Structural and mechanistic insights into azetidine-associated αKG-NHFe enzyme OkaE with multifunctional catalysis

Domain: chemistry
Published: 2026-02-17
Editorial outcome: minor_revision

Abstract
α-Ketoglutarate-dependentmononuclearnon-haemiron(αKG-NHFe)enzymesarecatalyticallyversatile,yetOkaEisuniqueforsynthesizingazetidineringsviaC–Cbondformation.Here,wereporttheunexpectedmultifunctionalityofOkaE,whichcatalyzessequentialoxidations.IsotopiclabellingstudiesdemonstratethatasecondO₂moleculeparticipatesinsequentialepoxidationandringcleavage,incorporatingtwooxygenatomswithinasinglecatalyticcycletoformthepreviouslyunknownstructure,neuokaramineIV.CrystalstructuresoftheOkaE•CoII•αKG•okaramineAcomplexunveilauniquemethionine–πinteractionnetworkthatfacilitatessubstratebinding.Mutationalandcrystallographicanalysessuggestthisnetworkfine-tunessubstrateorientationrelativetothemetallo-centre,activatingdistinctreactionpathwaysatthe3a-OHorC8apositions.QM/MMsimulationsindicatethatdynamicrotationoftheFeIV=Ospeciesinitiatesthecycle,enablingreactionbifurcation.ThisstudyelucidatesthestructuralandmechanisticbasisofOkaE’sreactivity,highlightingitspotentialasaprogrammablebiocatalystfornaturalproductdiversification.

Introduction
α-Ketoglutarate-dependentmononuclearnon-haemiron(αKG-NHFe)enzymesplaycrucialrolesinnaturalproductbiosynthesisthroughareactiveFeIV=Ospecies to selectively abstract hydrogen atom from substrate C–H bonds via a Hydrogen Atom Transfer (HAT), driving oxidations that generate structurally diverse and biologically significantnaturalproducts1,2.Thisenzymefamilyexhibitscatalyticversatility,including hydroxylation, ring fragmentation, C–C bond cleavage,epimerization,desaturation,endoperoxidation,andheterocycleformation3.While~70%ofreportedαKG-NHFeenzymescatalyseasingletypeofreaction1,3,4,theremaining30%enzymesexhibitmultifunctionality, contributing to diverse biological processes such as cellular metabolism, signaling, and biosynthesis of complex natural products1.ProminentexamplesofmultifunctionalαKG-NHFeenzymesinclude FtmOx15–9, AsqJ10, VioC11, SptF12, DnmT13, AusE14, PrhA14, DAOCS15, HppD16, EFE17, AndA18, and DabC19. These multifunctional enzymes exhibit catalytic versatility, often facilitating complex chemicaltransformationsthatyieldstructurallydiverseandbiologicallyactivemetabolites20,21.

Structural classification based on αKG coordination geometry reveals two distinct modes, distal-type and proximal-type, each accountingfor~50%ofknownαKG-NHFeenzymes1.Theproximal-typeenzymespositionthecatalyticferrylspeciesadjacenttothesubstratefordirectoxygeninsertiontypicalofsimplehydroxylation.However,indistal-typeenzymes,αKGcoordinatesinsuchawaythattheFeIV=Ospeciesispositionedawayfromthesubstrate,promotingmorecomplicatedreactionsviaemergingmechanisticmodelsinvolvingaminoacid radical-mediated process in the COX-like model of FtmOx1-catalysis5,7,αKGrotationinAsqJ-catalysis10,andFeIV=Ospeciesrotationin WelO5- and SyrB2-catalysis22,23. Notably, in distal-type αKG-NHFeenzymes, both substrate activation and access to multiple reactive sites are critical for enabling multifunctional catalysis, as demonstrated in AusE14, PrhA14, and SptF12. A deeper understanding of the catalytic mechanisms on these distal-type enzymes is crucial for advancing enzyme engineering,biocatalysis,anddrugdiscovery.

Theazetidinescaffoldisafour-memberedsaturatedcyclicamine(Fig.1a),andisfoundinrelativelyfewnaturalproductswithdistinctivephysiological functionalities24. Notable examples include quorum-sensing regulation azetidomonamides25, antifungal and anticancer vioprolides26, anti-Alzheimer’s disease candidate bonnevillamides27, insecticidal okaramines28, and anti-fungal polyoxins29. Beyond their natural occurrence, azetidines are increasingly valued as important pharmacophores in medicinalchemistry30, offering enhanced three-dimensionality31, favorable pharmacokinetic properties32,33, and reducedlipophilicity34,therebyaddressingcriticalchallengesindrugdesign. However, chemical synthesis of the azetidine ring remains particularlychallengingduetotheirhighstrainandintrinsicinstabilityrelativetofive-orsix-memberedN-heterocycles(Fig.1a).Conventionalsyntheticroutesofteninvolvedstrainedtransitionstates,high-energyintermediates, and multistep sequences to overcome significant kineticandentropicbarriers35,36.

In contrast,naturehasevolved specialized enzymes capable of assemblingthisstrainedringwithefficiencyandselectivity.Todate,only five biosynthetically validated azetidine-ring forming enzymes havebeenreported36,37.MostoperatethroughS-adenosylmethionine(SAM)-dependent intramolecular cyclization, including Class I methyltransferasesVioH38,NAS39,andAzeJ25,whichcatalyzeintramolecular C–N bond formation with concomitant release of 5′-methylthioadenosine (MTA)36. A mechanistically distinct case is the haem-oxygenase-likediironoxidasePolF,whichmediatesC–Ncyclizationinthebiosynthesisofpolyoxamicacid37(Fig.1b).

Strikinglydivergentfromthisparadigm,thebiosyntheticpathwayof okaramines, a class of complex indole alkaloids isolated from PenicilliumandAspergillusspecies28,involvesanαKG-NHFeenzyme,OkaE,capableofcatalyzingazetidineringformationviaC–Cformation(Fig.1c).Notably,OkaEistheonlycharacterizedmemberoftheαKG-NHFefamilycapableofmediatingthisunusualcyclization,positioningitasabiochemicalanomalywithsignificantmechanisticandpharmacophoreimplications40,41.Beyondazetidineringformation,OkaEalsofacilitatessubsequenthydroxylation,demonstratingmultifunctionality. However, despite Lin and co-workers’ elucidation of OkaE’s dual catalytic role42, key questions remain unresolved, particularlyconcerningthemechanismoftheFeIV=OmediatedazetidineringformationanditsdivergencefromclassicalαKG-NHFeoxidativechemistry.Inaddition,okaraminesexhibitpotentinsecticidalactivitybyselectivelyactivatingglutamate-gatedchloridechannels(GluCls)ininvertebrates,withoutaffectinghumanligand-gatedanionchannels43.Thisselectivitymakesokaraminespromisingleadsforinsecticidalpharmacophoredesign.GiventhedualfunctionalityofOkaEandtheunresolvedmechanisticquestionssurroundingitsreactivity,adeeperinvestigationofits catalytic mechanism and subsequent protein engineeringholdssignificantpotentialforadvancingbiocatalyticandinsecticidedesign.

Inthisstudy,wecharacterizedOkaEinvitroanduncovereditsmultifunctionalityandcatalyticpromiscuity.Inadditiontoazetidineringformationandhydroxylation,OkaEwasfoundtocatalyzeaseriesof oxidation reactions on the substrate okaramine A (1), including carboxylation, epoxidation, ring fragmentation, and alcohol dehydrogenation (Fig. 2b). Through an integrative approach combining isotopic labelling, X-ray crystallography, quantum mechanics/molecularmechanics(QM/MM)calculations,andproteinengineering,we decipheredthemechanistichierarchygoverningthismultifunctionalcatalysis.Keyinsightsincludedthefollowing:(i)theidentificationofconformationalchangesintheFeIV=OspeciesofOkaE;(ii)thedetermination of distinct hydrogen abstraction sites on the substrate, enablingbifurcatedreactionpathways;(iii)thecharacterizationofareactioninvolvingaseconddioxygenmolecule,asseeninendoperoxidaseslikeFtmOx1andNvfI;and(iv)elucidationofamethionine–π(Met–π)interactionnetworkthatmodulatessubstratepositioningandradical propagation. These insights, spanning atomic to molecular levels, established a unified framework explaining the multifunctionalityand catalytic plasticityofOkaE.Bymappingstructure-functionrelationshipsacrossmultiplecatalyticmodalities,thisworkprovides a blueprint for engineering next-generation biocatalysts capableoforchestratingcomplexsynthetictransformations.

Results
InvitrobiochemicalcharacterizationofOkaErevealsitsmultifunctionalcatalyticproperties

An understanding of the catalytic mechanismofOkaE is crucial for efficientlyconstructingtheassociateazetidinefunctionalitymoietyinokaramines.WeanalyzedtheaminoacidsequenceofOkaEderivedfromPenicilliumsimplicissimumATCC90288(BBB04331.1)alongside134reportedαKG-NHFeenzymesthatcatalyzewithsmallmolecules,using sequence similarity network (SSN) analysis (Supplementary Fig.1a;andSupplementarydataset1).AtanE-valuethresholdof10⁻⁷⁰,OkaE clustered with two multifunctional enzymes, AsqJ10 and FtmOx15–9 (Supplementary Fig. 1b). AsqJ catalyses stereoselective dehydrogenation and epoxidation in quinolone alkaloid biosynthesis10.FtmOx1catalysesendoperoxidation,dealkylation,anddehydrogenationreactionsoftheindolealkaloidfumitremorginB5–9.Structuralsimilarityanalysis(SSA)alsoconfirmedthatOkaEsharesthehigheststructuralhomologywithFtmOx1andAsqJ(Z-scores:36.1and40.3,respectively;SupplementaryFigs.1cand1d).WespeculatedthatOkaE might function as a multifunctional enzyme. To test this hypothesis,weclonedtheokaEgeneintothepASK-IBA3-plusvectortoexpress a C-terminal strep-tagged protein in Escherichia coli BL21(DE3),followedbyanaerobicpurificationwithinafewhours(See“Methods”sectionfordetails).Concurrently,okaramineA(1,112.3mg)wasisolatedfromP.simplicissimumATCC90288culturedin16kgofokaramediumat28°Cfor14days(See“Methods”sectionfordetails).

For functional characterization of OkaE, the anaerobically purified proteinwasemployedininvitrobiochemicalassaysusingokaramineA(1) as the substrate. Unexpectedly, these biochemical assays, conductedunderbothsingle-turnoverandmultiple-turnoverconditions,revealed the formation of four previously uncharacterized minor products,designatedasneuokaraminesI−IV(4−7),inadditiontothepreviously reported 12-deshydroxyl-okaramine E (2, minor product) andokaramineE(3,majorproduct)42(Fig.2a,tracesi,SupplementaryFig.2a).ThedivergenceinproductprofilesfromthefindingsofLin etal.42islikelyattributabletodifferingexperimentalmethodologies.In contrast to their work, which involved the aerobic purification and assay of a His-tagged enzyme from S. cerevisiae, our study utilized Strep-taggedOkaEthatwaspurifiedanaerobicallyfromE.coli.Under multiple-turnoverconditions,~70%ofokaramineA(1)wasconvertedtoproducts(SupplementaryFig.2a,traceii).Wespeculatedthattheincompleteconversionmightbeduetothelowsolubilityofthesubstrate.Totestthis,weintroduced3.6%(v/v)DMSOintothereactionsystem, which indeed promoted complete substrate consumption (SupplementaryFig.2a,traceiii).Toinvestigatethestoichiometryoftheco-substrateαKG,reactionswereperformedwithdifferentmolarratios of αKG to 1 (sourcedata are provided asa SourceData file). WhentheratioofαKG:1reached~1:1,compound1wasalmostcompletely consumed, indicating that a single equivalent of αKG is requiredfortheformationofmultipleproducts3–7(Fig.2b,traceiii).Additionally,increasingtheαKG:1ratioto2:1promotedthegradualconversionof3to4(Fig.2b,traceiv).However,whenthemolarratioofαKGto1wasincreasedto4:1,theproductdistributionremainedessentiallyunchanged(Fig.2b,tracev).

Toinvestigatewhetherproducts3–7areformedwithinasinglecatalyticturnoverorthroughmultiplesequentialreactionsinwhichintermediate products serve as new substrates, we varied the stoichiometric ratio of the co-substrate αKG to substrate 1. When the αKG:1 ratio was ~1:1, the compound 1 was almost completely consumed,yieldingaseriesofproducts(3–7)(Fig.2b,traceiii).Increasing the αKG:1 ratio to 2:1 promoted the gradual conversion of 3 to 4 (Fig.2b,traceiv).However,furtherincreasingtheratioto4:1didnotaltertheoverallproductdistribution(Fig.2b,tracev).UsingHR-ESI-MSand1DNMR(SupplementaryFigs.4and5;SupplementaryTable2),weidentified12-deshydroxyl-okaramineE(2)andokaramineE(3)intheP.simplicissimumATCC9028842,44,45.However,theneuokaraminesI−IV(4−7)werenotdetected.Subsequently,compounds4−7wereisolatedandpurifiedfromascale-upenzymaticreactionsystem,consistingof40parallelreactions(eachcontaining70μMOkaE,210μMsubstrate,2100μMFeII,700μMαKG,210μMascorbate).Theirstructureswereelucidated by comprehensive spectroscopic analyses, including HR-ESI-MS,1Dand2DNMR,aswellasECDandNMRcalculations.(SeetheMethods section and Supplementary Methods for details; Supplementarydatasets2and3).StructuralanalysisrevealedthattheNMRspectraofcompound4closelyresembledthoseof3,exceptforthereplacementofthehydroxymethylgroupatC12byacarboxylgroup(SupplementaryFigs.5and6;SupplementaryTables2and3).Comparison of the specific rotation of 3 ([α]20 +222.1) with 4 ([α]20 +233.6), together with their experimental ECD spectra (SupplementaryFig.11),indicatedthattheysharethesameabsoluteconfigurations 2S,3aS,8aR,11S. In contrast, 5 featured an intramolecular ether bridge between C3a and C12 (Supplementary Fig. 7; SupplementaryTable 4). Detailed NOESY correlations, coupling constants, dihedralangles,andNMRcalculationscombinedwithDP4+analysisestablished the relative configuration of 5 as 2S*,3aS*,8aR*,11R*,12R* (Supplementary Methods; Supplementary Figs. 12 and 13). Furthermore,thestrongagreementbetweenitsexperimentalECDspectrumandthecalculatedspectrumof(2S,3aS,8aR,11R,12R)−5(SupplementaryFig. 11; Supplementary Tables 8 and 9) confirmed the absolute configuration of 5 as 2S,3aS,8aR,11R,12R. Furthermore,thestrongagreementbetweenitsexperimentalECDspectrumandthecalculatedspectrumof(2S,3aS,8aR,11R,12R)−5(SupplementaryFig. 11; Supplementary Tables 8 and 9) confirmed the absolute configuration. Detailed NMR analysis of 6 confirmedasimilarstructuralskeletontothatof1,withcleavageoftheC−NbondbetweenN1andC8a,andtheintroductionofacarbonylgroupatC8a(SupplementaryFig.8;SupplementaryTable5).Notably,theplanarstructureof6wasidenticaltothatofokaramineR,previouslyisolatedfromP.simplicissimum ATCC 9028846. The absolute configuration of 6 was assignedas2S,3aS,basedonNMRspectroscopiccomparisonsandECDcalculations(SupplementaryFig.11;SupplementaryTables10and11).Similarly,theNMRspectralfeaturesof7alsocloselyresembledthoseof1.Adetailedanalysisrevealedthat7containedanadditionalepoxide ring connecting C11 and C12. Furthermore, the C−C bond cleavage betweenC8aandC3ain7resultedintheoxidationofthesecarbons,formingacarbonylgroupatC3aandanamido-carbonylgroupatC8a(Supplementary Fig. 9; Supplementary Table 6). 2D NMR analysis confirmedthatallotherstructuralfeaturesof7wereconsistentwiththose of 1. The absolute configuration of 7 was assigned as 2S,11R, based on NMR calculations (R² = 0.9986, RMSD=1.82), DP4+ probabilityanalysis(94.3%),andECDcalculations(SupplementaryFigs.11,14and15;SupplementaryTables12and13).

To determine whether compounds 4−7 were derived from enzyme-catalysedreactions,weperformedinvitroOkaEassaysusing2 or3asthesubstrates.Notably,OkaEefficientlyconvertedboth2and3 into 4 and 5, respectively, demonstrating its ability to catalyse a sequentialreactionseriesinvolvingazetidineringformation,hydroxylation,andcarboxylation,withokaramineA(1)servingastheinitialsubstrate (Fig. 2a, traces iii–vi). In this process, two putative intermediates(IM2andIMa )werenotdirectlycharacterizedduetoeitherlow yield or lability. However, their structures were inferred to be aldehyde-containingspecies(IM2)andgem-diolspecies(IMa )basedon HR-ESI-MS analysis (Supplementary Fig. 10) and their chemical transformation relationship with 2 and 3 (Fig. 2a, traces iii–vi; SupplementaryFig.2b).Biochemicalevidence(Fig.2a,tracesiii–vi)suggeststhat4islikelygeneratedthroughenzymaticoxidationofIM2, whereas5resultsfromnon-enzymatic,acid-promotedintramolecularhemiacetalformationofIM2.ThegeminaldiolIMa mayalsoundergodirectnon-enzymaticconversionto5.Furthermore,OkaEwasunabletofurthercatalyze5and6(Fig.2a,tracesvii–x).Compounds6and7 areproposedtooriginatefromdistinctreactionbranches,involvingadditional oxidative transformations triggered by cleavage of the C8a–N1 or C8a–C3a bonds, diverging from the pathway leading to compounds2–5.Collectively,ourinvitrostudiessuggestedpossiblereactionpathways catalysed by OkaE, as illustrated in Fig. 2c. The observed reaction complexity underscores the need for additional mechanisticstudiestofullyelucidatethemultifunctionalpropertiesofthisαKG-NHFeenzyme.

TheoverallarchitectureoftheOkaEstructureresemblesatypicalfoldingpatternoftheαKG-NHFeenzymefamily

Inordertoinvestigatestructure-function-functionrelationshipsofOkaE,crystalstructuresofOkaE•CoII(PDBID:9M2M),OkaE•CoII•αKG(PDBID:9J85),and OkaE•CoII•αKG•okaramine A (1) complex (PDB ID: 9M2L) were solved to resolutions of 2.20Å, 2.15Å and 2.55Å, respectively(SupplementaryTable14;Supplementarydataset4),wherecobaltwasusedasthemetal instead of iron to prevent possible reactions. In these structures,OkaEensembledasahomodimer,whichisconsistentwiththeSECdata(SupplementaryFig.16).IneachOkaEmonomer,sevenβstrands stacked antiparallelly to form a double-stranded β-helix (DSBH) core, surrounded by the remaining α-helices and loops (Fig.3a),andthisso-called“jelly-rollfold”resemblesacharacteristicfeatureofαKG-NHFeenzymefamily47.TheOkaEdimerwasformedbyaninternaltwo-foldsymmetryoperator,withanaveragedimerinterfaceof2,658Å2,accountingfor~18%ofthetotalsurfaceareaoftheprotein.Theprimary region involved in dimerization is the loop S264−L283whichinteractswithE63′−Y69′,W79′−D82′,Y238′−T242′aswellasG138′−I142′ from the adjacent monomer (Supplementary Fig.17). In addition,ashortα-helixregionQ143−P153interactedwiththesameregionfromtheadjacentmonomerinadditiontoP110′andH111′,andtheloopT232−P237couldcontributetodimerizationwithitscounterpartfromtheothermonomerthroughtwo-foldsymmetry,aswellaswithH139′−I142′(SupplementaryFig.17).Likeothermembersofthisenzymefamily,theactivesiteofOkaEwascomposedofresiduesfrombothmonomers.Thecobaltionischelatedbyacon-servedHx(D/E)x Htriad(H134,D136,andH210)andcoordinatedbythree additional water molecules to construct an octahedral geometry20(SupplementaryFig.18a).

StructuralanalysisoftheOkaE(cid:129)CoII(cid:129)αKG(cid:129)okaramineA(1)complexrevealsthekeyfeaturesoftheactivepocket

StructuralsuperimpositionrevealedthattheproteinsmostsimilartoOkaEareAsqJ10,FtmOx15andAndA18,withRMSDvaluesrangingfrom1.6Åto2.0Å.Althoughnoneoftheseproteinsshare>43%sequenceidentitywithOkaE,theyallbelongtothemultifunctionalαKG-NHFeenzymes and catalyse a series of diverse reactions, including epoxidation,endoperoxidation,hydroxylationand isomerization.Toelucidate the substrate specificity of OkaE and further explore the mechanism of the sequential oxidation, we further analysed the structure ofthe OkaE•CoII•αKG•okaramine A(1) complex. Because1 hasnegligiblesolubilityinwater,asoakingoflongdurationwasusedtoobtaincrystalsoftheOkaE•CoII•αKG•okaramineA(1)complex.ThemFo-DFcmapclearlyshowsastrongpositivedensityattheactivesite withashapeconsistentwith1,confirmingsubstratebinding(SupplementaryFigs.18band19a).

TheRMSDbetweenOkaE•CoIIandOkaE•CoII•αKG•okaramineA(1)complexis0.36Å,suggestingnomajorstructuralchangesuponsubstratebinding.Superimpositionofthesestructuresrevealedthattheproteinsmostsignificantrearrangementinducedbysubstratebindingoccurred in the lid regions of the substrate binding pocket, including Y69−P74,Q131−D136andR179−D189,whichshiftedslightlytowardthemetallo-centre when αKG and 1 were included (Supplementary Fig.20).Asaresult,thevolumeofthesubstratebindingpocketwasreducedby~10%(627Å3vs.559Å3,byCavitOmiX).Inaddition,theloopregionQ131−H137whichcontainsthehistidineresiduescoordinatingwith the cobalt ion moved ~1Å toward the substrates, potentially reinforcingtheinteractions.

ThesubstratebindingpocketofOkaEcanfurtherbedividedintotwosub-pocketsforαKGand1,consistentwithotherenzymefamilymembers1,3.Theco-enzymeαKGwasburiedinthedeepersub-pocket,whereitcoordinateswiththeCobaltioninabidentatemannerviaits1-carboxyl and 2-keto group to replace water molecules. Similar to AsqJ10, FtmOx15,7,9, and EasH48, αKG adopted a distal mode, the 1-carboxylatewastranstothe2ndhistidine(H210)intheHx(D/E)x Htriad,whilethe2-ketogroupwastranstoD136.Inaddition,αKGwasstabilizedby multiple polar interactions, including a salt bridge betweenitscarboxyltailandR223,ahydrogenbondwithT171,aswellasanotherhydrogenbondbetween1-caboxylateandQ131(Fig.3d).

Incontrast,substrate1waslocatedattheoutersub-pocketwhichwas more hydrophobic, with its indole head sandwiched between H134−G138ononesideandI269′−N271′fromtheadjacentmonomer(Fig.3e).Notably,OkaEcontainedtheresidueS270′thatformedanα,β-turnthrough a cis-peptide bond with I269′, wherethis position is oftenreplacedbyprolineresidueinothermembersofthisfamilytoformeithertrans(AsqJ,PDBID:6EOZ)orcis(FtmOx1,PDBID:4Y5T)peptidebondwiththeprecedingresidues.Asimilarnon-prolineresidueatthispositionwasalsoobservedinAndA(PDBID:5ZM3)whereanasparticacid(D270)formsatrans-bondwithK269,whereasinEasH(PDBID:5M0T),atruncatedloopformsashortβ-hairpinwithatrans-glycineatthecorner.Theconservationofthisunusualcis-peptideinOkaEacrossdifferentorganismssuggeststhatitmayplayacriticalroleincatalysis(SupplementaryFig.18e).Indeed,thisβ-turnundergoesa conformational shift, flipping toward the adjacent monomer upon substratebinding,therebynarrowingthecavityfortheindoleheadandislikelytocontributetosubstratespecificity.

TheC-ringtoE-ringof1wereroughlyco-planerwiththeindoleheadandinteractedwithQ131,Q132,andH134.Aweakp−πstackinginteraction existed between the carbonyl at C12′ on the D-ring and H134withadistanceof4.1Å(Fig.3f).Ontheotherhand,theindoletail(A-andB-rings)of1wasflippedalmost90°relativetothemajorplanetoaccommodatethepocketformedbyM64,M71,W79,N118,M122,L227,andT273′.AhydrogenbondbetweentheC3ahydroxylgroupand W79 oriented the reverse prenyl group at N8 of 1, where the reactionoccurred (Fig. 3e and Supplementary Fig. 18c). Crucially, a uniqueMet–πinteractionnetworkinvolvingresiduesM64-M71-W79-M122envelopedtheA-ringof1(Fig.3e),stabilizingtheindoletailinacatalyticallycompetentgeometryandorientatingC8aandO3a(oxygenatomon3a-OH)towardtheactivecenter(Fig.3f).Thisnetwork,coupledwithslightlylarger substrate pocket (Supplementary Fig. 18e), enabledOkaEtoaccommodatediverseoxidationintermediateswhilemaintainingregioselectivity,whichisakeystructuralfeatureunderlyingthemultifunctionalityofOkaE.

QM/MMcalculationsindicateFeIV=OspeciesrotationasaperequisitestepinOkaE-catalysis

GiventhatallavailablecrystalstructuresofOkaEdemonstrateadistal-typeαKGbindingmode,weobservedthatthepotentialO bindingandactivationsiteinOkaE,whichistypicallyoccupiedbyasolventmoleculeinthecrystalstructures,wasnotadjacenttothesubstratebindingpocket(Fig.3f).TheC8aandO3areactionsitesonokaramineA(1)were~6.9Åand6.5Å,awayfromabovepotentialoxygenbindingsite,respectively.Furthermore,αKGactedasaspatialbarrier,preventingdirectinteractionbetweenthesubstrateandtheoxygen-bindingsite.Consequently,thesubsequentformationoftheFeIV=Ointermediate,along with O addition and the oxidative decarboxylation of αKG, directedawayfromthereactionsiteC8aandO3a,renderingitinaccessible for subsequent chemical transformations. In our previous study on the endoperoxidation reaction catalysed by FtmOx1, we observedthepresenceofacrucialresidueY224,positionedbetweenthesubstrateandthemetallo-centre5.Furthermore,theFeIV=OspeciesoxidizestheactivesiteY224toformY224•,whichsubsequentlyactsasthecatalyticspeciesresponsiblefortheendoperoxidationreaction5.Additionally, in the investigation of the epoxidation mechanism of AsqJ, the αKG rotation and the potential oxo-hydroxo/water tautomerismoftheFeIV=Ointermediatehavebeenproposed10,49.ThekeystepseffectivelyrepositionedtheFeIV=Ospeciesnearthereactionsiteonthesubstrate,anddirectlyabstractedahydrogenatomtoinitiatethesubsequentreaction.However,inthecrystalstructureofOkaE,we did not observe residues such as tyrosine, cysteine, tryptophan, or phenylalanine located between the metallo-centre and substrate, which can serve as the catalytic species in the reaction cycle. We hypothesizedthattherotationofαKGorFeIV=OmayalsobeaperequisitestepinthecatalyticcycleofOkaE.

TofurthergaininsightsintothedetailedreactionmechanismofOkaE,aseriesofquantummechanicalcalculationswasperformed.Thegeneration ofFeIV=Ospecies and itsαKG-assisted oxygenactivation processhavebeenextensivelystudiedinothermembersoftheαKG-NHFeenzymes1,andwerevisitedthisprocessusingQM/MMcalculations (Supplementary Tables 15 and 16; Supplementary dataset 5). Figure4ashowstheQM/MM-calculatedenergyprofile.Thereactionbeganwiththedistaloxygen(O )ofsuperoxideattackingtheC2ofαKG(RC1),leadingtotheformationofthedecarboxylationintermediateInt1a.Thecalculatedenergybarrierforthisstepwas8.5kcal/mol. The subsequent O–O bond cleavage (Int1a→TS1b) produced succinate and the equatorial FeIV=O species (Int1b). This step encountered an energy barrier of 6.4kcal/mol, indicating that the transformationfromRC1toInt1bwasrelativelyeasy.Moreover,duringtheprocess,thereleaseofCO coincidedwiththecollapseoftheinitiallyuprightFe–O bond,settingthestageforthesubsequentO–OcleavageandtheformationoftheequatorialFeIV=Ospecies(Int1b).ThisprocesswasconsistentwithpreviousfindingsforanotherαKG-NHFeenzyme,WelO522wheretheconformationofFeIV=Ospeciesischanged.

As depicted in Fig. 4b, the distance between O and H8a decreased from 4.83Å (RC1) to 2.89Å (Int1b), and the distance between O and H3a(hydrogenatomon3a-OH)bothdecreasedfrom5.06Å (RC1) to 2.62Å (Int1b). Moreover, the Fe–O –H8a and Fe–O –H3a angles increased to optimal positions for the reaction, reaching147°and169°,respectively.ThesechangesprovidefurtherinsightintotheroleofFeIV=OinthecatalyticcycleofOkaE,allowingit to catalyse a variety of oxidation reactions. The QM/MM-calculated energy profile (Fig. 4a) indicated that the intermediate Int1b can proceedthrougheithertheH8a–HATor3a-OH–HATpathway,bothencounteringcomparableenergybarriersof21.2kcal/mol (Int1b→TSH8a) and 22.4kcal/mol (Int1b→TSH3a), respectively, producing thecorrespondingintermediatesTSH8aandTSH3a(SupplementaryFig.21).TheseresultssuggestthatthetwoHATpathwaysarekineticallycompetitiveandcouldleadtodistinctproductoutcomes.

Met−πinteractionsgovernreactionpathwayselection

Structural analysis of the OkaE•CoII•αKG•okaramine A (1) complex revealed an importantMet–π interaction network(M64-M71-W79-M122) thatstabilizesthesubstratewithinthebindingpocketofOkaE.Thisinteractionnetworkmaintainsprecisespatialorientationof1,enablingFeIV=OspeciestoselectivelyabstracthydrogenfromeitherC8aorO3apositionsandgovernaccesstodivergentoxidationpathways.Toshedlight on the functional role of this Met–π interaction network, we performedalaninescanningmutagenesisonM64,M71,W79,andM122(Fig.5a,b;SupplementaryTables17and18;sourcedataareprovidedasaSourceDatafile).Notably,bothH8a–HATand3a-OH–HATpathways remained active in OkaE -catalysed reactions; however the productprofilewassimplified,with6becomingpredominantproduct(Fig.5a,traceii).TheOkaE mutantfavoredtheH8a–HATpathway,producing6asthemajorproductand3and5astheminorproducts(Fig.5a,traceiii).Incontrast,theOkaE mutantretainedaproductdistributionalmostidenticaltoOkaE (Fig.5a,tracesiandiv),exceptfora~10%increaseinformatof3.TheOkaE mutationshiftedthe reactionpreferencetowardthe3a-OH–HATpathway,asevidencedbya~10% increase in 7 formation (Fig. 5a, trace v). Meanwhile, the H8a–HAT pathway exhibited a comparable increase (~10%) in production of 6 relative to OkaE (Fig. 5a, trace i). Collectively, these biochemical assays revealed pronounced alterations in product distribution, underscoring the essential role of these residues in modulatingcatalyticspecificity.

To elucidate the role of Met–π network residues in governing product selectivity during OkaE-catalysis, we determined the high-resolution crystal structures of the complexes OkaE •CoII•αKG•1 (2.44Å,PDBID:9UAD),OkaE •CoII•αKG•1(2.55Å,PDBID:9UAG),and OkaE •CoII•αKG•1 (2.50Å, PDB ID: 9UAH) (Supplementary dataset 4). Structural analyses confirmed a conserved αKG binding mode across all mutants, despite active-site perturbations (SupplementaryFig.22b).Localizedconformationalchangeswereobserved,includingsubtlechangesinsubstrateorientationandthedistancesbetweenthemetallo-centreandthereactionsitesC8aandO3aof1(Fig.5c–g).Theloopregion(residues69YQMPVP74)showed0.6–1.6ÅdisplacementscomparedtotheOkaE (SupplementaryFig.22c;andSupplementaryTables19–21),likelycontributingtoreducedcatalyticefficiencyoftheenzyme(Fig.5a,b).

In the OkaE mutant, loss of the methionine side chain causedtheindoletail(A-andB-rings)of1tobendtowardM71byabout8.5°(Fig.5c).IntheOkaE mutant,thedisruptionoftheinteractionbetweenthesidechainofM71andtheindoletailof1made the loop region (residues 69YQMPVP74) shift outward by 0.6Å without major changes in substrate alignment or pocket geometry(Fig.5c;andSupplementaryFig.22c).Nevertheless,thedistance between the metallo-centre and C8a was shortened by 0.1Å and the distance between the metallo-centre and O3a waslengthened by 0.2Å compared to OkaE (Fig. 5d, f), favoring hydrogen abstraction at C8a and promoting the formation of product 6 (Fig. 5a, b). Despite identical 5.9Å distances between the metallo-centre and O3a observed in the crystal structures of the OkaE and OkaE mutants,only OkaE was capable of producing 7, whereas OkaE was not. To clarify this discrepancy, we performed MD simulations for both mutants. The simulationsrevealed thatinOkaE ,theFe=O···H3a(hydrogenatom on 3a-OH) and Fe=O···H8a distances were 2.0±0.6Å and 2.7±0.4Å, respectively, whereas in OkaE these distances were 4.8±0.6Å and 2.6±0.3Å (Supplementary Fig. 23). These results explain the biochemical observations, indicating that OkaE maintains reactive proximity to both 3a-OH and H8a, thereby allowing access to dual abstraction pathways and formation of products 3–7. In contrast, the extended Fe=O···H3a distance in OkaE restricted 3a-OH–HAT pathway, leading exclusivelytothe formationofproducts3–6.

In the OkaE mutant, enzymatic reaction preference was restrictedtotheC8apositionduetoextensiverearrangementoftheMet–πnetwork.RemovaloftheindolylgroupofW79disruptedtheinteractionsbetweenM64andindoletailof1,causingan~120°rotationoftheM64sidechainalongitsCα –CβbondandtheformationofanewstackinginteractionwithA79andtheindoletailof1(Fig.5c).AnewhydrogenbondwasformedbetweensulfuratomofM64andthehydroxylgroupatC3a(Fig.5c).Thesechangesinducedan~3.4°bendoftheindoletailof1towardtheC−Gringsplaneandan~11.4°rotationoftheD−Eringstowardmetallo-centre(Fig.5d,g).Takentogether,these conformational shifts increased the distance between the metallo-centreandO3afrom5.7Åto6.4Å,whilethedistancebetweenthe metallo-centre and the C8a remained unchanged compared to OkaE ,preventingO3ahydrogenabstractionandenforcingC8aasthesolereactive site (Fig. 5d, g). As a result, the OkaE mutant redirected the enzymatic reaction preference from complex oxidations to a single C–N bond cleavagereaction.

Isotope-labellingexperimentstodeterminetheoxygensource

WhileourstructuralandmutagenesisanalysesdelineatedhowMet–πnetworkinOkaEspatiallyregulatesreactionsiteaccessibility,theoriginofoxygenatomsinitsmultifunctionalproductsremainedunresolved.Toaddressthis,isotopiclabellingexperimentswereconductedusing18O andH 18O(Fig.6;andSupplementaryFig.25;SupplementaryTable22;source data are provided asa SourceData file). Five biochemical reactions with different isotopic labelling oxygen sources were performed: (i) 18O-enriched 18O gas (98 atom % 18O); (ii) 18O-enrichedH 18O(98atom%18O);(iii)18O-enrichedH 18O(98atom%18O);(iv)50%18O-enriched18O gas(50atom%18O);(v)controlswithnaturalabundanceO andH O.TheresultsshowedthatbothO andH Ocontributedasoxygensourcesforcompounds3–6,withvaryingincorporationratios:~7:3,7:3,7:3,and3:7,respectively(Fig.6a−d).Incontrast,oxygenincompound7 predominantlyoriginatedfromO (Fig. 6e). For compounds 3−6, although the catalytic cycle of αKG-NHFeenzymeswasactivatedbyO ,theoxygenatominFeIII−OHandFeIV=Ospeciescanbeexchangedwithsolventwater50,51duringcatalysis. Alternatively, oxygen incorporation may occur via transient cationic intermediates49,51involvingwatermolecules.Forcompound7,biochemicalassaysrevealedthatitsyieldremainedunchangedwhen themolarratioofαKG:1wasincreasedfrom1:1to4:1(Fig.2b),suggestingthat7arisesfromasingleαKG-dependentcatalyticreactionratherthanfrommultipleroundsofoxidativecatalysis.Intraceivof7(Fig.6e),the+2peakwasnearlyundetectable,whereasthe+0and+4peaksappearedinalmostequalproportions(m/z551(+0):553(+2):555(+4)=46.7%:1.3%:52.0%),furthersupportingtheinvolvementofa second dioxygenmolecule withina single reactioncycle. Suchdual dioxygen engagement is exceptionally rare among αKG-NHFe enzymes5,51,highlightinguniquecatalyticplasticityofOkaE.TheconformationalflexibilityofMet–πnetworklikelyfacilitatesthisdualengagement,modulatingsolventaccessibilityforwaterexchangewhilealsoaccommodatingthebindingofaseconddioxygenmolecule.

ProposedcatalyticmechanismofOkaE

Based on structure-function analyses, QM/MM calculations, and isotope-labellingexperiments,weproposedacatalyticmechanismfortheconsecutiveoxidationreactionscatalysedbyOkaE,asdepictedinFig.7andSupplementaryFigs.26–30.TherapidadditionofO tothemetallo-centre generates the FeIV=O species. Prior to hydrogen abstraction,theFeIV=Ospeciesundergoesaconformationalrotationfromanaxialtoequatorialposition,aligningitselftowardtworeactionsitesC8aandO3aonsubstrate1.

In the proposed path a (Fig. 7a), the FeIV=O species directly abstracts a hydrogen atom from the C8a position of 1 (red arrow), producing a radical intermediate IM1. The radical subsequently transferstoC12(patha 1),facilitatingtheformationofanazetidineringbetweenC8aandC11.TheradicalatC12onintermediateIMa canbequenchedeitherbyareducingagent,suchasascorbate,yielding2,orviahydroxylreboundtoformthehydroxylatedproduct3.Once3isgenerated,anewreactioncycleinvolvinghydrogenabstractionatC12induces hydroxyl rebound (blue arrow). Alternatively, isotopic labellingexperimentssuggestthepossibilityofdirectelectrontransfertoFeIII−OH (green arrow), generating a carbocation at C12 of intermediateIMa .Thiscarbocationisthenhydrolyzedtoformthealdehyde hydrate IMa , which spontaneously dehydrates to yield the aldehyde IM2. Subsequent hydrogen abstractionat C12 of IM2, followedbyhydroxylationthrougheitherrebound(bluearrow)orcationmechanism (green arrow),completestheformation ofthe carboxyl compound neuokaramine I (4). Alternatively, IMa and IM2 readily undergo acid-catalysed intramolecular hemiacetalization, forming neuokaramineII(5)inanon-enzymaticprocess(magentaarrow).

Isotopiclabellingexperimentsfurtherindicatedtheinvolvementofwatermoleculesintheoxidationcycleleadingtotheformationof6.Two concurrent reaction pathways for C−N bond cleavage are proposedtogenerateneuokaramineIII(6)(Fig.7b).Inpatha 2 -i(brown arrow), hydroxyl rebound generates an unstable hemiacetal amine intermediateIMc 2 ,whichisthenhydrolysedtoform6.Inpatha 2 -ii(pinkarrow),theradicalatC8aofIM1isfurtheroxidizedbyFeIII−OH,forming an imide intermediate IMb via cation rearrangement and proton transfer. IMb subsequently undergoes spontaneous hydrolysis,cleavingtheC−Nbondandyieldingcompound6.

The formation of neuokaramine IV (7) (Fig. 7c) begins with hydrogenabstractionattheO3apositionof1(purplearrow,pathb), formingFeIII−OHandasubstrateradicalIMd.TheradicalthentransferstoC8a,generatingintermediateIMd ,whichleadstotheformationofacarbonylgroupatC3aandcleavageoftheC3a−C8abond.Before hydroxyl rebound at C8a with the FeIII−OH species, the C8a−radicalreactswithO toformaperoxylradicalIMd .Theresultingperoxyl radical attacks C11 to form an endoperoxide-containing intermediate IMd . Subsequently, FeIII−OH abstracts the hydrogen atom at C8a, leading to cleavage of the endoperoxide bridge and formationofanamido-carbonylgroupatC8a,alongsideanepoxide ring connecting C11 and C12, thereby yielding 7. This mechanistic blueprint resolves how a single iron centre orchestrates azetidine synthesis,hydroxylation,C–Ncleavage,andepoxidation,expandingthecatalyticrepertoireofnon-haemironenzymes.

Methods
Materials
NMRspectrawereacquiredonaBrukerAvanceDRX600spectrometer andBrukerAvanceHDX500-MHzspectrometerequippedwithaTCI cryoprobe (600MHz for 1H-NMR and 150MHz for 13C-NMR). NMR spectrawererecordedinpyridine-d (99.5atom%enriched,AICON), andacetone-d (99.8atom%enriched,Kanto).1H-NMRchemicalshifts (δ)werereferencedtoresidualpyridine-d (δ 8.74,7.58,7.22ppm), acetone-d (δ 2.05 ppm) and methanol-d (δ 3.31 ppm). 13C-NMR chemicalshiftswerereferencedtopyridine-d (δ 150.35,135.91,123.87 ppm),acetone-d (δ 206.26,29.84ppm)and methanol-d (δ 49.3 ppm). LC-HR-MS(ESI) measurements were obtained on Thermo Q Exactive Orbitrap mass spectrometer coupled with a Shimadzu LC-30AD UPLC equipped with a Waters ACQUITY BEH C18 column (2.1×100mm,1.7μmparticles).TheLC-MSanalysiswasrunusingan Agilent 1100 Series separation module with an Agilent G1956B MS detector.RP-HPLCwasperformedonanAgilent1260Seriesseparation module with a diode array detector. HPLC analysis was carried out using a C18 column (ACE C18-PFP, 4.6×250mm, 5μm, 1mL/min). SemipreparativeHPLCwascarriedoutusingaC18column(Agilent-ZORBAX-SB, 9.4×250mm, 5μm, 3mL/min). Protein-substrate complexeswerefirstscreenedforcrystallizationusingaCrystalGryphon autodispenser(ArtRobbinsInstruments,LLC),andoptimizedcrystals were sent to the Shanghai Synchrotron Radiation Facility (SSRF) to collectX-raydiffractiondataunderbeamlineBL02U1andBL19U1with a Pilatus3 6M image plate detector. Biological reagents, chemicals, andmediawerepurchasedfromstandardcommercialsourcesunless statedotherwise.

SequenceandstructuralsimilarityanalysisofαKG-NHFe enzymes
Atotalof135previouslyreportedαKG-NHFeenzymesequencesthat catalyse small molecules were collected (up to 25 April 2025) from published sources and public databases,including GenBank [http://www.ncbi.nlm.nih.gov/(accessedonApril25,2025)]and UniProtKB [http://www.uniprot.org/(accessedonApril25,2025)].Theseenzymes were categorized into 99 monofunctional and 36 multifunctional enzymesbasedontheircatalyticreactions.Sequencesimilaritynetworks(SSNs)wereconstructedusingpairwiseBLASTalignments,with FtmOx1asthequery.SSNswerethenvisualizedusingCytoscape3.6.0 atvariousE-valuethresholds.Additionally,thecrystalstructuresfrom the PDB database and models built by AlphaFold 3 for 36 multifunctional αKG-NHFe enzymes, along with 28 monofunctional enzymesclusteredwiththem,wereobtained.Astructuralsimilaritymatrix(SSM)wasconstructedbyperforminganall-against-allstructurecomparisonusingtheDaliserver63.

FermentationofP.simplicissimumATCC90288andpurification ofokaramines
P.simplicissimumATCC90288waspurchasedfromATCC,USA.This fungalstrainwasculturedonapotatodextroseagar(PDA)mediumat 28°Cfor3days.Theagarplugswereplacedinto250mLErlenmeyer flasks,eachcontaining100mLofpotatodextrosebroth(PDB).Flask cultures were incubated at28°C on a rotary shaker at220rpm for 3daysastheseedculture.320aseptic-bags,eachcontaining50gof freshokara,wereindividuallyinoculatedwith5mLofseedcultureand wereincubatedat28°Cfor14days.Then,theentireculturemasses wereextractedwith10Lacetonefourtimes,andthetotalresiduewas obtained by reduced pressure evaporation. The remaining aqueous phase was further extracted four times by EtOAc to yield a crude extract(164.0g).

The crude extracts were subjected to column chromatography (CC)oversilicagelelutedwithagradientofCH Cl -MeOH(100:0,50:1, 20:1,10:1,and0:100)togivefivefractions(A–E).FractionB(24.0g), dissolvedinCH Cl andMeOH(1:1),wasfractionatedbyMPLCCCover RP-18elutedwithMeOH–H O(from30:70to100:0,v/v)togiveseven sub-fractions(B1–B7).BasedoncharacteristicUVabsorptionandHR-ESI-MSspectra,theanalysisofthesefractionsrevealedtheexistenceof thetargetcompoundsokaramineA(1)and12-deshydroxyl-okaramine E(2)infractionB5andB6,respectively.Then,fractionB5wasfurther purified by semi-preparative RP-HPLC using a C18 column (Agilent-ZORBAX-SB,9.4×250mm,5μm)withaflowrateof3.0mL/minandan isocratic elution with 68% MeOH–H O to obtain 1 (112.3mg, t =37.2min).FractionB6wasalsopurifiedusingsemi-preparativeRP-HPLCwithanisocraticelutionwith66%MeOH–H O(0.1%formicacid) toafford2(10.2mg,t =24.8min).FractionCwasalsoseparatedby MPLCCCoverRP-18withagradientelutionofMeOH–H O(from30:70 to100:0,v/v),andthenwaspurifiedbysemi-preparativeRP-HPLCwith an isocratic elution 55% MeOH–H O (0.1% formic acid) to obtain okaramineE(3)(5.3mg,t =27.5min).

Recombinantplasmidconstruction
The okaE gene was inserted into the pASK-IBA3-plus vector, which placeditunderthecontrolofthetet-promoterandallowedforthe productionofC-terminallystrep-taggedOkaE.Theprimersusedfor theconstructionofplasmidsforsite-directedmutagenesisstudiesare listed inSupplementaryTable 17. The plasmid of the WT OkaEwas usedasthetemplateforPCR-basedsite-directedmutagenesis.After digestion of the PCR products with DpnI to remove the wild-type plasmid,thereactionmixturewasusedforBluntingKinationLigation andthentransformedintoE.coliBL21(DE3).Followingplating,individual colonies were selected, plasmids were extracted, and their identitieswereconfirmedbyDNAsequencing.

OverexpressionandpurificationoftheOkaEprotein
The recombinantplasmidofOkaEanditsmutantsweretransformedinto E.coliBL21(DE3)cells(InvitrogenInc.,USA)forproteinoverexpression. A single colony was picked into Luria-Bertani (LB) medium supplemented with 100μg/mL ampicillin and incubated at 37°C overnight. Culture(10mL)and39.2mgFe(NH )(SO )•6HOwereplacedin1LLB medium supplemented with 100μg/mL ampicillin. The mixture was incubatedat37°CuntilOD reach0.6~0.8andthencooledto20°C. TheoverexpressionoftheOkaEproteinwasinducedbyaddinganhy-drotetracyclinetoafinalconcentrationof500μg/Lat20°C,andincubatedforanadditional16hbeforeharvesting.

Purification was performed at 4°C. In a typical anaerobic purification,wetcellpastewasresuspendedinanaerobicbuffer(100mM Tris-HCl and 50mM NaCl, pH 7.5) in an anaerobic Coy chamber. Lysozyme (0.5mg/mL final concentration), EDTA (5mM final concentration)andascorbate(5mMfinalconcentration)werethenadded into the cell suspension, and the mixture was incubated on ice for 30minwithgentleagitation.Thecellsweredisruptedbysonication (43cyclesof3sbursts)usingaVCX130ultrasonicprocessor(Sonics& Materials,Inc.,Newtown,USA).Thesupernatantandthecelldebris wereanaerobicallyseparatedbycentrifugationat4°Cfor40minat 39,000 × g. The resulting supernatant was mixed with Strep-Tactin resinandincubatedonicefor30min.Afterthecelllysatewasdrained bygravity,thecolumnwaswashedwithwashingbuffer(100mMTris-HCl,50mMNaCl,pH7.5)untiltheOD readoutofeluatewasless than0.05.TheOkaEproteinwaselutedwithelutionbuffer(2.5mM desthiobiotinin100mMTris-HCland50mMNaCl,pH7.5).Theeluted proteinwasconcentrated,flashfrozenwithliquidnitrogen,andstored at−80°C.PurifiedproteinswereexaminedbySDS-PAGE.

EnzymaticreactionofOkaEusingcompounds1−3,5,and6as substrate
Theenzymaticreactionswereperformedunderanaerobicconditions inaCoychamber.Forsingle-turnoverreactions,theanaerobicreactionmixture(100μL,100mMTris-HCland50mMNaCl,pH7.5)contained70μMokaramineA(1),70μMOkaE,140μMFeII,70μMαKG, and210μMascorbate(orw/oascorbate).Formultiple-turnoverreactions,theanaerobicreactionmixturecontained210μMokaramineA (1),70μMOkaE,420μMFeII,210μMαKG,210μMascorbate(orw/o ascorbate), and DMSO (3.6% final concentration, or w/o DMSO). A negative control reaction was prepared using boiled protein. The anaerobicreactionmixturesweretransferredoutoftheCoychamber, andthereactionswereinitiatedbyadding300μLoxygen-saturated buffer (1.25mM of oxygen), followed by incubation at room temperaturefor15min.Theenzymaticreactionswerequenchedbyadding 400μLethylacetate(EA),followedbycentrifugationat13,000×gfor 1min to remove precipitated protein. The EA layer was carefully separated for further analysis,with the extraction performed twice. ThecombinedEAlayerswereconcentratedbyrotatoryevaporationin vacuo, and the residues were re-dissolved in 60μL of acetonitrile (ACN)forLC-MSanalysis.

LC–MSanalyseswerecarriedoutonanACEC18-PFPcolumn(4.6× 250mm,5µm)usingH₂O(solventA)andACN(solventB)ataflowrate of1.0mL/min.Thegradientwasprogrammedasfollows:0.0–15.0min, B increased from 36.0% to 36.2%; 15.0–27.0min, B increased from 42.0% to 49.0%; 27.0–32.0min, B increased from 49.0% to 55.0%; 32.0–34.0min,Bheldat55.0%;and34.0–39.0min,Brampedto99.0% forcolumnwashing.Thereactionsetupproceduresof12-deshydroxyl-okaramineE(2),okaramineE(3),neuokaramineII(5)andneuokaramineII(6),andLC-MSanalysesofenzymaticproductswerethesame astothoseofokaramineA(1).

PurificationofneuokaramineⅠ−Ⅳ(4−7)fromenzymaticreactionofOkaE
Toobtainenoughnovelproductsforstructuraldetermination,multiplelarge-scaleenzymaticreactionswereperformed.Theanaerobicreactionmixture(4mL,in100mMTris-HCland50mMNaCl,pH7.5)contained210μMokaramineA(1),70μMOkaE,2100μMFeII,700μMαKG, 210μMascorbateandDMSO(3.6%finalconcentration).Themixturewassealedindouble-neckedflask,andthereactionwasinitiatedbyadding10mLofoxygen-saturatedbuffer(1.25mMofoxygen). The reaction was incubated at room temperature for 30min. The enzymaticreactionwasquenchedbyadding14mLEA,theprecipitated proteinwasremovedbycentrifugationat13,000×gfor10min,and theEAlayerwascarefullyseparated.Theextractionwasrepeatedusing an additional 14mL of EA. The combined EA extracts were concentratedbyrotatoryevaporation,andtheresidueswerere-dissolved ina1:1mixtureofCH Cl andACN.

Subsequently,theresidueswerefractionatedbypreparativeRP-HPLCusingaC18column(Agilent-ZORBAX-SB,9.4×250mm,5μm)withaflowrateof3.0mL/minwithagradientelution(0−32min,ACN was increased from 34% to 53.2% in H O and then the column was eluted for 5min with 99% ACN in H O) to obtain four sub-fractions(F1–F4).

FractionF1waspurifiedbysemi-preparativeRP-HPLCwithaC18 column(ACEC18-PFP,4.6×250mm,5μm)withaflowrateof1.0mL/min withagradientelution(During0−20min,ACNwasincreasedfrom25% to35%in0.1%HCOOH-containingHO,andthenduring20−30min,ACN was increased from 35% to 40% in 0.1% HCOOH-containing HO) to obtainneuokaramineI(4,5.6mg,t =29.2min).

Fraction F3 was purified by semi-preparative RP-HPLC on a C18 column(ACEC18-PFP,4.6×250mm,5μm)withaflowrateof1.0mL/min withagradientelution(During0−27min,ACNwas33%inHO,andthen during27−40min,ACNwasincreasedfrom36%to53%inHO)toobtain neuokaramineII(5,2.8mg,t =37.3min).

Fraction F4 was purified by semi-preparative RP-HPLC on a C18 column(ACEC18-PFP,4.6×250mm,5μm)withaflowrateof1.0mL/min withagradientelution(During0−28min,MeOHwas62%inHO,and thenduring28−40min,MeOHwasincreasedfrom62%to65%inHO)toobtain neuokaramineIII(6,3.2mg,t =39.3min),andneuokaramineIV (7,6.3mg,t =25.8min).

ECDcalculations
Theelectroniccirculardichroism(ECD)calculationswereconducted usingtheGaussian09programpackage64.Conformationalsearches wereperformedusingSYBYL-X2.1basedontheMMFF94molecular mechanicsforcefield,withanenergywindowof10kcal/mol65,66.All low-energyconformersweresubsequentlyoptimizedandsubjectedto frequency calculations at the B3LYP/6-31G level of theory using Gaussian 09. Time-dependent density functional theory (TD-DFT) calculationsforECDspectrawerethencarriedoutattheωB97XD/6-311+G(d,p) level, incorporating the IEFPCM solvation model with methanol as solvent. Theoretical ECD spectra were generated in SpecDisV1.71usingaGaussianfunction67.ThecalculatedECDcurves wereobtainedbyBoltzmannweightingofindividualconformers accordingtotheirGibbsfreeenergies.

NMRcalculations
Optimizedconformerswithinanenergywindowof5kcal/mol,withno imaginaryfrequencies,wereselectedforgauge-independentatomic orbital(GIAO) calculations of13C-NMR chemicalshifts using density functional theory (DFT) atthemPW1PW91/6-311+G(d,p)level,incorporating the PCM solvation model. The resulting 13C-NMR shielding constantsofindividualconformerswereBoltzmannweightedbased ontheirrelativeGibbsfreeenergies.Tetramethylsilane(TMS)was calculatedatthesametheoreticallevelandusedasthechemicalshift reference.Agreementbetweenexperimentalandcalculated13CNMR datawasassessedusingtheimprovedprobabilityDP4+method68.

18O andH 18OlabelingexperimentusingokaramineA(1)as substrate
These enzymatic reactions were performed in an anaerobic Coy chamber.WaterexchangewithH 18O(98atom%18O)wasperformed throughconcentrationandre-dilutionoftheproteinsolutionwiththe Trisbufferpreparedinlabeledwatertoreachlabelinglevelsof~80%. FeIIandαKGhadtobedissolvedintheH 18Obeforeadditiontothe anaerobicreactionsystem.Theanaerobicreactionmixture(400μL,in H O)containing70μMokaramineA(1),70μMOkaE,140μMFeII,and 70μMαKG.18O gas(98atom%18O)or50%18O gas(50atom%18O) was injected into the reaction mixture. The reaction mixtures were incubatedfor10minatroomtemperature,andquenchedbyadding 400μLEA,theprecipitatedproteinwasremovedbycentrifugationat 13,000×gfor2min.TheEAlayerwascarefullyseparatedforfurther analysis,withtheextractionperformedtwice.ThecombinedEAlayers wereconcentratedbyrotatoryevaporationinvacuo.Afterward,the residuewasre-dissolvedin60μLofacetonitrileandsubjectedtoLC-HR-MS(ESI) analysis on a C18 column (ACE C18-PFP, 4.6×250mm, 5μm)withaflowrateof1.0mL/min.TheflowrateintotheHR-MS(ESI) module was adjusted to ~0.35min/mL by the post-column shunt method(During0−32min,ACNwasincreasedfrom34%to53.2%in 0.1%HCOOH-containingH Oandthenthecolumnwaselutedfor5min with 99% ACN in 0.1% HCOOH-containing H O). The data are summarizedinSupplementaryTable22.Allreactionswereperformedin triplicate.Dataarepresentedasmeanvalues,andtheerrorbarsindicate standarddeviations(SD).

Complexformation,crystallization,anddatacollection
Apo OkaE incubated with metal cobalt in a molar ratio of 1:3 was concentratedto10mg/mLforinitialscreeningusingthesitting-drop vapor-diffusionmethod.Rod-likecrystalswereobservedafter2days fromawellcontaining0.1Mimidazole,0.1MMESmonohydrate,pH= 6.5,20%PEG500MME,10%PEG20,000,0.02MD-glucose,0.02MD-mannose,0.02MD-galactose,0.02ML-fucose,0.02MD-xylose,and 0.02 M N-acetyl-D-glucosamine. Crystals were optimized by varying thepHaswellasprecipitantconcentrationtoyieldafinallargerod-shapedcrystalafter7daysatroomtemperature.

Toobtaintheprotein•αKG•okaramineA(1)complex,crystalsof wild-type OkaE•CoII and its mutants (M64A, M71A and W79A) were transferredtoanewsoakingbuffercontainingthesamecrystallization buffersupplementedwithoptimizedconcentrationsofokaramineA (10mM for wild-type OkaE, OkaE and OkaE , 20mM for OkaE ),5mMαKGand10%DMSO,andincubatedat20°Cforupto 72h.Thesoakedcrystalswerecryoprotectedbyaddingglycerolinto crystallizationsolutiontoreachfinalconcentrationsof5%(v/v).The complex crystals were then flash cooled in liquid nitrogen for data collection. Diffraction datasets were collected on the beamlines BL18U1,BL19U1,andBL02U1attheShanghaiSynchrotronRadiation Facility(SSRF)at100K.Crystallographicdatasetswereintegratedand scaled using XDS69. The crystal parameters and the data collection statisticsaresummarizedinSupplementaryTables14and19–21.

Structuredeterminationandanalysis
The crystals of OkaE•CoII, OkaE•CoII•αKG, as well as OkaE•CoII•αKG•okaramineA(1)complexbelongstothespacegroupof P222 where two dimers of OkaE formed an asymmetric unit. The structuresofOkaE•CoII,OkaE•CoII•αKGandOkaE•CoII•αKG•okaramine A(1)complexweresolvedbymolecularreplacementusingPhasera implemented in the Phenix/CCP4 suite of programs70, with the apo modelgeneratedusingAlphaFold271.Theinitialsolutionwasfurther improvedusingiterativecyclesofmanualmodelbuildinginCOOT72 followed by refinement using phenix.refine73,74. After ligands were placed, potential sites of solvent molecules were identified by the automatic water-picking algorithm in COOT and phenix.refine. The positions of these automatically picked waters molecules were checked manually during model building. Figures were generated using PyMOL (Schrödinger, LLC) and UCSF Chimera75, and surface electrostaticswerecalculatedwithAPBS76.

SystemsetupandMDsimulations
ThecrystalstructureofOkaE(PDBID:9M2L)wasusedasthestarting model, and only chain C was retained for subsequent simulations. ProtonationstatesoftheconservedHx(D/E)x Htriad(H134,D136,and H210)weredeterminedbasedonpK calculationsusingH++77,complementedbymanualinspectionoflocalhydrogen-bondingnetworks. HistidineresiduesH137,H134,H210andH282wereprotonatedattheδ position,whereasH19,H42,H92,H101,H111,H140,H178,H205,H215 andH222wereprotonatedattheεposition.Allglutamateandaspartate residues were assigned in their deprotonated forms. Missing hydrogenatomswereaddedusingtheLEAPmoduleimplementedin Amber1878.ThegeneralAMBERforcefield(GAFF79)wasappliedtothe substratesokaramineA(1),whiletheαKGcoordinatedFeIII-superoxo complex(RC1)wasparametrizedusingtheMCPBapproach80.FiveNa+ ions were added to neutralize the total charge of the system. The resultingneutralsystemwassolvatedinacubeofTIP3Pwatermoleculeswithabufferdistanceof15Å81.Proteinresiduesweredescribed usingtheAmberff14SBforcefield82.

After setup, energy minimization was first carried out using a combinationofsteepestdescentandconjugategradientalgorithms. The system was then gradually heated from 0 to 300K under a canonical ensemble for 0.2ns, applying a weak restraint of 15kcal/ (mol∙Å2). Subsequently, a 1ns density equilibration was performed undertheNPTensembleat300Kand1.0atmtostabilizethesystem density.Allrestraintsontheproteinwerethenremoved,followedby anadditional4nsofNPTequilibrationusingaLangevinthermostat and a Berendsen barostat. A 50ns production MD simulation was finallyconducted.Allcovalentbondsinvolvinghydrogenatomswere constrained using the SHAKE algorithm83. Representative structures extracted from the equilibrated MD trajectory were used as initial modelsforsubsequentQM/MMcalculations.

QM/MMmethodology
QM/MMcalculationswerecarriedoutusingtheChemShellpackage84, inwhichTurbomole85wasemployedasthequantummechanical(QM) engine and DL_POLY86 as the molecular mechanics (MM) engine. Residues located within 12.0Å of the Fe atom were included in the active region. An electronic embedding87 scheme was adopted to accountforpolarizationeffectsoftheenzymeenvironmentontheQM region.TheQM/MMinterfacewastreatedusinghydrogenlinkatoms88 in combinationwithacharge shiftmodel89.FortheQMregion,the B3LYP90 functional was applied, which has been widely used for mechanisticstudiesofαKG-NHFeenzymes.Geometryoptimizations and frequency calculations were performed using the all-electron Def2-SVP basis set (labeled as B1). Single-point energy refinements were subsequently performed using the larger Def2-TZVP basis set (labeled as B2). Final reported energiesconsistedofelectronicenergies evaluated at the B3LYP/B2 level together with Grimme’s D3BJ dispersioncorrection91.Consistwithpreviouscomputationalstudies ofαKG-NHFeenzymes,suchasSyrB292,WelO522,ScoE93,andEFE17,the ground state is the quintet state. Therefore, the RC1 species was modeled in the quintet state in this study. Geometry optimizations wereconductedusingtheDL-FINDoptimizer94,andallminimawere fullyoptimizedwithoutsymmetryconstraints.Transitionstateswere initially identified as maxima along the reaction coordinates, with potentialenergysurfacescansperformedusingastepsizeof0.02Åin thevicinityofthetransitionregions.Alltransitionstates(TSs)were locatedbyrelaxedpotentialenergysurface(PES)scansfollowedbyfull TSoptimizationsusingtheP-RFOalgorithm.Frequencyanalyseswere finallycarriedouttoconfirmthatalloptimizedTSsandminima exhibited1and0imaginaryfrequencies,respectively.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript reports the structural and mechanistic insights into the α-ketoglutarate (αKG)-dependent non-heme iron\nenzyme OkaE that builds the azetidine ring in okaramine biosynthesis. In vitro reconstitution of OkaE with okaramine A (1)\nproduced okaramine E (3), 12-dehydroxy-okaramine E (2), and four new congeners, neokaramines I–IV (4–7). Crystal\nstructures of OkaE•Co²⁺, OkaE•Co²⁺•αKG, and OkaE•Co²⁺•αKG•okaramine A revealed a homodimeric double-stranded β-\nhelix fold, a distal-type αKG binding mode, and placement of substrate atoms C8a and O3a near the metal center within a\npocket framed by M64, M71, M122, and W79. QM/MM calculations supported the distal αKG geometry and showed substrate\npositioning that shortens Op–H8a and Op–H3a distances (≈4.8→2.9 Å and 5.1→2.6 Å). Site-directed mutagenesis indicated\nthat M71A biases H8a hydrogen-atom transfer (HAT), W79A reduces overall reactivity, and M122A modestly enhances\nformation of 7 via O3a-HAT; structures of M64A, M71A, and W79A complexes corroborate these product profiles. Isotope-\n18O-labeling showed mixed O₂/H₂O incorporation into 3–6, consistent with hydroxyl-rebound and/or cationic pathways,\nwhereas 7 incorporates only O₂, implying rare capture of a second O₂ within a single catalytic cycle. Together, the data\nsupport a mechanism in which H8a-HAT drives azetidine formation followed by sequential\noxidations to give 2–6, while a possible alternate O3a-HAT pathway channels\nperoxidation/epoxidation to yield 7. Overall, the study elucidates the structure-function relationships of OkaE and demonstrates its potential to be reprogrammed to tailor and\ndiversify complex natural products. Specific comments are listed as follows,\n1. Figure 4. In vitro assays showed that M64A converts substrate 1 to 7, whereas M71A did not (Figure 4A,B). The proposed\npathway for 7 formation involves abstraction of the C3a–OH proton. However, the crystal structures place the C3a–OH at the\nsame distance from the metal center in both M64A and M71A (~5.9 Å). How do the authors explain these divergent\noutcomes with the similar C3a–OH–metal distances?\n2. Page 5, Line 117, the authors mentioned “four previously uncharacterized minor products, designated as neuokaramines\nI–IV (4–7),” but on Page 6, Lines 140–141 stated that “the structure of 6 was identical to that of okaramine R.” In reference\n43, the stereochemistry at C-3a of okaramine R was not determined in reference 43, while the present study showed the\nconfiguration of C-3a OH on the structure (Scheme 1). Please provide how to verify the stereochemistry of C-3a OH of\ncompound 6 or if it could be okaramine R reported in reference 43.\n3. For structure elucidation of the enzymatic products, please provide detailed stereochemical descriptions of compounds 4–\n7, especially for protons like H-2, H-12, and H-11. Please explain how the stereochemistry of compounds 4–7 based on\nNOESY or other spectroscopic data.\n4. Is there any evidence to support that IM2 is involved in the conversion of 3 to 4 or 5?\n5. Figure 4, the in vitro experiment of M71A with 1 showed several additional signals compared to those in WT with 1 (e.g.\nretention time around 14 min and 33 min). Did the authors have any idea what the products may be and if they are related to\nokaramines?\n6. For Figure 5, please provide the corresponding mass spectra in SI.\n7. Page 11 to 12, the contents described the structure rearrangement of OkaE•CoII and OkaE•CoII•αKG•okaramine A (1)\ncomplex (for example, the conformational shift of the beta-helix formed by I269-S270 ). It is suggested to add Figures in the\nmain text or SI to show the comparison and structure superimposition figure of OkaE•CoII and OkaE•CoII•αKG•okaramine A\n(1) complex.\n8. Page 30, line 595, “Table S8” should be “Table S7”.\n9. Please check the stereochemistry of C-2 of the structures shown in Figure 1. Some structures did not show the\nstereochemistry properly.\n10. In the Experimental section, please clearly specify which procedures were performed under aerobic versus anaerobic\nconditions.\nSupporting Information:\n1. Figure S11: The current legend for Figure S11 mentioned “Peak 1” (void) and “Peak 2” (OkaE), but these peaks are not\nlabeled or indicated in the figure. Please revise the legend to match the actual presentation of the chromatogram.", "reviewer_2": "The manuscript describes the multifunctionality of OkaE, an αKG-dependent non-haem iron enzyme, in azetidine formation\nand subsequent scaffold diversification. While the authors report in vitro assays, crystallography, and QM/MM simulations,\nthe study lacks novelty since the catalytic versatility of this enzyme family is well documented, and the presented findings do\nnot substantially advance mechanistic understanding. Furthermore, the work is not thorough: the mechanistic evidence is\ninsufficiently supported, the structural interpretations are speculative. Overall, the manuscript does not reach the standard of\noriginality or rigor required for publication.\nComments:\nLine 120: Regarding the observed differenced from Lin et al. results, differences in reaction conditions should be clearly\ndescribed in the main text.\nLine 127: I believe it is unlikely that only one eq. of aKG is needed for 4, 5, 7.\nLine 152: The biochemical evidence should be clearly presented.\nFig4a: The authors assume that 3,4,5,6 are generated through H8b-HAT, and 7 is formed via OH3a-HAT. However, no\nexperimental evidence is provided to correlate the site of HAT with the product structures.\nFig 6a: Formation of a primary cation Ima3 should be less likely.\nFig 6c: The mechanism shown in 6c involving second O2 is not fully supported by the results. What would happen if the\nauthors use a mixture of 16O2 and 18O2? Product with 16O/18O should be formed if the hypothesis is correct.\n1. The QM/MM calculations mainly address the formation of the Fe(IV)=O species; additional simulations on the later stages\nof the reaction would provide deeper mechanistic understanding.\n2. For compound 7, determining the oxygen stoichiometry would provide more solid evidence supporting the proposed\ninvolvement of two dioxygen molecules in a single catalytic cycle.\n3. In the 18O-labeling experiments, using a mixture of 18O2 and 16O2 could provide critical insights into the incorporation of\ntwo oxygen atoms during the formation of 7.\n4. The map levels should be indicated in the legends of Supplementary Figures S13 and S16.\n5. In Figure 2, the meaning of the black dashed lines in panels d and f should also be clarified in the legend.", "reviewer_3": "“Structural and Mechanistic Insights into Azetidine-associated αKG NHFe Enzyme OkaE with Multifunctional Catalysis” by\nWang, Yu, Liu, Zhang, Zhu, Yan, and Liu et al. is about OkaE, a α-Ketoglutarate-dependent mononuclear non-haem iron\nenzyme that can synthesize a pharmacophoric motif azetidine ring via unique C–C bond formation. The authors find that\nOkaE also performs sequential oxidations such as hydroxylations, epoxidation, and ring cleavages. The authors find that not\none but two oxygen molecules are involved for sequential epoxidation and ring cleavage. The authors use a host of\nexperimental techniques (isotopic labeling, X-ray crystallography, and protein engineering), along with QM/MM calculations\nto understand the reactivity of OkaE.\nAlthough this article is well written and sound, I have a few concerns that should be addressed prior to publication of the\narticle. I commend the authors on a comprehensive work with the methods being described in a very reproducible way. I\nurge the authors to consider my suggestions below and upload their simulation data so that it can be reused by the\ncommunity..\n1. The authors mention that OkaE undergoes HAT. Are the authors sure about this, or do they mean Proton-Coupled\nElectron Transfer (PCET) in the general sense? https://onlinelibrary.wiley.com/doi/full/10.1002/anie.201805511. Here, HAT\nmay imply a mechanistic picture that the authors are not fully implying. There should be a clarification on this point.\n2. How did the authors choose their E-value threshold for the sequence similarity analysis? Is this just a strict cutoff that finds\nthe most similar sequences? If so, why not a multiple sequence alignment?\n3. The authors mention that the substrate binding pocket size is reduced by 10% when they analyze their structures. Is this a\nfair analysis? Static structures may not indicate the true reduction in the effective pocket size which may be influenced by the\ndynamics. Is the spread of this pocket size minimal? The authors may have an answer from their QM/MM simulations.\n4. There are some typos that should be fixed. Page 12 line 227, “coplaner”. Page 18 line 329: ‘to addressing this’\n5. In figure 4b, why do the bars not all add up to 100%? At a minimum, shouldn’t the wild type bars add up to 100%?\n6. The isotope labeling experiments in this work are beautiful. However, I do not follow why the alphaKG ratio being altered\nto 4:1 indicates the involvement of the second dioxygen. This should be explained in a bit more detail for readers. The\nspeciation of the second oxygen must be different if it is what the readers indicate. What is the form of this hypothesized\nsecond oxygen species likely to be?\n7. When altering between axial and equatorial conformations for the iron oxo, there may be a transition state:\nhttps://pubs.acs.org/doi/full/10.1021/acscatal.2c01143. How would OkaE overcome such a barrier in the proposed\nmechanism? Some commentary on this would be helpful.\n8. I urge the authors to upload their data to zenodo instead of making their data available upon request. This will facilitate the\nreuse of the authors’ work.\nThe article is well written. Thus, I’d like to commend the authors on this effort."}, "author_response": "Responses to reviewers’ comments:\nReviewer #1:\n1. The manuscript reports the structural and mechanistic insights into the α-\nketoglutarate (αKG)-dependent non-heme iron enzyme OkaE that builds the azetidine\nring in okaramine biosynthesis. In vitro reconstitution of OkaE with okaramine A (1)\nproduced okaramine E (3), 12-dehydroxy-okaramine E (2), and four new congeners,\nneokaramines I–IV (4–7). Crystal structures of OkaE•Co²⁺, OkaE•Co²⁺•αKG, and\nOkaE•Co²⁺•αKG•okaramine A revealed a homodimeric double-stranded β-helix fold, a\ndistal-type αKG binding mode, and placement of substrate atoms C8a and O3a near the\nmetal center within a pocket framed by M64, M71, M122, and W79. QM/MM\ncalculations supported the distal αKG geometry and showed substrate positioning that\nshortens Op–H8a and Op–H3a distances (≈4.8→2.9 Å and 5.1→2.6 Å). Site-directed\nmutagenesis indicated that M71A biases H8a hydrogen-atom transfer (HAT), W79A\nreduces overall reactivity, and M122A modestly enhances formation of 7 via O3a-HAT;\nstructures of M64A, M71A, and W79A complexes corroborate these product profiles.\nIsotope-18O-labeling showed mixed O₂/H₂O incorporation into 3–6, consistent with\nhydroxyl-rebound and/or cationic pathways, whereas 7 incorporates only O₂, implying\nrare capture of a second O₂ within a single catalytic cycle. Together, the data support a\nmechanism in which H8a-HAT drives azetidine formation followed by sequential\noxidations to give 2–6, while a possible alternate O3a-HAT pathway channels\nperoxidation/epoxidation to yield 7. Overall, the study elucidates the structure-function\nrelationships of OkaE and demonstrates its potential to be reprogrammed to tailor and\ndiversify complex natural products.\nResponse:\nWe appreciate the reviewer's comprehensive summary and positive reception of\nour work.\n2. Figure 4. In vitro assays showed that M64A converts substrate 1 to 7, whereas\nM71A did not (Figure 4A, B). The proposed pathway for 7 formation involves\nabstraction of the C3a–OH proton. However, the crystal structures place the C3a–OH\nat the same distance from the metal center in both M64A and M71A (~5.9 Å). How do\nthe authors explain these divergent outcomes with the similar C3a–OH–metal distances?\nResponse:\nWe thank the reviewer raised for the critical insight. We agree that static crystal\nstructures cannot capture the full spectrum of conformational dynamics during catalysis.\nTo probe these dynamics, we conducted MD simulations on the M64A and M71A\n1\ncomplexes (see Figure 1 below). These results revealed a key structure difference: in\nthe M64A mutant, the Fe=OH3a distance is 2.0 ± 0.6 Å, which is conducive for\nhydrogen abstraction. In contrast, this distance is significantly longer in the M71A\nmutant (4.8 ± 0.6 Å), while the Fe=OH8a distances remained similar in both (M64A:\n2.7 ± 0.4 Å; M71A: 2.6 ± 0.3 Å). These simulations data directly explain the\nbiochemical results. The shorter Fe=OH3a distance in M64A facilitates the\nabstraction necessary to form compound 7, whereas the increased distance in M71A\nrenders this reaction unfavorable. We have incorporated the MD simulation results and\nrevised the corresponding text in the updated manuscript (Lines 340–347, Page 17) and\nthe revised SI document (Figure S22) to clarify this issue.\nFigure 1 in this response letter: Distance distributions between O atom in\nFe(IV)=O species and H3aandH8a during 50 ns MD simulations. a Distance\nprofiles for the M64A complex. b Distance profiles for the M71A complex.\n3. Page 5, Line 117, the authors mentioned “four previously uncharacterized minor\nproducts, designated as neuokaramines I–IV (4–7),” but on Page 6, Lines 140–141\nstated that “the structure of 6 was identical to that of okaramine R.” In reference 43, the\nstereochemistry at C-3a of okaramine R was not determined in reference 43, while the\npresent study showed the configuration of C-3a OH on the structure (Scheme 1). Please\nprovide how to verify the stereochemistry of C-3a OH of compound 6 or if it could be\nokaramine R reported in reference 43.\nResponse:\nWe apologize for the imprecise description of compound 6. The compound was\ninitially designated as new because, while its planar structure is identical to okaramine\nR, its absolute configuration on C3a was determined for the first time in this study.\nfollowing evidence. First, considering its biosynthetic origin from OkaE-catalyzed\noxidations of okaramine A (1), an epimerization at C3a through a hydrogen atom\ntransfer (HAT) pathway is mechanistically unlikely. This is corroborated by the ternary\ncrystal structure, which shows the H3a atom in substrate 1 oriented on the opposite\nplane relative to the metallo-centre.\nTo definitively confirm the configuration, we compared the experimental ECD\nspectrum of 6 with the calculated spectra for the two possible stereoisomers,\n(2S*,3aS*)-6a and (2S*,3aR*)-6b. The experimental ECD spectrum showed a good\nmatch with the calculated spectrum of the (2S*,3aS*) isomer (6a; see Figure 2 below),\nthereby firmly establishing the S configuration at C3a. We have incorporated these ECD\ndata as Figure S11 in the revised SI document and updated the relevant discussions in\nthe main text (Lines 161–164, Page 7) and the SI document (Supplementary Methods,\nPages 6 and 7) to clarify this point.\nExp CD of6\nCal ECD of6b\n40\n∆  0\n250 300 350 400\n-40\n-80 Wavelength (nm)\nFigure 2 in this response letter: Calculated and experimental ECD data for\ncompound 6.\n4. For structure elucidation of the enzymatic products, please provide detailed\nstereochemical descriptions of compounds 4–7, especially for protons like H-2, H-12,\nand H-11. Please explain how the stereochemistry of compounds 4–7 based on NOESY\nor other spectroscopic data.\nResponse:\nWe thank the reviewer for pointing this out. We apologize for the lack of clarity\nin our original description. Firstly, compound 4 was characterized as the carboxylated\nderivative of 3. This structural assignment is supported by the close similarity of their\nNMR data and specific rotations ([α]20 + 132.7 for 2; [α]20 + 222.1 for 3; [α]20 +\nD D D\n233.6 for 4). Moreover, the nearly identical experimental ECD spectra of 2–4 (see\nFigure 3 in this response letter) provide additional evidence that they share the same\nabsolute configuration. The relevant sections have now been revised accordingly in the\n3\nmain text (Lines 152–154, Page 7) and in the SI document (Supplementary Methods,\nPage 5).\nExp CD of3\n 0\n∆\n300 400 500\n-30\n-60 Wavelength (nm)\nFigure 3 in this response letter:The CD spectra comparison of compounds 2–4.\nThe stereochemical assignment of compound 5 has been clarified in the revised SI\ndocument (Supplementary Methods, Pages 5 and 6). The NOESY correlations of δ\nH\n3.31 (H, br s, H11)/δ 1.91 (3H, s, H13), δ 0.81 (3H, s, H14)/δ 5.85 (H, br s, H12)/δ\nH H H H\n6.85 (H, d, J = 7.7 Hz, H7), combined with the rigid framework formed by rings A–C,\nH, and I, indicated that the C8aC11 bond is α-oriented. Consequently, H11 and H13\nwere assigned β-orientations, while H12 and H14 were α-oriented (see Figure 4 in this\nresponse letter). The comparable dihedral angles H2–C2–C3–H3A (≈ -35.0°) and H2–\nC2–C3–H3B (≈ -156.0°), and coupling constants (3J = 9.3 Hz; 3J = 8.0 Hz),\nH2, H3A H2, H3B\nwhich were similar to those of compound 31-3, together with the NOESY correlations\nof δ 2.98 (H, dd, J = 8.0, 14.0 Hz, H3B)/δ 7.49 (H, d, J = 6.7 Hz, H4),\nH H\nH2, H3A and 3a-OH share α-orientation, whereas H3B and the C3a–C3b bond are β-\noriented (see Figure 4 in this response letter). To further confirm the relative\nconfiguration of C2, NMR chemical shift calculations and DP4+ probability analysis\nwere performed for two possible isomers, (2S*,3aS*,8aR*,11R*,12R*)-5a and\n(2R*,3aS*,8aR*,11R*,12R*)-5b. The calculated chemical shifts of 5a exhibited an\nexcellent agreement with the experimental data, showing a higher correlation\ncoefficient (R2 = 0.9983), a lower root-mean-square deviation value (RMSD = 1.78)\n(Figure 5 in the response letter), and a DP4+ probability of 100% (Figure 6 in the\nresponse letter). Moreover, comparison of the experimental ECD spectrum of 5 with\nthe calculated spectra for (2S*,3aS*,8aR*,11R*,12R*)-5a and its enantiomer\n(2R*,3aR*,8aS*,11S*,12S*)-5c revealed an excellent match with that of 5a (Figure 7\nin the response letter), unambiguously confirming the absolute configuration of 5 as\n2S,3aS,8aR,11R,12R. The corresponding data and analysis have been incorporated into\nthe main text (Lines 154–159, Page 7) the SI document (Supplementary Methods, Pages\n5–6).\n200 y = 0.9566x - 0.3653 20\nR² = 0.9983\nRMSD = 1.78\n)m\n150\nδ\nCorr 10\np p (\nδ\npxE\n100\nδ\nδ\n=\n- Exp 0\n50 ∆-10\n0 -20\n0 50 100 150 200 233a3b45677a8a910111213141'2'4'5'6'6a'7a'8'9'10'11' 11a' 11b'12'13'14'\nδCal (ppm) Carbon position\n200 y = 0.9767x - 2.7484 20\nR² = 0.9901\nRMSD = 4.30\n)m\n150\nδ\nCorr 10\np p (\nδ\npxE\n100\nδ\nδ\n=\n- Exp 0\n50 ∆-10\n0 -20\n0 50 100 150 200 233a3b45677a8a910111213141'2'4'5'6'6a'7a'8'9'10'11' 11a' 11b'12'13'14'\nδCal (ppm) Carbon position\nFigure 5 in this response letter: 13C NMR calculation results of two possible\nisomers of 5a and 5b. a Two possible isomers of 5a and 5b. b Linear correlation\nplots of predicted versus experimental 13C NMR chemical shift. c Relative errors\nbetween the predicted δ of two potential structures and recorded δ .\nC C\n5a (Isomer 1) and 5b (Isomer 2).\n6\nCal ECD of5a\n∆  0\n200 300 400 500\n-30\n-60 Wavelength (nm)\nFigure 7 in this response letter:Calculated and experimental ECD data for\ncompound 5.\nThe stereochemical assignment of compound 6 has been clarified as (2S, 3aS)-6,\nconsistent with our clarification in response to Comment #3. This assignment was\nestablished based on its chemical transformation pathway and supported by the\ncomparison between the experimental and calculated ECD spectra. The relevant data\nare provided in Figure S11 of the revised SI, with corresponding revisions in the main\ntext (Lines 161–164, Page 7) and the SI (Supplementary Methods, Pages 6 and 7).\nThe absolute configuration of compound 7 has been assigned as (2S,11R)\nstereoisomer based on NMR calculations, DP4+ probability analysis, and ECD\ncalculations. To determine its relative configuration, two stereoisomers (2S*,11S*)-7a\nand (2S*,11R*)-7b (Figure 8a in the response letter) were evaluated using the GIAO\nmethod at the mPW1PW91/6-311+G(d,p) level with the PCM model. Among these,\nisomer 7b showed the closest agreement with the experimental NMR data (R² = 0.9986,\nRMSD = 1.82, DP4+ probability = 94.30%; see Figures 8b, 8c, and 9 in this response\nletter). To further establish the absolute configuration of 7, ECD calculations were\ncarried out for the two enantiomers (2S*,11R*)-7b and (2R*,11S*)-7c. Comparison\nwith the experimental ECD spectrum indicated a superior match for 7b (Figure 10 in\nthis response letter), thereby confirming the absolute configuration of 7 as 2S,11R.\nThe corresponding data and analysis have been added to the main text (Lines 167–170, Page\n8) and the revised SI (Supplementary Methods, Page 7).\n7\n250 y = 0.9427x + 0.4077 30\nR² = 0.9982\n20\n)m p p ( δ pxE 1 1 0 5 0 0 δ δ = - E C x o p\nrr\n10 0\nδ\n50 ∆\n-10\n0\n-20\n0 50 100 150 200 250\nδCal (ppm)\n233a3b45677a8a91011\nC\n12\na\n1\nr\n3\nb\n1\no\n4\nn\n1 '\npo\n2'\nsi\n4\nt\n'\nio\n5'\n6'6a'7a'8'9'10'11' 11a' 11b'12'13'14'\n250 y = 0.9457x - 0.0168 30\nR² = 0.9986\n20\n)m p p ( δ pxE 1 1 0 5 0 0 δ δ = - E C x o p\nrr\n10 0\nδ\n50 ∆\n-10\n0\n-20\n0 50 100 150 200 250\nδCal (ppm)\n233a3b45677a8a91011\nC\n12\na\n1\nr\n3\nb\n1\no\n4\nn\n1 '\npo\n2'\nsi\n4\nt\n'\nio\n5'\n6'6a'7a'8'9'10'11' 11a' 11b'12'13'14'\nFigure 8 in this response letter:13C NMR calculation results of two possible\nisomers of 7a and 7b. a Two possible isomers of 7a and 7b. b Linear correlation\nplots of predicted versus experimental 13C NMR chemical shift. c Relative errors\nbetween the predicted δ of two potential structures and recorded δ .\nC C\n7a (Isomer 1) and 7b (Isomer 2).\nCal ECD of7c\n40\n∆  0\n200 300 400\n-40\n-80 Wavelength (nm)\nFigure 10 in this response letter: Calculated and experimental ECD data for\ncompound 7.\nThe absolute configurations of compounds 4–7 were determined based on\nspecific rotation data, ECD spectroscopy, and 13C NMR calculations. A concise\nsummary of this analysis has been added to the revised main text. To maintain the focus\non the main findings，full stereochemical details are provided in the revised SI\ndocument (Supplementary Methods, Pages 5–7; Figures S11–S15).\n5. Is there any evidence to support that IM2 is involved in the conversion of 3 to 4 or\n5?\nResponse:\nWe appreciate the reviewer’s valuable question regarding this intermediate. Our\ninterpretation is supported by the following evidence and reasoning:\nFirst, time-course analysis captured the transient formation of species (IM2) with\na mass consistent with an aldehyde intermediate (detected as [M + H]+ at m/z 535.2335,\ncalculated for C H O N +, 535.2340; Figure S10 in the revised SI document).\n32 31 4 4\nSecond, although its inherent lability prevented isolation of IM2, its proposed role\nis strongly supported by established mechanistic paradigms. Specifically, the oxidation\nof hydroxyl or methyl groups to carboxylic acids, often proceeding through an aldehyde\nintermediate, is well-documented for several enzyme classes, including αKG-NHFe\nenzymes4, CYP450s5, and flavoprotein oxidases6 (Figure 11 in this response letter).\nThus, we proposed that IM2 serves as a key intermediate in the conversion of 3 to\n4. Additionally, the geminal diol IMa may also undergo direct non-enzymatic\n4\nconversion to 5, independent of IM2. This refined mechanistic scheme has been\nincorporated into the revised manuscript (Lines 176–181, Page 8), and Figures 1 and 6\nhas been updated accordingly to reflect these pathways.\na hydroxyl group into a carboxylic acid. a CYP450 enzyme XiaM. XiaM catalyzes\nthe three-step hydroxylation of preindosespene to produce indosespen5. b\nFlavoprotein oxidase AAO. AAO catalyzes the oxidation of 5-hydroxymethylfurfural\n(HMF) to 2,5-formylfurancarboxylic acid (FFCA) via the intermediates 2,5-\ndiformylfuran (DFF) and 2,5-hydroxymethylfuran carboxylic acid (HMFCA)6. c\nAlpha-KG-NHFe enzymes TET. TET catalyzes the stepwise oxidation of 5-\nmethylcytosine (5mC) residues in nucleic acids to 5-hydroxymethylcytosine (5hmC), 5-\nformylcytosine (5fC), and 5-carboxycytosine (5caC)4.\n6. Figure 4, the in vitro experiment of M71A with 1 showed several additional signals\ncompared to those in WT with 1 (e.g. retention time around 14 min and 33 min). Did\nthe authors have any idea what the products may be and if they are related to\nokaramines?\nResponse:\nWe have analyzed the two additional peaks (t = 14.254 min, t = 32.498 min)\nR R\nnoted by the reviewer. However, due to their very low abundance, it was not feasible\nto isolate sufficient quantities for full structural characterization.\nTo address the reviewer’s query, we provide a detailed analysis of these\nuncharacterized compounds from the M71A reaction using LC-HR-MS experiments.\nAs shown in Figure 12a and 12b below, both unknown compounds exhibited\ncharacteristic UV absorption peaks at 230 nm, 280 nm, and 370 nm, similar to those of\n11\ncompounds 2 and 3, suggesting they are likely okaramine analogues. Based on the LC-\nHR-MS data, their molecular formulas are tentatively assigned as C H O N (t =\n32 33 4 4 R\n14.254 min) and C H O N (t = 32.498 min), respectively.\n32 31 4 4 R\nNevertheless, the minute quantities prevented definitive determination. We have\nincluded this analysis in the revised SI document (Figure S23). As these compounds\ndo not appear to represent novel chemical transformations, and given the substantial\nresources required for large-scale fermentation and isolation, we have not pursued their\nfurther characterization at this stage. We hope this response satisfactorily addresses the\nreviewer’s question.\nFigure 12 in this response letter: LC-MS analysis of unidentified products with\nelevated abundance in the OkaE mutant reaction. a UV spectrum of the\nM71A\nproduct at t = 14.254 min compared with that of product 3. b UV spectrum of the\nR\nproduct at t = 32.498 min compared with that of product 2. c HR-ESI-MS spectrum\nR\nof the product at t = 14.254 min. d HR-ESI-MS spectrum of the new product at t =\nR R\n32.498 min.\n7. For Figure 5, please provide the corresponding mass spectra in SI.\nResponse:\nWe thank the reviewer for pointing this out. As shown in the figure below, the\ncorresponding mass spectra for Figure 5 have now been incorporated into the revised\nSI as Figure S24.\n2 2 2 2 2 2 2 2 2 2\nisotopic purity of the H 18O water was approximately 80% after mixing with water\n2\nexchanged protein solution. The 50% 18O gas used in the experiment contained a 1:1\n2\nisotopic mixture of 18O (50 atom%) and 16O (50 atom%). Oxygen atoms incorporated\n2\nfrom 18O are marked with a red background, while those derived from H 18O are\n2 2\nmarked in blue.\n8. Page 11 to 12, the contents described the structure rearrangement of OkaE•CoII\nand OkaE•CoII•αKG•okaramine A (1) complex (for example, the conformational shift\nof the beta-helix formed by I269-S270). It is suggested to add Figures in the main text\nor SI to show the comparison and structure superimposition figure of OkaE•CoII and\nOkaE•CoII•αKG•okaramine A (1) complex.\nResponse:\nWe thank the reviewer for this valuable suggestion. As now shown in Figure S19\nof the revised SI document (Figure 14 in this response letter), the structural\n13\nsuperimposition of OkaE•CoII and OkaE•CoII•αKG•okaramine A (1) complexes\nvisually highlights the conformational shifts in the key residues discussed in the new\nversion of our manuscript (Lines 235–239, Page 12).\nFigure 14 in this response letter:Structural comparison of OkaE•CoII and\nOkaE•CoII•αKG•okaramine A (1) complex. Superimposition of the apo OkaE•CoII\nstructure (green) and the ternary OkaE•CoII•αKG•1 complex (cyan).\n9. Page 30, line 595, “Table S8” should be “Table S7”.\nResponse:\nRevised as suggested.\n10. Please check the stereochemistry of C-2 of the structures shown in Figure 1. Some\nstructures did not show the stereochemistry properly.\nResponse:\nThe stereochemistry of C2 in the structures of Figure 1 has been corrected in the\nupdated version of our manuscript.\n11. In the Experimental section, please clearly specify which procedures were\nperformed under aerobic versus anaerobic conditions.\nResponse:\nWe have revised the Experimental section to clearly indicate which procedures\nwere performed under aerobic versus anaerobic conditions (Lines 577–579, Page 29).\n12. The current legend for Figure S11 mentioned “Peak 1” (void) and “Peak 2” (OkaE),\nbut these peaks are not labeled or indicated in the figure. Please revise the legend to\nmatch the actual presentation of the chromatogram.\nResponse:\nThe figure legend of Figure S16 has been updated accordingly to match the\nchromatogram in the revised SI document.\nReviewer #2:\n14\n1. The manuscript describes the multifunctionality of OkaE, an αKG-dependent non-\nhaem iron enzyme, in azetidine formation and subsequent scaffold diversification.\nWhile the authors report in vitro assays, crystallography, and QM/MM simulations, the\nstudy lacks novelty since the catalytic versatility of this enzyme family is well\ndocumented, and the presented findings do not substantially advance mechanistic\nunderstanding. Furthermore, the work is not thorough: the mechanistic evidence is\ninsufficiently supported, the structural interpretations are speculative. Overall, the\nmanuscript does not reach the standard of originality or rigor required for publication.\nResponse:\nWe thank the reviewer for the constructive comments. We have carefully\nconsidered the critiques regarding the novelty and thoroughness of our study. While we\nacknowledge that the αKG-dependent non-haem iron enzyme family is known for its\ncatalytic versatility, we believe our work on OkaE provides several new insights that\nsubstantially advance the field. Below, we address the specific concerns point by point,\nand we hope the reviewer will find our response and the additional clarifications in the\nrevised manuscript persuasive.\n(1) Regarding novelty issue on “…the study lacks novelty since the catalytic\nversatility of this enzyme family is well documented, and the presented findings do\nnot substantially advance mechanistic understanding…”:\nWe apologize for the insufficient emphasis on these aspects in the original\nmanuscript. Our study on OkaE presents several key advances, as summarized below:\ni) OkaE represents a new paradigm in enzymatic scaffold diversification. Unlike\npreviously reported systems by Lin group3, OkaE enzyme is a single enzyme\ncapable that catalyzes multiple distinct chemical steps, including hydroxylation,\ncyclization, and possibly rearrangement, to assemble and diversify a complex\nazetidine-containing scaffold. This function versatility provides a new biosynthetic\nmodel for scaffold diversification in natural product biosynthesis.\nii) First atomic-resolution view of azetidine formation. By integrating of\ncrystallography with QM/MM simulations, we provide the first atomic-level\ninsight into azetidine ring formation within this enzyme family, addressing a novel\nand unresolved mechanistic question.\niii) Discovery of a potential new peroxygenase-like activity in non-heme iron\nenzyme. Our isotopic labeling experiments reveal that OkaE incorporates an\noxygen molecule to form an endoperoxide during catalysis. This function trait,\nobserved in only a few αKG-dependent non-haem iron enzyme such as FtmOx17-\n15\n11and NvfI12, suggests that OkaE may represent a third functionally distinct system\ncapable of endoperoxide formation. This finding not only expands the mechanistic\nrepertoire of this enzyme family but also highlights OkaE as a promising starting\npoint for engineering novel biocatalysts that exploit peroxide-bridge intermediates\nfor selective C‒H bond activation.\niv) The Met–π interaction network controls pathway bifurcation and offers a new\nengineering principle. The structure of OkaE in complex with okaramine A\nreveals a unique the Met–π interaction network (M64-M71-W79-M122) that envelops\nthe A-ring of the substrate. Site-directed mutagenesis demonstrates that this\nnetwork is critical structural determinant of reaction bifurcation: the M71A variant\nshifts selectivity toward the H8a–HAT pathway, yielding predominantly 6,\nwhereas W79A disrupts bifurcation entirely. This provides decisive experimental\nevidence that Met–π network is not merely structural but functionally essential.\nFurthermore, this study offers the first example of leveraging a Met–π interaction\nnetwork to engineer catalytic pathway bifurcation for natural product\ndiversification.\nv) A distal αKG-binding mode enables novel oxygen activation. The OkaE enzyme\nand substrate complex exhibits a distal-type αKG-binding mode that prevents\nconventional rebound hydroxylation. QM/MM calculations support a feasible\nazetidine-formation pathway and reveal that rotation of the FeIV=O species initiates\ntwo competing HAT channels (3a-OH–HAT and H8a–HAT), rationalizing the\nproduct diversity. This study expands the repertoire of distal-type αKG enzymes\nand provides quantitative computational validation of their potential for non-\ncanonical oxygen activation mechanisms, offering new prospects for predicting\nand engineering novel enzyme functions.\n(2) Regarding structural interpretations issue on “…the work is not thorough: the\nmechanistic evidence is insufficiently supported; the structural interpretations are\nspeculative…”:\nIn response, we have conducted additional experiments that substantially\nstrengthen the support for our proposed mechanism. While we believe our original\nstructural interpretations remain well-founded, the newly acquired data enhanced\nmechanistic validation:\ni) The electron density for the substrate and the coordinating residues is clear and\nunambiguous. A mFo-DFc maps have been provided in the SI document (Figures\nS18 and S21).\n16\nii) The proposed roles of active site residues are directly inferred from the observed\nhydrogen-bonding and electrostatic interactions in the structure. The A–E rings of 1\nadopt a catalytically competent conformation in the binding pocket formed by M64,\nM71, W79, N118, M122, L227, and T273. Especially, a unique Met–π interaction\nnetwork involving residues M64-M71-W79-M122 envelops the A-ring of 1, orientating\nactive sites C8aand O3a toward the active centre. Furthermore, these proposed roles\nare consistent with and supported by the outcomes of our alanine scanning\nmutagenesis of residues M64, M71, W79, and M122.\niii) As detailed in our response to Comment #4, QM/MM calculations support the\nassignment that 3, 4, 5, and 6 are generated through H8a–HAT pathway, whereas 7\nis formed via 3a-OH–HAT, thereby providing a theoretical basis for the observed\nproduct divergence.\niv) Following the reviewer’s suggestion, mixed 16O /18O labeling experiments were\n2 2\nperformed. The resulting isotopic distribution supports the involvement of a second\nO molecule in the formation of 7, as further elaborated in our response to Comment\n2\n#7.\nIn summary, we believe the revised manuscript presents a comprehensive and\nnovel mechanistic study of OkaE. The combination of in vitro biochemistry, structural\nbiology, and computational modeling provides a compelling and multi-faceted\nmechanism. We have thoroughly revised the text to better highlight the novelty and\nmechanistic insights of our work and are confident that the manuscript in its current\nform meets the standards for publication. We sincerely thank the reviewer’s valuable\nsuggestions, which have significantly improved the quality and clarity of our\nmanuscript.\n2. Line 120: Regarding the observed differenced from Lin et al. results, differences\nin reaction conditions should be clearly described in the main text.\nResponse:\nWe thank the reviewer for pointing this out. Specifically, the differences observed\nbetween the OkaE-catalyzed reaction profile and that reported by Lin et al.3 likely arise\nfrom variations in protein expression and assay conditions. In our study, Strep-tagged\nOkaE was rapidly purified under anaerobic conditions from E. coli BL21(DE3) and\nassayed using precisely controlled stoichiometric ratios of cofactors (FeII, αKG, and\nascorbate). In contrast, Lin et al. employed His-tagged OkaE expressed in S. cerevisiae,\npurified under aerobic conditions, and assayed with cofactors and co-substrates\nsupplied in large excess. The differences in reaction conditions between our study and\n17\nthat of Lin et al. are now explicitly described in the revised manuscript (Lines 127–130,\nPage 6).\n3. Line 127: I believe it is unlikely that only one eq. of aKG is needed for 4, 5, 7.\nResponse:\nWe thank the reviewer for the insightful comments. The reviewer is correct to note\nthat one equivalent of KG is stoichiometrically insufficient to support the formation\nof multiple products (4, 5, and 7). We apologize for the lack of clarity on this point in\nour original manuscript. Our data indicates that the single equivalent of KG is\npartitioned among competing catalytic pathways at a key mechanistic branching point.\nThis is supported by supplemental experiments demonstrating that the product ratio of\n4 and 5 increases when the KG equivalent is raised from 1:1 to 4:1 relative to the\nsubstrate. The initial condition (1:1 equivalent) was selected for the presented assay to\nbest represent the native partitioning observed under stoichiometric turnover conditions.\nWe have revised the manuscript to clarify this rationale (Lines 139–144, Page 7).\n4. Line 152: The biochemical evidence should be clearly presented.\nResponse:\nWe thank the reviewer for this important question. In our biochemical assays\n(Figure 1a, traces iii–iv), using compounds 2 and 3 as substrates, we detected the\nformation of a putative intermediate (IM2) along with the product 4. Based on LC-HR-\nMS and NMR analyses, we propose that compound 4 is formed through a stepwise\noxidation process, beginning with the hydroxyl group of 2, leading to the aldehyde-\ncontaining intermediate IM2, and subsequently to the carboxylic acid product 4.\nNotably, compound 5 possesses a newly formed classical hemiacetal moiety, which is\nmost plausibly generated via a non-enzymatic, acid-promoted intramolecular\ntransformation of the aldehyde-containing intermediate IM2. The corresponding\ndescription has been revised in the main text (Lines 176–181, Page 8).\n5. Fig4a: The authors assume that 3, 4, 5, 6 are generated through H8a-HAT, and 7 is\nformed via OH3a-HAT. However, no experimental evidence is provided to correlate\nthe site of HAT with the product structures.\nResponse:\nWe thank the reviewer for raising this point. We agree that experimental\nconfirmation of the HAT sites would be ideal. Given the current challenges in\nsynthesizing okaramine substrates2, the total chemical synthesis of isotopically labeled\nokaramine A is not currently feasible, making direct isotopic labeling experiments\ndifficult to pursue at this stage.\nthe H8a–HAT and 3a-OH–HAT pathways. As shown in the figure below, the distance\nbetween O and H8a decreased from 4.83Å (RC1) to 2.89 Å (Int1b), and the distance\np\nbetween O and H3a (hydrogen atom on 3a-OH) both decreased from 5.06 Å (RC1) to\np\n2.62 Å (Int1b). Moreover, the Fe–O –H8a and Fe–O –H3a angles increased to optimal\np p\npositions for the reaction, reaching 147° and 169°, respectively. These changes provide\nfurther insight into the role of FeIV=O in the catalytic cycle of OkaE, allowing it to\ncatalyse a variety of oxidation reactions. The QM/MM-calculated energy profile (Fig.\n3a) indicates that the intermediate Int1b can proceed through either the H8a–HAT or\n3a-OH–HAT pathway, both encountering comparable energy barriers of 21.2 kcal/mol\n(Int1b→TSH8a) and 22.4 kcal/mol (Int1b→TSH3a), respectively, producing the\ncorresponding intermediates TSH8a and TSH3a. These results suggest that the two\nHAT pathways are kinetically competitive and could lead to distinct product outcomes.\nBy combing these computational results with biochemical assays, we can infer which\nHAT pathway leads to which specific products.\nThe relevant sections in both the main text (Lines 302, 303 and 306–310, Page 16;\nFigure 3) and SI document (Figure S20) have been revised to include these findings.\nspecies through αKG activation and subsequent hydrogen abstraction from C8a\nand O3a. a QM(UB3LYP/B2)/MM relative energies (kcal/mol) for this process. The\ndispersion corrections are included in the relative energies. b QM(UB3LYP/B2)/MM-\n20\noptimized geometries of three key species involved in the reaction. Key distances are\ngiven in Å.\n6. Fig 6a: Formation of a primary cation IMa3 should be less likely.\nResponse:\nWe appreciated the reviewer for raising this important question. As correctly noted,\nthe generation of an unstable primary carbocation is generally disfavored under typical\nchemical conditions. We have revised the corresponding description (Lines 408–413,\nPage 22) and updated Figure 6 accordingly in the revised manuscript.\n7. Fig 6c: The mechanism shown in 6c involving second O is not fully supported by\n2\nthe results. What would happen if the authors use a mixture of 16O and 18O ? Product\n2 2\nwith 16O/18O should be formed if the hypothesis is correct.\nResponse:\nFollowing the reviewer’s suggestion, we performed the mixed 16O /18O\n2 2\nbiochemical reaction and analyzed the products by LC-HR-MS. The measured isotopic\ndistribution was as follows: m/z 551 (+0) : 553 (+2) : 555 (+4) = 46.7% : 1.3% : 52.0%\n(see Figure 16e, trace iv in this response letter).\nNotably, the +2 peak is nearly absent, while the +0 and +4 peaks occur in nearly\nequal abundance. This pattern indicates that the first O molecule activates the oxidation\n2\nreaction but does not rebound to the substrate to form 7; otherwise, a significant +2\nisotopic signal would have been observed. Instead, our data are consistent with the\nincorporation of a second O molecule, which gives rise to the observed +0 or +4 mass\n2\ndistribution.\nIn addition, biochemical assays show that the yield of 7 is unchanged when the\nmolar ratio of αKG:1 is increased from 1:1 to 4:1. This result confirms that 7 is formed\nin a single αKG-dependent catalytic event involving two O molecules, rather than\n2\nthrough multiple sequential oxidative turnovers.\nWe are pleased to report that the additional experiments, conducted as the\nreviewer recommended, have provide strong experimental validation for our proposed\nhypothesis. The corresponding discussion has been revised in the updated manuscript\n(Lines 374–378 and 383–388, Page 20). As recommended, we have also replaced\nFigure 5 in the revised main text and the new Figure S24 and Table S23 have been\nadded into the new version of SI document.\n2\n(black vs red and blue traces). f Diagram of the incorporation ratio of dioxygen and\nwater-derived oxygen in compounds 37. The biochemical assays of OkaE were\nconducted under the following conditions: (i) 18O + H O, (ii) O + H 18O, (iii) 18O +\n2 2 2 2 2\nH 18O, (iv) 50% 18O /O + H O, (v) O + H O. The isotopic purity of the H 18O water\n2 2 2 2 2 2 2\nwas approximately 80% after mixing with water exchanged protein solution. The 50%\n18O gas consisted of 50 atom% 18O and 50 atom% 16O. Oxygen atoms incorporated\n2\nfrom 18O are shown in red, while those derived from H 18O are indicated in blue.\n2 2\n8. The QM/MM calculations mainly address the formation of the Fe(IV)=O species;\nadditional simulations on the later stages of the reaction would provide deeper\nmechanistic understanding.\nResponse:\nWe appreciated the reviewer for this helpful suggestion. We performed QM/MM\nsimulations for both the H8a–HAT and 3a-OH–HAT pathways. As summarized in\nFigure 15 in this response letter (see response to Comment #5), the calculated energy\nbarriers are 21.2 kcal/mol for H8a–HAT and 22.4 kcal/mol for 3a-OH–HAT. These\nresults indicate that both hydrogen atom transfer (HAT) pathways are kinetically\ncomparable and may lead to distinct product outcomes.\nWe fully acknowledge the importance of investigating the complete reaction\npathway. However, modeling the late-stage transformation, particularly the\nrearrangement and cyclization steps following the initial HAT, presents significant\ncomputational challenges. These include the need to account for multiple\n22\nconformational states, the complexity of potential reaction coordinates, and the\nsubstantial resources required for adequate sampling of such a large system. While\nthese steps are beyond the scope of the present study, we agree with the reviewer that\nthis is a valuable direction for future investigation, and we are actively considering\nways to address these challenges in subsequent work.\n9. For compound 7, determining the oxygen stoichiometry would provide more solid\nevidence supporting the proposed involvement of two dioxygen molecules in a single\ncatalytic cycle.\nResponse:\nWe thank the reviewer for this valuable suggestion. We agree that direct\ndetermination of the oxygen stoichiometry would provide stronger evidence for the\nproposed mechanism. However, such analysis is experimentally challenging for the\nOkaE-catalyzed system. The reaction mixture contains multiple low-abundance and\nlabile okaramine analogues (e.g., peaks at t = 7.82, 8.81, and 14.25 min; Figure 17 in\nR\nthe response letter), as well as structurally similar products that are difficult to separate\nand quantify accurately. These factors prevent a reliable stoichiometric measurement\nof O consumption in this multi-step enzymatic process.\n2 2\nlabeling with αKG-dependent biochemical assays to elucidate the two-oxygen\nincorporation mechanism leading to the formation of compound 7.\nWe are actively performing protein engineering of OkaE to simplify the reactions\nand make compound 7 the dominant, or even the sole, product for accurate\ndetermination of its oxygen stoichiometry in future studies.\nFigure 17 in this response letter:In vitro biochemical characterization of OkaE. a\nHPLC profiles of OkaE-catalysed reactions in vitro using compounds 1, 2, 3, 5, and 6\nas the substrate (i−x). b Substrate consumption and product profile analysis of OkaE\n23\nusing different proportions of αKG (i−v). The asterisk (*) denotes unidentified\nokaramine analogues due to their low yield or instability.\n10. In the 18O-labeling experiments, using a mixture of 18O and 16O could provide\n2 2\ncritical insights into the incorporation of two oxygen atoms during the formation of 7.\nResponse:\nWe thank the reviewer for this important point. As noted in our response to\nComment #7, we have performed mixed 16O and 18O labeling experiments, which\n2 2\nprovide additional insights into oxygen incorporation during the formation of 7.\n11. The map levels should be indicated in the legends of Supplementary Figures S13\nand S15.\nResponse:\nThe contour level of the mFo-DFc map used in these two figures was ± 3.0σ. We\nhave updated the figure legends of Figures S18 and S21 with the correct map level\nshown in the revised SI document.\n12. In Figure 2, the meaning of the black dashed lines in panels d and f should also be\nclarified in the legend.\nResponse:\nWe apologies for the confusion caused by different dashed lines in this figure. As\nshown in Figure 18 in this response letter, the black dashed lines indicate polar contacts\nbetween residues and/or compounds (e.g., hydrogen bonds and salt bridges). In panel\ne, additional cyan dashed lines are used to depict the Met–π interaction network. In\npanel f, the yellow dashed lines are included to highlight the distances from the O -\n2\nbinding site to key atoms of the compound and the purple dashed lines are used to depict\nthe cationπ interaction.\nThe Figure 2 and its corresponding legend have been updated accordingly in the\nrevised version of the manuscript (Lines 220–222, Page 11).\narchitecture of OkaE is shown as a functional dimer with one monomer color-coded\nbased on secondary structure (shown as stereo images). b Active site of the\nOkaE•CoⅡ•αKG complex. c Active pocket of the OkaE•CoⅡ•αKG•okaramine A (1)\ncomplex is shown in electrostatic mode. d–f Close-up views of the active site of the\nOkaE•CoⅡ•αKG•okaramine A (1) complex. The water molecule is modeled based on\nthe binary structure, as the resolution of ternary is insufficient to resolve water\nmolecule. Residues around the active pocket are displayed as gray sticks, the Cobalt\natom is shown as pink sphere. Okaramine A (1) is displayed as orange sticks. The\nαKG is displayed as tint sticks. The water molecule is shown as red sphere. Two loop\nregions (H134–G138 and I269–N271) are displayed as cartoons. The cyan dashed\nlines are used to depict the Met–π interaction network. The yellow dashed lines are\nincluded to highlight the distances from the O -binding site to key atoms of the\n2\ncompound and the two purple dash lines are used to depict the cation-π interaction.\n1. “Structural and Mechanistic Insights into Azetidine-associated αKG NHFe\nEnzyme OkaE with Multifunctional Catalysis” by Wang, Yu, Liu, Zhang, Zhu, Yan,\nand Liu et al. is about OkaE, a α-Ketoglutarate-dependent mononuclear non-haem iron\nenzyme that can synthesize a pharmacophoric motif azetidine ring via unique C–C bond\nformation. The authors find that OkaE also performs sequential oxidations such as\nhydroxylations, epoxidation, and ring cleavages. The authors find that not one but two\noxygen molecules are involved for sequential epoxidation and ring cleavage. The\nauthors use a host of experimental techniques (isotopic labeling, X-ray crystallography,\nand protein engineering), along with QM/MM calculations to understand the reactivity\nof OkaE.\nAlthough this article is well written and sound, I have a few concerns that should\nbe addressed prior to publication of the article. I commend the authors on a\ncomprehensive work with the methods being described in a very reproducible way. I\nurge the authors to consider my suggestions below and upload their simulation data so\nthat it can be reused by the community.\nResponse:\nWe thank the reviewer for this positive evaluation and for recognizing the\nreproducibility of our methods. In accordance with the reviewer’s suggestion, we have\nuploaded the simulation data to a public repository so that it can be reused by the\ncommunity (https://doi.org/10.5281/zenodo.17485598).\n2. The authors mention that OkaE undergoes HAT. Are the authors sure about this,\nor do they mean Proton-Coupled Electron Transfer (PCET) in the general\nsense? https://onlinelibrary.wiley.com/doi/full/10.1002/anie.201805511.Here, HAT\nmay imply a mechanistic picture that the authors are not fully implying. There should\nbe a clarification on this point.\nResponse:\nWe thank the reviewer for raising this important point. OkaE follows the classical\ncatalytic paradigm of αKG-dependent non-heme iron enzymes, in which the high-spin\n(S = 2) FeIV=O intermediate cleaves substrate C–H bonds via a Hydrogen Atom\nTransfer (HAT) mechanism13. As noted by the reviewer, the distinction between these\nmechanisms is illustrated in the Angew. Chem. Int. Ed. paper14 cited by the reviewer:\nTauD-J (a canonical αKG-dependent non-heme iron enzyme) was analyzed as a model\nfor HAT, whereas lipoxygenase (with high-spin (S = 5/2) FeIIIOH) was used as an\n26\nexample of concerted PCET. To clarify this mechanistic context, we have revised the\ncorresponding context in the updated manuscript (Lines 42 and 43, Page 2).\n3. How did the authors choose their E-value threshold for the sequence similarity\nanalysis? Is this just a strict cutoff that finds the most similar sequences? If so, why not\na multiple sequence alignment?\nResponse:\nThe E-value threshold was chosen based on established practice in BLAST-based\nsequence similarity searches, where increasingly stringent cutoffs are commonly\napplied to balance sensitivity and specificity. Specifically, we examined three\nthresholds (10−30, 10−50, and 10−70) to ensure that our results were not dependent on a\nsingle value. This approach allowed us to focus on highly similar sequences, thereby\nenabling reliable structural comparison and mechanistic interpretation. While multiple\nsequence alignment is indeed a powerful complementary method, its primary utility lies\nin identifying conserved motifs and residue-level variation across a broader set of\nhomologs. Our specific aim here was instead to capture the most closely related\nhomologous sequences to guide structural modeling and functional inference. For this\npurpose, E-value–based filtering provides a more direct and appropriate framework.\n4. The authors mention that the substrate binding pocket size is reduced by 10% when\nthey analyze their structures. Is this a fair analysis? Static structures may not indicate\nthe true reduction in the effective pocket size which may be influenced by the\ndynamics. Is the spread of this pocket size minimal? The authors may have an answer from their\nQM/MM simulations.\nResponse:\nTo address the question, we performed 50 ns molecular dynamics (MD)\nsimulations of both the apo OkaE and the ternary OkaE complex, using the crystal\nstructure as the initial conformation. As shown in the figure below, the calculated\npocket volumes were 657 ± 135 Å³ for the apo form and 705 ± 124 Å³ for the ternary\ncomplex, corresponding to an approximate increase of 7.3 ± 2 %. Although the crystal\nstructures of OkaE enzymes suggested a ~10% reduction in pocket volume upon\nsubstrate binding, the MD simulations revealed that conformational rearrangements of\nthree flexible loop regions (Y69–P74, Q131–D136, and R179–D189) collectively lead\nto a more relaxed and accessible substrate-binding pocket in the ternary state. The\ncorresponding discussion has been revised in the updated manuscript (Lines 235–239,\nPage 12).\nternary OkaE during 50-ns MD simulations. Time-dependent changes in pocket\nvolume for apo OkaE (black) and the ternary OkaE complex (red) over 50 ns of\nmolecular dynamics simulations.\n5. There are some typos that should be fixed. Page 12 line 227, “coplaner”. Page 18\nline 329: ‘to addressing this’.\nResponse:\nWe have gotten these items revised as the reviewer suggested in the new version\nof our manuscript.\n6. In figure 4b, why do the bars not all add up to 100%? At a minimum, shouldn’t the\nwild type bars add up to 100%?\nResponse:\nThis apparent discrepancy is due to the detection of several additional minor\nproducts (e.g., peaks at retention times of ~14 min and 33 min) that exhibit UV\nabsorption features similar to those of okaramines, as shown in Figure 12 of the\nresponse letter. These minor peaks, along with residual substrate 1, were integrated in\nour quantification but were not displayed in Figure 4b. As a result, the stacked bars for\ncompounds 3–7 do not sum to 100%. We have revised the legend of Figure 4b to clarify\nthis point and provide a more accurate representation of product ratios (Lines 361–366,\nPages 19–20).\n7. The isotope labeling experiments in this work are beautiful. However, I do not\nfollow why the alphaKG ratio being altered to 4:1 indicates the involvement of the\nsecond dioxygen. This should be explained in a bit more detail for readers. The\nspeciation of the second oxygen must be different if it is what the readers indicate. What\nis the form of this hypothesized second oxygen species likely to be?\nWe thank both reviewers for their valuable comments on the isotope labeling\nexperiments, which prompted us to conduct additional experiments to further elucidate\nthe mechanism. As shown in figure below, αKG-dependent assays showed that the yield\nof compound 7 remains unchanged when the molar ratio of αKG:1 is increased from\n1:1 to 4:1, indicating that 7 is generated in a single αKG-dependent catalytic event that\nrequires two O molecules, rather than through multiple sequential oxidative turnovers.\n2 2\nlabeling experiments. The resulting mass distribution showed a negligible +2 signal,\nwhile the +0 and +4 peaks are predominant. This pattern indicates that the first O\n2\nmolecule activates the oxidation but does not rebound to form 7; otherwise, a +2 peak\nwould have been detected. Instead, the incorporation of a second O molecule accounts\n2\nfor the observed +0 or +4 mass distribution, consistent with a mechanism in which two\nO molecules are engaged within a single αKG-dependent catalytic cycle (see also our\n2\nresponse to Comment #5 from Reviewer 2).\nWe have clarified these points in the revised manuscript (Lines 373–378 and\n383–388, Page 20). Figure 5 has been replaced, and Figure S24 and Table S23 have\nbeen added into the new version of SI document.\nOkaE-catalyzed formation of compound 7. a Varying 16O /18O incorporation\n2 2\nlevels in compound 7 was observed (black vs red and blue traces). b Substrate\nconsumption and product profile analysis of OkaE using different proportions of αKG\n(i−v). c Proposed mechanism for the formation of compound 7.\n8. When altering between axial and equatorial conformations for the iron oxo, there\nmay be a transition state: https://pubs.acs.org/doi/full/10.1021/acscatal.2c01143. How\nwould OkaE overcome such a barrier in the proposed mechanism? Some commentary\non this would be helpful.\nResponse:\nWe thank the reviewer for raising this insightful point regarding the potential\nenergy barrier between axial and equatorial conformations of the FeIV=O species, as\nhighlighted in the reference study15. We agree that conformational transitions can\npresent meaningful energy barriers in some non-heme iron enzyme systems.\nIn the case of OkaE in our study, our QM/MM calculations indicate that the\nequatorial conformation of the FeIV=O species is formed directly following OO\n30\ncleavage from Int1a, without proceeding through axial conformation. Specifically, the\nNFeOp angle (where N is from H210 and Op is the oxo group) changes from 100.1°\nin the reactant complex (RC) to 160.7° in Int1a. This significant angular shift occurs\nconcurrently with decarboxylation and its facilitated by a hydrogen-bonding interaction\nbetween the substrate’s 3a-OH group and the Op atom. This interaction appears to\ndirect the oxo group into the equatorial position during the formation of Int1a, thereby\navoiding the need for a subsequent axial-to-equatorial transition.\n9. I urge the authors to upload their data to zenodo instead of making their data\navailable upon request. This will facilitate the reuse of the authors’ work.\nResponse:\nWe appreciated the reviewer for giving this important suggestion. In accordance\nwith the recommendation, we have uploaded our data to Zenodo to ensure open\naccessibility and facilitate reuse by the community\n(https://doi.org/10.5281/zenodo.17485598).\n10. The article is well written. Thus, I’d like to commend the authors on this effort.\nResponse:\nWe sincerely thank the reviewer’s encouraging comments on our manuscript. We\nhave also taken into account these constructive suggestions in our revision, which we\nbelieve have helped improve the quality of the work and its alignment with the journal's\nstandards.\n1 Hayashi, H., Asabu, Y., Murao, S. & Arai, M. New Okaramine Congeners,\nOkaramines D, E, and F, from Penicillium simplicissimum ATCC 90288.\nBiosci., Biotechnol., Biochem. 59, 246-250 (1995).\n2 Song, X., Wang, Q., He, Z. & Wang, M. Structure, nuclear magnetic resonance\ndata, biological activity, and synthesis of the okaramine family compounds. J.\nAgric. Food. Chem. 73, 985-999 (2025).\n3 Lai, C.-Y. et al. Biosynthesis of complex indole alkaloids: elucidation of the\nconcise pathway of okaramines. Angew. Chem. Int. Ed. 129, 9606-9610 (2017).\n4 Tamanaha, E., Guan, S., Marks, K. & Saleh, L. Distributive processing by the\niron(II)/α-ketoglutarate-dependent catalytic domains of the TET enzymes is\nconsistent with epigenetic roles for oxidized 5-methylcytosine bases. J. Am.\nChem. Soc. 138, 9345-9348 (2016).\n5 Zhang, Q. et al. Carboxyl formation from methyl via triple hydroxylations by\nXiaM in xiamycin A biosynthesis. Org. Lett. 14, 6142-6145 (2012).\n6 Carro, J. et al. 5-hydroxymethylfurfural conversion by fungal aryl-alcohol\noxidase and unspecific peroxygenase. FEBS. J. 282, 3218-3229 (2015).\n7 Zhu, G. et al. Dissecting the mechanism of the nonheme iron endoperoxidase\nFtmOx1 using substrate analogues. JACS Au 2, 1686-1698 (2022).\n8 Lin, C.-Y. et al. Use of noncanonical tyrosine analogues to probe control of\nradical intermediates during endoperoxide installation by verruculogen\nsynthase (FtmOx1). ACS Catal. 12, 6968-6979 (2022).\n9 Yan, W. et al. RETRACTED ARTICLE: Endoperoxide formation by an α-\nketoglutarate-dependent mononuclear non-haem iron enzyme. Nature 527, 539-\n543 (2015).\n10 Dunham, N. P. et al. Hydrogen donation but not abstraction by a tyrosine (Y68)\nduring endoperoxide installation by verruculogen synthase (FtmOx1). J. Am.\nChem. Soc. 141, 9964-9979 (2019).\n11 Wu, L., Wang, Z., Cen, Y., Wang, B. & Zhou, J. Structural insight into the\ncatalytic mechanism of the endoperoxide synthase FtmOx1. Angew. Chem. Int.\nEd. 61, e202112063 (2022).\n12 Mori, T., Zhai, R., Ushimaru, R., Matsuda, Y. & Abe, I. Molecular insights into\nthe endoperoxide formation by Fe(II)/α-KG-dependent oxygenase NvfI. Nat.\nCommun. 12, 4417-4428 (2021).\n13 Bollinger Jr., J. M. et al. 2-Oxoglutarate-Dependent Oxygenases Ch. 3, 95-122\n(The Royal Society of Chemistry, 2015).\n14 Klein, J. E. M. N. & Knizia, G. cPCET versus HAT: A Direct Theoretical\nMethod for Distinguishing X–H Bond-Activation Mechanisms. Angew. Chem.\nInt. Ed. 57, 11913-11917 (2018).\n15 Vennelakanti, V., Mehmood, R. & Kulik, H. J. Are Vanadium Intermediates\nSuitable Mimics in Non-Heme Iron Enzymes? An Electronic Structure Analysis.\nACS Catal. 12, 5489-5501 (2022).\nReviewer#2:\n1. The authors have responded to reviewers' concerns and the MS is now acceptable for\npublication.\nResponse: We thank the reviewer for their positive assessment and for confirming that\nthe manuscript is now acceptable for publication. We appreciate the reviewer’s time\nand constructive evaluation of our revisions.\nReviewer#3:\n1. The authors have addressed my concerns. -Aditya Nandy (I chose to sign my review).\nResponse: We sincerely thank the reviewer, Dr. Aditya Nandy, for the careful review\nand for acknowledging that all concerns have been satisfactorily addressed. We are\ngrateful for the reviewer’s insightful comments, which have helped to improve the\nquality of the manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have responded to reviewerrs' concerns and the MS is now acceptable for publication.", "reviewer_3": "The authors have addressed my concerns. -Aditya Nandy (I chose to sign my review)."}, "author_response": ""}]

Decision letter