2.1ReactionscatalysedbyAj_EasHhomologues.a)HydroxylationofapeptidesubstratebyCp_EasH.b)Twostepoxidationsof4’-methoxycyclopeptincatalyzedbyAsqJ.

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SI-2.2SequencealignmentofAj_EasH,Cp_EasHandAsqJSecondary structure elements of the Aj_EasH and AsqJ crystal structures are displayed (top and thebottom line). Numbering corresponds to Aj_EasH residues. Identical and similar amino acids arehighlightedinredandyellow,respectively.Aj_EasHiron-bindingaminoacids(H131,E133andH209)areindicatedbybluedots,andAsqJactivesiteaminoacids(H134,D136andH211)areindicatedbygreendots. The “lid” region (residues 52-72) is underlined by a blue line. Alignment was generated usingClustalW.10 The similarity coloring scheme used is based on physico-chemical properties. SecondarystructuredepictionwasaddedusingESPript3.11

Lid

Lid

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SI-2.3SDS-PAGEanalysisoftheAj_EasHfractionscollectedduringgelfiltrationchromatography.TheexpectedsizeofEasHis32.5kDaafterremovaloftheGST-tag.(M)RunBluePrestainedmarkers(Expedeon);(1-9)Proteinfractionscollectedduringgelfiltrationchromatography.

SI-2.4Co-elutionofcycloclavineauthenticstandardwithanAj_EasHassayproduct.ThefigureshowsUPLC/MStracesatselectedionmonitoringofm/z239(massoftheexpectedenzymaticproduct,cycloclavine4)ofa)cycloclavine4authenticstandardandb)arepresentativeinvitroassayproduct.Theassayswereperfomedwithsubstrate,chanoclavinealdehydeI1,EasA,EasHandEasGandcofactors(FeSO4,L-ascorbicacid,NADP+andaKG)asdescribedinsectionSI-1.3.

31

110

66

21.5

6.5

M123456789

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SI-2.5InhibitionofEasHbydivalentmetalcations.The figure shows UPLC/MS traces at selected ion monitoring of m/z 239 (mass of the expectedenzymaticproduct,cycloclavine4)of invitroassays inwhichdifferentdivalentcationswereaddedtothereaction.Theassayswereperfomedwithsubstrate,chanoclavinealdehydeI,EasA,EasHandEasGandcofactors(FeSO4,L-ascorbicacid,NADP+andaKG)asdescribedinsectionSI-1.3,exceptthatvariousdivalentmetalswere added at the start of the enzymatic reaction, as described below. a)Wild typeenzyme (positive control) with no additional divalent metal added; b) Addition of 1 mM of NiCl2; c)Additionof1mMofZn(CH3CO2)2;d)Additionof1mMofCoCl2,e)Additionof1mMofCuSO4.Inhibitionofcycloclavine4formationbydivalentmetalcationshasbeenobservedinentriesb-e.

SI-2.6B-factorsofthe“lid”region(residues52-72)Main-chain B-factors are averaged for each residue of the final model and plotted against residuenumber.Thetracesforthetwosubunitsintheasymmetricunitareoverlaidclearlyillustratingthatthelidregionsarethemostmobileinbothcases.

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SI-2.7ComparisonofmonomersofAj_EasHhomologuesThemaindifference is inthe loopregion(magenta).aKG isrepresentedasspheres(carbonatomsareshowningreenandoxygenatomsinred;theironisshowninorange).

Aj_EasH(PDB 5M0T)

Cp_EasH(PDB 4NAO)

AsqJ(PDB 5DAP)

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SI-3Supportingtables

TableS1.PrimersusedinthisstudyMutant Forwardprimer ReverseprimerY65A CATGAAGCAATGGGAATTGGGTCAAAAA

TCCGCCCAAGAATCTTACTTGGCTGACCCAATTCCCATTGCTTCATG

Y69A CATGAAGCAATGGGAATTGGGTCAAAAATCCTACCAAGAATCTGCCTTGGCTGGTATGAG

TGACCCAATTCCCATTGCTTCATG

Partofthelid:64-69toA

CATGAAGCAATGGGAATTGGGTCAAAAAGATGACCCACATTTTTGGTTGGCTGGTATGAG

TGACCCAATTCCCATTGCTTCATG

E133A CTGAACCAGGTTACCATGGTCAAGAATTGCATAGAGCACATGATGGTATTCCAATCTG

CAATTCTTGACCATGGTAACCTGGTTCAG

D135A CTGAACCAGGTTACCATGGTCAAGAATTGCATAGAGAACATGCTGGTATTCCAATCTG

CAATTCTTGACCATGGTAACCTGGTTCAG

Q128A CTTCTTCTGTTTTGGAAACTGAACCAGGTTACCATGGTGCAGAATTGCATAGAGAACATG

CCTGGTTCAGTTTCCAAAACAGAAGAAG

131-133HREtoHQA

CAGGTTACCATGGTCAAGAATTGCATCAAGCACATGATGGTATTCCAATCTGTACT

CAATTCTTGACCATGGTAACC

131-133HREtoHRA

CAGGTTACCATGGTCAAGAATTGCATAGAGCACATGATGGTATTCCAATCTGTACT

CAATTCTTGACCATGGTAACC

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TableS2.CrystallographystatisticsDatacollection

Beamline I02,DiamondLightSource Wavelength(Å) 0.9795 Detector Pilatus6M Resolutionrange(Å) 47.47–2.20(2.26–2.20) SpaceGroup P212121 Cellparameters(Å) a=40.25,b=100.17,c=

148.88 Totalno.ofmeasuredintensities 185,902(13,000) Uniquereflections 31,397(2,300) Multiplicity 5.9(5.7) MeanI/s(I) 8.7(1.3) Completeness(%) 99.7(99.5) Rmergea 0.151(1.413) Rmeasb 0.166(1.558) CC½c 0.996(0.539) WilsonBvalue(Å2) 34.6Refinement Resolutionrange(Å) 47.47–2.20(2.26–2.20) Reflections:working/freed 29,876/1,521 Rwork/Rfreee 0.218/0.244(0.357/0.364) Ramachandranplot:

favoured/allowed/disallowedf(%)97.4/2.6/0.0

R.m.s.bonddistancedeviation(Å) 0.009 R.m.s.bondangledeviation(°) 1.38 No.ofproteinresidues:chainA/chainB

(ranges)287(7-293)/279(9-287)

No.ofAKG/Fe2+/Zn2+/watermolecules 3/2/4/107 MeanBfactors:protein/AKG/

Fe2+/Zn2+/water/overall(Å2)51/52/33/61/40/51

PDBaccessioncode 5M0T

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Valuesinparenthesesarefortheouterresolutionshell.aRmerge=∑hkl∑i|Ii(hkl)-áI(hkl)ñ|/∑hkl∑iIi(hkl).bRmeas=∑hkl[N/(N-1)]1/2×∑i|Ii(hkl)-áI(hkl)ñ|/∑hkl∑iIi(hkl),whereIi(hkl)istheithobservationofreflectionhkl,áI(hkl)ñistheweightedaverageintensityforallobservationsiofreflectionhklandNisthenumberofobservationsofreflectionhkl.cCC½isthecorrelationcoefficientbetweensymmetryequivalentintensitiesfromrandomhalvesofthedataset.dThedatasetwassplitinto"working"and"free"setsconsistingof95and5%ofthedatarespectively.Thefreesetwasnotusedforrefinement.eTheR-factorsRworkandRfreearecalculatedasfollows:R=å(|Fobs-Fcalc|)/å|Fobs|,whereFobsandFcalcaretheobservedandcalculatedstructurefactoramplitudes,respectively.fAscalculatedusingMolProbity.12

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