Unlocking enzymatic deracemization of structurally diverse allenes by designed photoenzymes

Domain: neuroscience
Published: 2026-01-27
Editorial outcome: minor_revision

Abstract
Precisethree-dimensionalarrangementsofatomsandfunctionalgroups governmolecularreactivityandbiologicalfunction,makingstereocontrola centralgoalinmodernorganicandmedicinalchemistry.Yetreconcilingbroad substrategeneralitywithhighenantioselectivityremainsacorelimitationof enantioselectivecatalysis,motivatingthepursuitofprivilegedchiralcatalysts. Herewedesignanew-to-naturephotoenzymethatunlocksenzymaticdera- cemizationofstructurallydiverseallenes,anentropicallydisfavoredprocess inaccessibletonaturalbiocatalysts.Structure-guideddesigncreated substrate-specificinteractionnetworks,deliveringhighenantioenrichmentfor axiallychiralalleniccarboxylicacids,esters,andamidesviatripletenergy transferunderaerobicconditions.X-raycrystalstructuresof enzyme–substratecomplexesrevealtailoredchiralpocketsthataccom- modatedistinctsubstratesanddirectenantiocontrol,rationalizingthe breadthandselectivityobserved.Thisworkreconcilesgeneralitywithhigh selectivityonanenzymaticplatform,establishesderacemizationasanevol- vableroutetoaxiallychiralmolecules,andbroadensactivationstrategiesfor stereochemicalediting.

Introduction
Controlling the three-dimensional arrangement of functional atoms andgroupsintargetmoleculesisacentralpursuitinpharmaceutical andsyntheticchemistry1–3.Asymmetriccatalysisisthepreferredsyn- theticstrategyforaccessingchiraltargets,utilizingchiralligandsor catalyststogovernactivityandstereoselectivity4–6.However,perfor- manceisoftenconstrainedbyfactors,suchaselectronicdistribution, steric/confinement effects, covalent/non-covalent interaction networks7–9.Duetothesereasons,substratevariationsoftendisrupt keystericconstraintsorligand-/catalyst-substrateinteractions,com- promising performance in reactions such as asymmetric hydrogenation10–12, epoxidation13,14, cyclopropanation15,16, and synth- esisofaxiallychiralallenes17–24.Theselimitationsfrequentlynecessi- tate tailor-made catalytic systems for specific substrates or entirely new reaction paradigms, as exemplified by asymmetric allene functionalization(Fig.1a)17–19,whereallenesareimportantstructural motifs in natural products, pharmaceuticals, agrochemicals and ligands, or as versatile building blocks in organic synthesis25–27. Recentlydevelopedphotocatalyticderacemizationofalleneslikewise faces similar substrate-dependent constraints20–24. Notably, chiral thioxanthonephotocatalystsarelargelyconfinedtoallenamides20–22, whereas Pd/chiral phosphine systems target 1,3-disubstituted aryl allenes(Fig.1b)23,24.Enzymecatalysisoffershighchemo-,regio-,and stereoselectivity28–30, but enzymatic construction of axially chiral allenesremainsnascent.Sofar,onlyafewstudieshavereportedthe use of naturally occurring allene-acetylene isomerases, lipases, or P450pyr monooxygenase through 1,3-prototropic rearrangement31, kinetic resolution32–34 or desymmetrichydroxylation35 (Fig. 1c), each withverynarrowsubstratescope.Accordingly,designingprivileged chiral catalysts that reconcile the tension between selectivity and generality for the synthesis of structurally diverse, enantioenriched productsremainsanactiveandunmetgoal.

To overcome the inherent substrate limitations of asymmetric catalysisandnaturalenzymes,engineeringorrepurposinganenzyme and scaffold protein has emerged as a promising strategy. These approaches enable tailored biocatalysts with broadened substrate scope to convert substrates that differ markedly from their native counterparts, allowing for responsive adaptation to substrate variations36–39. This flexibility obviates repeated redesign of chiral ligands or catalysts typically required in conventional chemical methodologies. Moreover, enzyme mechanisms can be rewired to generate new-to-nature catalytic activities that transcend natural functional boundariesfor the synthesisofchiralcompounds40–49.In addition,theincorporationofexogenousfunctionalmoieties,suchas photoactivenoncanonicalaminoacids(ncAAs),intoscaffoldproteins can create programmable platforms for unprecedented catalytic functionstoproducechiralcompounds50–58.Despitetheseadvances, theapplicationofnon-naturalbiocatalyticstrategiestochiralallene synthesis, particularly enzymatic deracemization, remains under- explored.Thisislargelyduetothethermodynamicallydisfavoredof deracemization,constraintsimposedbymicroscopicreversibility,and theneedofanexternaldrivingforce20,59–61.Althoughseveralchemical deracemization methods have been established by Bach60,62,63 and others64–67, enzymatic deracemization is currently absent from the catalyticrepertoireofnaturalenzymes.

Toaccessstructurallydiverseaxiallychiralallenesviaenzymatic deracemizationwithinaconfinedenzymeactivesite,weendeavoredto designanengineeredphotoenzymethatharnesseslightasanexternal driving force to enable a new-to-nature synthetic pathway with high enantioenrichment. Guided by this rationale, we incorporated the photoactive noncanonical amino acid 4-benzoylphenylalanine (BpA) into a protein scaffold, generating variants that harvest light and accommodatediverseallenes.Throughstructure-basedrationaldesign, weachieveoxygen-tolerantderacemizationofallenicacids,esters,and amides with high enantioenrichment via triplet-energytransfer (EnT) underaerobicconditions(Fig.1d).Furthermore,X-raycrystalstructures of photoenzyme–substrate complexes reveal tailored chiral pockets that rationalize the observed enantioselectivity across axially chiral allenes. Collectively,this establishes a new-to-nature photoenzymatic deracemizationstrategyforconstructingchiralmolecules.

Results
Photoenzymaticderacemizationofalleniccarboxylicacids
Webeganbytargetingthederacemizationofalleniccarboxylicacids,a transformationthat,toourknowledge,hasnotbeenrealizedbyche- mical or enzymatic methods. After screening our previously con- structed CTB10-based photoenzyme library54, we identified CTB10- W13BpA(photosensitizerBpAinstalledataminoacidW13,PEC1.0)as thepromisingvariantforthemodelsubstratealleniccarboxylicacid rac−1a upon 365nm irradiation (Fig. 2a and Supplementary Fig. 1, Supplementary Tables 1–3), affording (S)-1a with 12% enantiomeric excess(e.e.).Theseresultsestablishproofofconceptforenzymatic deracemizationofallenes.

Toimproveenantioselectivity,wedocked(R)−1aintothecrystal structureofPEC1.0inlinewithourderacemizationmodel.Thedock- ingresultsrevealedalackofkeystabilizinginteractionsintheactive site—mostnotably,hydrogenbondstothesubstratecarboxylateinthe catalyticpocket(Fig.2bandSupplementaryFig.2).Thisdeficitcor- related with weak substrate binding affinity, as demonstrated by kineticanalysis(K =243.56±18.8μM,SupplementaryTable4,Sup- m plementaryFig.3).Guidedbythepropertiesofthecarboxylgroupin the substrate, we pursued Strategy 1: rationally mutating residues proximaltothecarboxylatetohydrogen‑bonddonorswithfavorable geometries (Fig. 2b). This potentially built a new hydrogen bond interactionnetworkbetweenthecarboxylgroupandtheseresidues. Thus,guidedbymoleculardockingsimulations(Fig.2bandSupple- mentaryFig.2),threeproximalresidues(Q72,H111,andM117)were mutatedtoLysorArg,potentiallyintroducinghydrogen-bonddonor groups(ammonium/guanidiniumdonors)withextendedsidechains (Supplementary Table 5). This yielded two variants with enhanced enantioselectivity: PEC1.0-H111K (27% e.e.) and PEC1.0-M117K (50% e.e.). However, the combined mutant PEC1.0‑H111K‑M117K did not further improve selectivity (4% e.e.). We therefore advanced PEC1.0‑M117KasPEC2.0forfurtherengineering.

Next,guidedbythewell-establishedinversecorrelationbetween EnT efficiency and donor-acceptor (D–A) distance68, we sought to reorientresiduesadjacenttothesubstrateorBpAtoshortentheD–A distance and thereby improve activity (Strategies 2 and 3).We first engineeredresiduesnearthesubstratetooptimizethesubstrate–BpA geometry (Strategy 2). Molecular docking analysis of (R)−1a with PEC2.0revealedthatH111ispositioneddirectlyabovethesubstrate’s phenylring(Fig.2candSupplementaryFig.4),suggestingthatdraw- ingH111closertothesubstratecouldimproveBpA-substrateproxi- mity.FurtherstructuralanalysisrevealedthatH111engagestwowater- mediatedhydrogenbondswithN36andT107(Fig.2c),likelybiasing H111awayfromthesubstrate.WethereforemutatedN36andT107to hydrophobic residues (N36L, T107V) to disrupt these interactions whilemaintainingstericbulk(SupplementaryTable6).PEC2.0‑T107V (designatedPEC3.0)exhibitedmarkedlyimprovedenantioselectivity (87%e.e.)(Fig.2a),whilePEC2.0-N36LandthedoublemutantPEC2.0- N36L-T107V showed greatly diminished enantioselectivity (Supple- mentaryTable6).OthermutationsintroducedattheN36andT107 sitesresultedinreducede.e.values,whichfurthercorroboratesthe designprinciple(SupplementaryTable6).Then,wetargetedresidues beneathBpA(L68,M69,andQ72)toreducethesubstrate–BpAdis- tance by introducing bulkier side chains (Strategy 3) (Fig. 2d and Supplementary Fig. 5). All variants, however, displayed severely reduced or abolished enantioselectivity (Supplementary Table 7), indicatingthesepositionsarecrucialformaintainingaproductiveBpA conformation.Tomodulatethedistancemoresubtly,wedisrupteda Q72–N76hydrogenbondidentifiedbydockingthroughsubstituting N76withVal,Leu,orMet(Fig.2dandSupplementaryFig.5).Among these(SupplementaryTable8),PEC3.0-N76V(PEC4.0)deliveredthe higheststereoselectivity(91%e.e.)(Fig.2a),andthenwasselectedfor substrate-scopeevaluation.

Substratescopeinvestigationsofalleniccarboxylicacidsand allenicesters
Next,weevaluatedthecatalyticperformanceofPEC4.0towardapanel of allenic carboxylic acids under optimized conditions (Supplemen- tary Tables 9–12). Exceptional stereoselectivity (up to 99% e.e.) for both the unsubstituted substrate ((S)-1b) and its ortho-substituted derivatives ((S)−1c–(S)-1e) was observed, demonstrating remarkable tolerancetoelectroniceffectsatthisposition(Fig.3andSupplemen- taryFig.6).Ameta-fluorosubstituentlikewisemaintainedhighenan- tioselectivity ((S)−1f, 98% e.e., Supplementary Fig. 6). In contrast, stericallydemandingmeta-substituents(chloro,methyl,ordimethyl) markedlydiminishedcatalyticefficiency((S)-1g–(S)−1i,Supplementary Fig. 6). Steric sensitivity was even more pronounced at the para- position. Replacing fluorine with bulkier halogens (chloro, bromo, iodo)reducedenantioselectivityto62%((S)-1j),19%((S)−1k),and10% e.e.((S)-1l)(SupplementaryFig.6),respectively.

Toelucidatethestructuralbasisofthestericconstraintsimposed bythissubstituent,wedeterminedthecrystalstructureofthePEC4.0- (R)−1ecomplexat1.78Åresolution(PDB:9VMC;Fig.3a,andSupple- mentary Fig. 7, Supplementary Table 13). Crystallographic analysis revealed that, consistent with Strategy 1 (Fig. 2b), M117K forms the designed hydrogen bond with the substrate carboxylate, while Q98 participatesinawater‑mediatedhydrogen‑bondnetworkthatbridges thebenzophenonecarbonylandthecarboxylate(Fig.3a).Asintended in Strategy 2,the interaction network involving residue 107 is abol- ished,andthecentroid-centroiddistancebetweenthephenylringof H111 and that of (R)-1e decreases from 4.5Å in PEC2.0 to 4.2Å in PEC4.0(SupplementaryFig.8).InlinewithStrategy3,theD–Adis- tance decreased from 4.55Å in PEC1.0 to 4.11Å in PEC4.0 (Supple- mentaryFig.9).Theseengineeredinteractionsoptimallypositionthe substratewithinthebindingpocket,therebyenhancingthecatalytic performanceofthePECvariants,asdemonstratedbykineticanalysis (Supplementary Table 4, Supplementary Fig. 3), showing a 23-fold increaseink anda67-foldenhancementink /K . cat cat m

Further structural analysis of residues flanking the substrate phenylringindicatedthatF114andA115,positionednearthemeta-and para-positions (Fig. 3b), likely underlie the limited tolerance to substituents at these positions.Thus,site-directed mutagenesis was performedonF114(toGly,Ala,Met)andA115(toGly)usingthepara- iodoalleniccarboxylicacidrac−1lasamodelsubstratetoenlargethe chiralpocket(SupplementaryTable14),potentiallyimprovingacces- sibilityfordiversearylsubstituents.Twovariantsshowedimproved performance:PEC4.0-A115G(44%e.e.)andPEC4.0-F114M-A115G(98% e.e.; designated PEC5.0). We then evaluated these enzymes against otherstericallyhinderedsubstrates.Experimentalresultsshowedthat both variants accommodated increasing steric demand (Fig. 3c). PEC4.0-A115G delivered excellent enantioselectivity for moderately hinderedsubstrates,includingmeta-chloro((S)-1g),meta-methyl((S) −1h),meta-dimethyl((S)-1i),para-chloro((S)−1j),andpara-methyl((S)- 1m),werederacemizedbyPEC4.0-A115G,rangingfrom77%to95%e.e. PEC5.0outperformedforbulkiersubstituents,suchaspara-bromo((S) −1k)andpara-trifluoromethyl((S)−1n)),achieving84%and93%e.e., respectively. Notably, the enzymatic system also maintained high stereoselectivityacrossdiversesubstratemodifications,includingthe replacementofthebenzeneringwithathiophenemoiety((S)−1o)and thesubstitutionofthetert-butylgroupwithanisopropylgroup((S) −1p). By contrast, replacing tert-butyl with a more rigid cyclohexyl group(S−1q)ledtoamarkeddropinselectivity,consistentwithsteric encumbrance.

Subsequently, catalytic activity was observed for allenic esters whenPEC4.0wasused,albeitwithlowerstereoselectivitycomparedto thecorrespondingalleniccarboxylicacids((S)-2a,20%e.e.;(S)-2b,15% e.e.;(S)-2c,8%e.e.),likelyattributabletodisruptionoftheessential hydrogen-bondingnetworkbytheestergroup.Followingcosolvent optimization(SupplementaryTable15),weengineeredtheenzymeto enhance enzyme–substrate interactions. Guided by the molecular dockingof(R)-2awithPEC4.0(SupplementaryFig.10),site-directed mutagenesiswasconductedatresiduessurroundingtheestergroup and the phenyl ring, specifically I15,Y52, Q72, M86, and K117 (Sup- plementary Table 16). The engineered variant PEC4.0-Q72R-M86K demonstrated significantlyenhancedstereoselectivity,affording(S)- 2awith52%e.e.,alongwithimprovedenantiocontrolforsubstrates2b and2c(47%and48%e.e.,respectively).

Photoenzymaticderacemizationofallenamides
Given the differing activities of the engineered photoenzymes on allenicacidsversusesters,weaskedwhethertheywouldretainactivity toward allenamides. However, experiments showed that the engi- neered PEC variants exhibited a nearly complete loss of catalytic activity toward the model amide substrate rac-3a (Supplementary Table17).Thissuggeststhatdifferencesinpolarity,chargestate,and hydrogen-bonding capacity between amide and carboxylate groups disruptthesubstrate-specificinteractionnetworks,necessitatingtheir redesignforallenamides.Tominimizetheexperimentaleffort,wefirst screened our previously established, compact CTB10-based photo- enzymelibraryratherthanthefullsetofCTB10-W13BpAvariants.Nine variantsfromthisfocusedlibrarydisplayedcatalyticactivitytoward rac-3a (Fig. 4a, Supplementary Table 17). Among them, CTB10- M40BpA (designated PEA1.0) delivered the highest performance, producing (S)-3a with 50% e.e (Fig. 4b and Supplementary Fig. 1, SupplementaryTable18).However,subsequentconventionalprotein engineering,includingstructuralsimulationsofPEA1.0(Supplemen- taryFig.11),moleculardockingofPEA1.0with(R)-3a(Supplementary Fig.11),andalaninescanningofresidueswithin4Åofthesubstrate (SupplementaryTable19),failedtoimprovethestereoselectivity.The likelyreasonforthisisfrominaccuraciesmodelingthenoncanonical aminoacidBpAduringsimulations.

To resolve ambiguities from modeling, we solved the crystal structureofPEA1.0at1.77Åresolution(PDB:9IR1;Fig.4c,andSup- plementary Fig. 12, Supplementary Table 13). The asymmetric unit containsadimer,andBpAadoptsdistinctconformationsinthetwo monomers (Fig. 4c, and Supplementary Fig. 12), which may explain why conventional engineering failed to improve activity. Structural analysis indicates that the conformational dynamics of BpA40 are modulated by proximal residues M86 and H111 (within 4Å), which adopt different rotamers across chains (Fig. 4c and Supplementary Fig. 13). We hypothesized that mutating M86/H111 could restrict BpA40 flexibility and stabilize a defined chiral pocket to enhance enantioselectivity. Guided by considerations of steric effects, π–π interactions,hydrogen bonding,andpolarity,wesubstitutedM86and H111 with Leu, Phe, His, Gln, Asp, Arg, Thr, or Ile (Supplementary Table20).Amongthese,M86Ideliveredhighperformance,achieving 91% e.e. Accordingly, PEA1.0‑M86I (PEA2.0) was advanced for substrate-scopeevaluation.

We nextevaluated PEA2.0 acrossa broadpanel of allenamides underoptimizedconditions(Fig.4d,SupplementaryTables21–23).It showed that para-substituted phenyl substrates bearing methyl or halogengroups(F,Cl,Br,I)generallyaffordedhighstereoselectivity, giving(S)-3bto(S)-3fwithupto96%e.e.Introducingabulkypara- trifluoromethyl group moderately reduced stereocontrol, providing (S)-3g with 78% e.e. Meta-substituted substrates ((S)-3h-(S)-3j) also delivered high enantioselectivity, up to 93% e.e. As expected from stericeffects,bothmono-ortho-methylanddi-ortho-chlorosubstituted substratesslightlyreducedthederacemizationefficiency,yielding86% e.e. ((S)-3k) and 71% e.e. ((S)-3l), respectively. PEA2.0 also accom- modated heteroaromatic substrate, producing the thiophene- modifiedallenamide(S)-3mwith86%e.e.Replacingtert-butylwitha smallerisopropylmaintainedhighstereoselectivity((S)-3n,95%e.e.), whereas a rigid cyclohexyl group severelyreduced stereoselectivity ((S)-3o,2%e.e.).Tomitigatestericencumbrancearoundthecyclohexyl group,residuesI15,V17,andM117weredownsizedbasedondocking analysis (Supplementary Fig. 14, Supplementary Table 24). PEA2.0‑V17A was the most effective variant, increasing the stereo- selectivityof(S)-3oto19%.Additionally,thederacemizationofrac-3b remained effective using PEA2.0 cell lysate, affording (S)-3b with a comparable81%e.e.Notably,scale-upreactionsretainedcomparable performance,delivering(S)-3bwith90%e.e.and85%isolatedyield. Overall,theseresultsdemonstratethebroadsubstrategeneralityand highenantioselectivityoftheengineeredphotoenzymeplatform.

Mechanisticstudyofphotoenzymaticderacemizationofstruc- turallydiverseallenes
Building on the successful deracemization of structurally diverse allenes catalyzed by CTB10-based photoenzymes, we undertook mechanistic studies to elucidate the enzymatic basis of deracemizations.First,theresultedPEC5.0andPEA2.0variantswere co-crystallized or soaked with the respective racemic substrates to investigate whether they could assess enantiomer discrimination in the active site. Co-crystallization of rac−1n with PEC5.0, which was solvedat1.75Åresolution(PDB:9WZW;Fig.5a,SupplementaryFig.15, and Supplementary Table 13), featuring an octameric complex dis- playinga7:1enantiomericratioof(R)-to(S)-isomers(Supplementary Fig.15).Interestingly,the(R)-enantiomerevenpopulatedthreedistinct conformationsina4:2:1ratiowithinthechiralcavity,withD–Adis- tances of 3.90Å, 4.97Å, and 5.14Å, respectively (Supplementary Fig. 15), whereas the (S)-substrate adopted a single conformation, characterized by a significantly longer D–A distanceof 5.80Å (Sup- plementaryFig.15).Theseresultsindicatethatthishighoccupancyof (R)-configuration and their shorter D-A distances ensure high stereoselectivity throughout the reaction. Moreover, thisconforma- tional diversity also highlights the exceptional versatility of the enzyme’schiralcavityinaccommodatingmultiplesubstrategeome- tries,surpassingtheadaptabilitytypicallyobservedforconventional chemicalchiralcatalysts.

In crystals of PEA2.0 soaked with rac-3n, which was solved at 2.24Åresolution,onlythe(R)-3nenantiomerwasobserved,withno electron density for the (S)-enantiomer (PDB: 9W6Z; Fig. 5b, and SupplementaryFig.16,SupplementaryTable13).Structuralanalysisof PEA2.0-(R)-3nrevealedenhancedinteractionsbetweenBpAandsur- roundingresiduescomparedtoPEA1.0(SupplementaryFig.17),lead- ingtoimprovedconformationalstability.Furthermore,thesubstrate- binding network in PEA2.0 is significantly strengthened relative to PEA1.0.ResidueN36stabilizestheallenamidesubstratethroughdual hydrogen-bondinginteractionswithitsamidemoiety,whileF84and I108formπ-alkylinteractionswiththeisopropylgroupandphenylring of the substrate, respectively (Fig. 5b, and Supplementary Fig. 17). Theseinteractionscollectivelycontributetoamorerigidchiralpocket andfavorabledonor–acceptorgeometry.

RelativetoPEC4.0andPEA1.0(SupplementaryFigs.18and19), structure-guided remodeling of the chiral binding pockets yielded well-definedsubstrate–enzymeinteractionnetworksthatpositionthe allenic double bonds of (R)−1n and (R)-3n at 3.9Å in pronounced PEC5.0and3.6ÅinPEA2.0fromtheBpAcarbonyl,respectively.This favorabledonor–acceptorproximityenablesefficientEnT(Figs.5a,b), andsupportsthehighphotochemicalquantumyield(Φ=0.38;Sup- plementaryFig.20).Together,thesecrystallographicdataunderscore apronouncedstereochemicalpreferencefor(R)-configuredsubstrates and provide a structural rationale for efficient deracemization via optimizedexcitationgeometryanddonor–acceptoralignment.

Next, to further prove the crystallographic results and probe whetherbothenantiomersengagethecatalyticcycle,enantiopureR- andS-substratesweresubjectedtophotoenzymecatalysis.R−1nandR- 3nwereefficientlyconvertedtotheircorrespondingS-isomers,giving 89%and98%yield,respectively,whereasnodetectableformationofR- configuredproductwasobservedwhenS−1norS-3nservedassub- strates(Fig.5c,andSupplementaryTables25and26).Theseoutcomes revealaclearreactivitybias:R-enantiomersreactpreferentially,while S-enantiomersare disfavored, consistent with the proposed derace- mizationmechanism20–24,54.Moreover,theseengineeredbindingcav- ities in PEC5.0 and PEA2.0 optimized substrates’ orientation, significantlyenhancingcatalyticefficiency,asevidencedby80.5-fold and 9.5-fold increases in k /K (Fig. 5c, and Supplementary cat m Fig.21and22),respectively.

Integratingthecrystallographicandkineticdata,wethenpropose thatCTB10-basedphotoenzymesachieveenantiomericenrichmentof axiallychiralallenesviainitialenantioselectivediscriminationbetween (R)-and(S)-substrates(Fig.5d).Followingthischiraldiscrimination, selectiveEnToccursfromphotoexcitedBpAtothe(R)-substrate,as supportedbyquenchingwithtripletquenchers2,5-dimethyl-2,4-hex- adieneandperylene(SupplementaryTable27).Uponenergyabsorp- tion,the(R)-substrateundergoesdouble-bondcleavagetoforma1,2- diradical intermediate20–22, which then dissociates from the photoenzyme promoted by the excess energy that results from EnT20–22. The free intermediate recombines insolution to a racemic mixtureof(R)-and(S)-enantiomers.The(R)-enantiomerisfunneled backintothecatalyticcycleandcontinuouslyconsumed,whilethe(S)- enantiomer accumulates, driving progressive enrichment of the S- configuration.Throughthisstereoselectivecycle,thedesignedpho- toenzymes deracemize structurally diverse allenes with high effi- ciency.Overall,structure-basedrationaldesignallowstheengineering of substrate-specific interaction networks,enabling efficient derace- mizationofstructurallydiverseallenes.

Methods
Generalinformation
Allchemicalreagentswereobtainedfromcommerciallysuppliersand used without additional purification. The pEVOL-pBpF plasmid was purchasedfromHunanFenghuiBiotechnology.(S)-2-amino-3-(4-ben- zoylphenyl)propanoic acid was sourced from Bidepharm; MultiF SeamlessAssemblyMixfromABclonalBiotechnology;andPrimeSTAR MaxDNAPolymerasefromTakara.Oligonucleotidesweresynthesized by Azenta Life Sciences and Yi Xin Life Technologies, while DNA sequencing was performed by Azenta Life Sciences. Photochemical reactions were performed in a custom-designed photoenzyme screening platform54 and a WATTCAS photoreactor (WP-TEC −1020LC). Reaction products were analyzed and quantified using reverse-phaseHPLCwithaWaters2695separationmodule.e.e.values were determined by chiral HPLC analysis on a Waters 2996 system equippedwithachiralstationaryphase,employinganisopropanol/n- hexanemobilephasesystem.Thesynthesisofallenesubstrateswas performed under ambient conditions unless otherwise noted, with reaction progress monitored by thin-layer chromatography (TLC). Structural characterization was performed through comprehensive spectroscopic analysis. Nuclear magnetic resonance (NMR) spectra wererecordedatambienttemperatureusingaBrukerspectrometer operating at 400MHz. High-resolution mass spectrometry (HRMS) data were obtained using a Waters Maldi Synapt UPLC-MS system equippedwithanelectrosprayionization(ESI)source.

ConstructionofCTB10-basedphotoenzymeexpression plasmids54
TheoriginalCTB10gene(GenBank:MZ323244.1)wassubclonedusing seamlesscloningandassemblyintoapET-21b(+)vectorcontaininga C-terminal His6-tag. The Trp13BpA and Met40BpA mutations were introduced by replacing the Trp13 and Met40 codons with a TAG codoninPCRandseamlesscloningtoyieldpET-21b(+)_PEC1.0orpET- 21b(+)_PEA1.0. Subsequent point mutations in PEC1.0, PEA1.0, and theirderivativevariantswereconstructedusingthesameprotocol.

Proteinexpression50,51
ForexpressionofPEC1.0orPEA1.0andtheirvariants,bothpET-21b(+) constructandpEVOL-pBpFplasmidwereco-transformedintochemi- callycompetentE.coliBL21(DE3)cellsfollowedwithLBagarplates cultivation (100mgL-1 ampicillin and 34mgL-1 chloramphenicol). A single colony was inoculated into 5mLofLBmediumwiththesame antibioticsandincubatedat37°Cand200rpmfor12h.Theovernight culturewasthentransferredinto500mLof2×YTmediumcontaining theidenticalantibiotics.Cellsweregrownat37°Cand115r.p.m.until theopticaldensityat600nm(O )reached0.6.Proteinexpression D600 was induced by adding 0.1mM IPTG, 0.02% L-arabinose, and 1mM BpA, followed by incubation at 18°C and 115rpm for 16h. Subse- quently, cell pellets were collected by centrifugation (4,000×g for10min).

Proteinpurification
Cell pellets were resuspended in lysis buffer (50mM Tris, 500mM NaCl,pH8.0,10mMimidazole,and10%(v/v)glycerol)andlysedusing a high-pressure homogenizer at 4°C. The lysates were clarified by centrifugation(40,000×gfor30min),andtheresultingsupernatants were subjected to affinity chromatography using Ni-NTA agarose (Cytiva).Targetproteinswereelutedwithelutionbuffer(50mMTris, 300mM NaCl, pH 8.0, 250mM imidazole, and 10% (v/v) glycerol). Thenthebufferexchangeofelutedproteinswasperformedbya5mL HiTrapdesaltingcolumn(Cytiva)withSECbuffer(25mMTris,150mM NaCl, pH 8.0) for use in photocatalytic experiments. Proteins were aliquotedandflash-frozeninliquidnitrogenthenstoredat-80°C.For crystallization, the eluted proteins were further purified by gel- filtration (Hiload 16/600 Superdex 200pg column from GE Health- care)withSECbufferandhomogeneousproteinswereconcentrated toappropriateconcentrationforcrystallization.

Generalprocedureforthescreeningofphotoenzymemutants
A reaction mixture (200μL) containing the purified photoenzyme (10mol%relativetosubstrate)andracemicsubstrate(0.2mM)inPBS buffer(pH7.4)withDMSO(5%–10%v/v)ina96-wellplatewasirra- diatedusinga365nmLEDfor15–30min.Themixturewasagitated continuouslyat200rpmonanorbitalshakerwithinanicebath.Fol- lowing irradiation, the mixture was extracted three times with dichloromethane (200μL per extraction). The combined organic extractswereconcentrated,andtheresultingresiduewasdissolvedin 250μLisopropanolfore.e.determination.Chiralnormal-phaseHPLC analysisofcompounds1aand2awasperformedusinganAD-Hchiral columnwithisopropanol/hexane(2%v/v)asthemobilephaseataflow rateof1mLmin-1,UVdetectionat215nm,andacolumntemperature of25oC.Forcompound3a,anOD-Hcolumnwasemployedwithiso- propanol/hexane (4% v/v) as the mobile phase, with all other para- metersremainingconstant.

Generalprocedureforthederacemizationofallenescatalyzed byCT10-basedphotoenyzmes
DeracemizationreactionsweresetupbymixingpurifiedCTB10-based photoenzyme(10mol%relativetosubstrate)withracemicsubstrate (0.2mM) inPBS (pH 7.4) containing 5%–10% (v/v) DMSO,to a total volume of 200μL in glass vials. Photochemical reactions were per- formedinaWATTCASWP-TEC-1020LCphotoreactorat10°Cunder 365–370nm irradiation (276mWcm-2) for 15–30min. For yield and determination,reactionswerequenchedwith200μLacetonitrileand analyzed by reverse-phase HPLC. For enantiomeric excess, reaction mixtures were extracted with dichloromethane (3 × 200μL); the combined organic layers were concentrated, redissolved in 250μL isopropanol,andanalyzedbynormal-phasechiralHPLCusingachiral stationaryphaseandanisopropanol/n-hexanemobilephase(Supple- mentaryTables28and29).

Generalprocedureforderacemizationusingcelllysate
PEC4.0celllysatewaspreparedbyresuspendingfrozencellsinPBS buffer (50mL, pH 7.4, OD =46.3) and lysing them using a high- 600 pressure homogenizer. The lysate was centrifuged (40,000×g, 30min,4°C),andtheresultingsupernatantwascollectedasthecrude enzyme solution for photocatalysis. The deracemization of rac-3b (0.2mM,200μL)wasconductedunderconditionsidenticaltothose employedforthepurifiedenzyme,withtheexceptionthat40μLof lysate was substituted for the purified enzyme. All other reaction parametersandworkupproceduresremainedunchanged.

Generalprocedureforthescale-upderacemizationreaction
Thescale-upreactionwasconductedina250mLround-bottomflask containingrac-3b(10mg),purifiedPEC4.0(10mol%),andPBSbuffer (215mL)withDMSO(5%v/v).Themixturewasirradiatedfor30min usinga365nmLEDlampequippedwithcoolingfansinanicebath. After irradiation, the solution was extracted with ethyl acetate (3 × 150mL). The combined organic layers were concentrated under reducedpressureandpurifiedbycolumnchromatographytoyieldthe targetcompound.Thee.e.wasdeterminedbyHPLCusingtheestab- lishedanalyticalmethod(SupplementaryTable28).

Datacollectionandanalysis
X-ray diffraction data for PEC4.0-(R)-1e, PEC5.0-1n, PEA1.0, and PEA2.0-(R)-3n were collectedattheShanghaiSynchrotronRadiation Facility(SSRF)usingbeamlinesBL10U2(forPEC4.0-(R)-1eandPEA2.0- (R)-3n)andBL19U1(forPEC5.0-1nandPEA1.0).Dataprocessingwas performedusingautoPXforPEC4.0-(R)-1e,HKL-3000forPEC5.0-1n, HKL-2000forPEA1.0,andXDSforPEA2.0-(R)-3n.Molecularreplace- ment was performed using the Se-CTB10 structure (PDB 9IKU) as searchmodel.AllstructureswererefinedwithMolrep-autoMRfrom theCCP4suite,Coot,andphenix.refine.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study presents a protein engineering approach aimed at the deracemization of axially chiral allenes through the\ndevelopment of designed photoenzymes. By integrating the photosensitive non-canonical amino acid BpA into a CTB10\nprotein scaffold, the authors successfully created tunable catalytic sites that facilitate enantioselective triplet energy transfer\nunder aerobic conditions. The authors further enhanced catalytic performance and stereocontrol through iterative structure-\nguided mutagenesis, which introduced hydrogen-bonding interactions, reoriented substrate-adjacent residues to optimize\ndonor-acceptor distances, and fine-tuned the BpA microenvironment. Crystallographic analysis revealed selective binding of\nthe (R)-enantiomer within the chiral pocket and illuminated the mechanism of energy transfer leading to double-bond\nisomerization via a biradical intermediate. This system demonstrated versatility, accommodating a variety of allenic\ncarboxylic acids, esters, and amides and achieving enantiomeric excess values of up to 98% for specific substrates. This\nwork illustrates the potential of integrating rational design, genetic code expansion, and photochemistry to generate\nnovel enzymatic functions for deracemization synthesis, providing a programmable and mild pathway to obtain chiral allenes.\nThis is an important advance in energy transfer photoenzyme catalysis. I support the publication of this study after the\nauthors address the following concerns:\n1. Benzophenone was employed as the photosensitizer for the activation of allenes through energy transfer at 365 nm. It is\nrecommended that the UV-Vis spectra of model substrates be examined to determine if there is any background excitation of\nsubstrates that could potentially compromise enantioselectivity.\n2. The mechanistic depiction in Figure 5d illustrates how enantiomeric enrichment was achieved through the\nenantioselective discrimination between (R)- and (S)-substrates. However, the authors state that following energy\nabsorption, the (R)-substrate undergoes double-bond cleavage to generate a 1,3-biradical intermediate, which subsequently\ndissociates from the photoenzyme. This free intermediate then recombines in solution. Given the short lifetime of triplet\ndiradicals, this scenario may be unlikely. The authors should clarify this situation in relation to the time scale of allene\ndiradicals and diffusion kinetics.\n3. The effects of triplet quenchers remain inconclusive (see Table S24). There is a recent study that explores the impact of\nvarious triplet quenchers on the enantioselectivity of photoenzyme catalysis (Nat. Catal. 2025, 10.1038/s41929-025-01433-\n3). The authors are encouraged to reference this study to perform extra quenching experiments to support their mechanistic\nproposal.\n4. The use of purified photoenzymes is noted. What would be the outcome if cell lysates containing the overexpressed\nphotoenzyme were utilized?\n5. It is impressive that the evolved photoenzymes achieved >90% enantiomeric excess within 30 minutes, indicating\nexcellent enantioinduction and high enzyme kinetics; however, 10 mol% of purified enzyme was used. I recommend\nreducing the enzyme loading and calculating the turnover number of the enzyme.\n6. The field of photobiocatalysis is rapidly evolving. Recent reviews on this topic should be included to provide an updated\noverview of this area: Angew. Chem. Int. Ed. 2025, e202419262; Chem. Soc. Rev., 2025, 54, 5157. Additionally, recent\nnotable studies on artificial energy transfer photoenzymes should be cited: Nat. Chem. 2025, 17, 1083; Nat. Catal. 2025, 8,\n822; Nat. Catal. 2025, 10.1038/s41929-025-01433-3; J. Am. Chem. Soc. 2024, 146, 19030.", "reviewer_2": "In this MS, the authors reported an artificial photoenzyme to enable the deracemization of diverse allenes, which is a\nthermodynamically challenging transformation beyond the scope of natural enzymes. Structure-based design facilitated\nprecise substrate interactions, achieving exceptional enantiocontrol in the synthesis of chiral allenic derivatives through\noxygen-tolerant triplet energy transfer catalysis. This study merges broad substrate generality with enzymatic precision for\nallene deracemization, demonstrates evolvable biocatalytic pathways to axial chirality, and expands the toolbox for\nstereochemical reprogramming via photoenzymatic activation. Overall, the manuscript is very well-written, demonstrating a\nhigh level of clarity and precision in its arguments and presentation. Hence, I recommend the publication of this manuscript\nin Nature Communications after minor revisions.\nSome minor points to be addressed: 1. In the Figure. 2c and Table S6, just by adding the single mutation T107V, the ee\nvalue increased from 50 to 87. Have other mutations at position 107 been performed? How effective were they?\n2. In Supplementary Information “12 General procedure for screening of photoenzyme mutants”, the author mentioned that\n“The reaction system, with a total volume of 200 μL, was prepared in 96-well plates, containing 0.2 mM racemic substrate,\npurified photoenzyme (10 mol%)”. For a reaction in a 96-well plate with 0.2 mM substrate, how was 10% pure enzyme\nextracted?\n3. It is strongly recommended to include scale-up examples to illustrate the isolated yields. Isolated yields and the\ncorresponding 1H NMR spectra needed to be attached.\n4. In Fig.5 and the main text, it should be “1,2-diradical intermediate” rather than “1,3-biradical intermediate” or “1,3-diradical\nintermediate”.\n5. Figure 4d, DMSO:PBS = 1:19 rather than 19:1.\n6. Reviews on photobiocatalysis, like Chin. J. Chem. 2024, 42, 3553-3558, and recent biocatalytic allenes synthesis\n(10.1021/jacs.5c08146) should be cited.\nOther comments:\n1. The HPLC traces of sustrate1c and 1f: the signal-to-noise ratio is too low. Please increase the concentration of the\nreaction substrate and repeat the test.\n2. On line 368, the citation page number needs to be corrected.\n3. On lines 375, 390, and 406, the journal volume number should be bolded.\n4. On line 447, it should be “Ed.” rather than “Edit.”.\n5. Fig. S30, the peaks in 13C NMR spectrum shouldn’t be integrated.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_4": "This manuscript describes the rational engineering of a photoenzyme to produce axially chiral allenic acids and amides. The\nthree design strategies are logically conceived and yield promising results. The crystal structures are well refined, and the\nelectron density for the bound ligands is clearly defined. I have several suggestions for minor revision:\n1. Lines 21 and 319: The authors state that the enzyme also affords high enantioenrichment for axially chiral allenic carboxylic esters. However, the reported e.e. values for these ester substrates are only 16–20%. The authors should\ntherefore refrain from making this claim.\n2. Electron density maps: All maps shown are 2Fo–Fc. Are these omit maps? If not, the authors should provide composite\nomit 2Fo–Fc maps to confirm ligand placement.\n3. PDB codes: Please include the PDB accession codes for all deposited structures in the main text.\n4. Line 168: Strategy 2 is intended to bring H111 closer to the substrate, but the authors did not demonstrate this with the\ncrystal structure of PEC4.0-(R)-1e. This should be clarified or supported with structural evidence.\n5. Line 189: The authors suggest that the ester group may disrupt the hydrogen-bonding network. Should they also examine\nwhether the reduced stereoselectivity could arise from steric clashes introduced by the ethyl group?\n6. Line 229: Please elaborate on how the M86I mutation restricts the flexibility of BpA40. Additionally, could altered\nsubstrate–enzyme interactions also contribute to enhanced enantioselectivity?\n7. Lines 253–257: The authors should clarify whether the analysis presented here is consistent with the high e.e. value of\n93%.\n8. Supporting information line 270-280: the concentrations of the ligands as well as the incubation or soaking time are not\nprovided. Please include these details to ensure reproducibility."}, "author_response": "Response: We sincerely thank the reviewer for the positive evaluation of our work. We greatly appreciate their support for publication. We have carefully considered all comments and suggestions and have addressed each point thoroughly in the revised manuscript to further improve its clarity and scientific impact.\nQ1. Benzophenone was employed as the photosensitizer for the activation of allenes through energy transfer at 365 nm. It is recommended that the UV-Vis spectra of model substrates be examined to determine if there is any background excitation of substrates that could potentially compromise enantioselectivity.\nResponse: We thank the reviewer for this insightful suggestion. As response, we measured the UV-Vis absorption spectra of representative substrates rac-1a and rac-3a (Fig. R1). Both substrates exhibit absorption exclusively below 300 nm, indicating that direct photoexcitation of the substrates does not occur under the reaction conditions. The corresponding spectra have been provided in the the Supporting Information.\nQ2. The mechanistic depiction in Figure 5d illustrates how enantiomeric enrichment was achieved through the enantioselective discrimination between (R)- and (S)-substrates. However, the authors state that following energy absorption, the (R)-substrate undergoes double-bond cleavage to generate a 1,3-biradical intermediate, which subsequently dissociates from the photoenzyme. This free intermediate then recombines in solution. Given the short lifetime of triplet diradicals, this scenario may be unlikely. The authors should clarify this situation in relation to the time scale of allene diradicals and diffusion kinetics.\nResponse: We appreciate the reviewer's insightful comments concerning the mechanistic rationale for diradical dissociation.\nAccording to the literature on diradical reactions (Angew. Chem. Int. Ed. 2020, 59, 21640-21647; Chem. Eur. J. 2002, 8, 2464-2475), the 1,2-diradical intermediate would be expected to yield exclusively the R-isomer when recombined within a chiral photoenzyme environment. In contrast, recombination in an achiral environment can lead to the formation of racemic mixtures, thereby facilitating enrichment of the S-isomer, which is consistent with the experimental outcomes. Furthermore, previous studies have reported the dissociation of this diradical species from a chiral environment. Bach et al. reported its decay to a racemic mixture (1:1 ratio of R- and S-isomers) (Nature 2018, 564, 240–243). Subsequent density functional theory calculations by the same group further indicate that the diradical possesses a symmetric, achiral structure (J. Am. Chem. Soc. 2021, 143, 11209–11217). Therefore, they suggest that the intermediate exhibits a sufficient lifetime to allow dissociation from the catalytic environment and that excess energy derived from energy transfer drives rapid dissociation of the complex.\nUpon energy absorption, the (R)-substrate undergoes double-bond cleavage to form a 1,2-diradical intermediate, which then dissociates from the photoenzyme promoted by the excess energy that results from EnT.\nQ3. The effects of triplet quenchers remain inconclusive (see Table S24). There is a recent study that explores the impact of various triplet quenchers on the enantioselectivity of photoenzyme catalysis (Nat. Catal. 2025, 10.1038/s41929-025-01433-3). The authors are encouraged to reference this study to perform extra quenching experiments to support their mechanistic proposal.\nResponse: We thank the reviewer for emphasizing the critical role of triplet quenchers in elucidating the reaction mechanism. In response, we performed supplementary experiments to investigate the effect of perylene as a triplet quencher on reaction activity. As detailed in the Supporting Information, the addition of perylene significantly reduced the e.e. in the deracemization of rac-3n (Table R1). This quenching effect provides further support for the proposal that the photoenzymatic deracemization proceeds via an energy-transfer mechanism. The relevant experimental data and descriptions have been incorporated into the main text and the Supporting Information accordingly.\nTable R1. Effect of triplet-state quencher perylene on the deracemization of rac-3n.\nEntry Triplet quencher e.e. (%)\n1 none 98\n2 3 equiv of perylene 88\n3 5 equiv of perylene 83\n4 10 equiv of perylene 65\n5 50 equiv of perylene 29\nQ4. The use of purified photoenzymes is noted. What would be the outcome if cell lysates containing the overexpressed photoenzyme were utilized?\nResponse: We thank the reviewer for raising this important methodological consideration. In response to the suggestion, we performed the deracemization of rac-3b using a lysate of PEA2.0，\n3\nwhich afforded (S)-3b with 81% e.e. These results have been incorporated into the main text, and experimental details are provided in the Supporting Information. The description in the main text was as follows:\nAdditionally, the deracemization of rac-3b remained effective using PEA2.0 cell lysate, affording (S)-3b with a comparable 81% e.e. Notably, scale-up reactions retained comparable performance, delivering (S)-3b with 90% e.e. and 85% isolated yield.\nQ5. It is impressive that the evolved photoenzymes achieved >90% enantiomeric excess within 30 minutes, indicating excellent enantioinduction and high enzyme kinetics; however, 10 mol% of purified enzyme was used. I recommend reducing the enzyme loading and calculating the turnover number of the enzyme.\nResponse: We thank the reviewer for the valuable suggestion regarding enzyme loading optimization and turnover efficiency evaluation.\nAs you suggested, we performed catalyst loading studies for the PEA4.0-catalyzed deracemization of rac-1a across a range of 2-14 mol% (Table R2). The results show that the e.e. increased progressively from 24% at 2 mol% to 91% at 10 mol% loading. Further increasing the loading to 12 or 14 mol% did not improve the e.e. Turnover number (TON) analysis indicated that TON values were higher at lower catalyst loadings, but decreased as the catalyst loading increased. Therefore, considering that 10 mol% offers a balance between reaction performance and catalyst efficiency, this loading was selected as the optimal catalyst amount for subsequent studies. These data have been incorporated into the Supporting Information.\nTable R2. Screening of catalyst loading for PEC4.0-catalyzed deracemization of rac-1a.\nEntry Catalyst loading (mol%) e.e. (%) TON\n1 2 41 10\n2 4 62 8\n3 6 74 6\n4 8 84 5\n5 10 91 5\n4\n6 12 90 4\n7 14 90 3\nQ6. The field of photobiocatalysis is rapidly evolving. Recent reviews on this topic should be included to provide an updated overview of this area: Angew. Chem. Int. Ed. 2025, e202419262; Chem. Soc. Rev., 2025, 54, 5157. Additionally, recent notable studies on artificial energy transfer photoenzymes should be cited: Nat. Chem. 2025, 17, 1083; Nat. Catal. 2025, 8, 822; Nat. Catal. 2025, 10.1038/s41929-025-01433-3; J. Am. Chem. Soc. 2024, 146, 19030.\nResponse: We sincerely appreciate the reviewer's valuable guidance on updating the literature context for this rapidly evolving field. We have incorporated all suggested references into the revised manuscript.\nTo Reviewer #2\nIn this MS, the authors reported an artificial photoenzyme to enable the deracemization of diverse allenes, which is a thermodynamically challenging transformation beyond the scope of natural enzymes. Structure-based design facilitated precise substrate interactions, achieving exceptional enantiocontrol in the synthesis of chiral allenic derivatives through oxygen-tolerant triplet energy transfer catalysis. This study merges broad substrate generality with enzymatic precision for allene deracemization, demonstrates evolvable biocatalytic pathways to axial chirality, and expands the toolbox for stereochemical reprogramming via photoenzymatic activation. Overall, the manuscript is very well-written, demonstrating a high level of clarity and precision in its arguments and presentation. Hence, I recommend the publication of this manuscript in Nature Communications after minor revisions.\nResponse: We sincerely thank the reviewer for the insightful evaluation and positive endorsement of our work. We are grateful for their recognition of the study's significance in expanding stereochemical reprogramming tools and their recommendation for publication in Nature Communications. As your comments, we have carefully addressed all points to further enhance the manuscript.\nQ7. In the Figure. 2c and Table S6, just by adding the single mutation T107V, the ee value increased from 50 to 87. Have other mutations at position 107 been performed? How effective were they?\nIn response, we performed additional mutagenesis at residue 107 (Table R3). Activity assays revealed that all mutations resulted in reduced e.e. compared to PEC2.0, confirming that the improvement in e.e. is highly specific to the valine substitution at this position. These results have been incorporated into the main text and the Supplementary Information.\nTable R3. Effect of the T107 mutation on the photoenzymatic deracemization of rac-1a.\nEntry CTB10-based photoenzymes e.e. (%)\nQ8. In Supplementary Information “12 General procedure for screening of photoenzyme mutants”, the author mentioned that “The reaction system, with a total volume of 200 μL, was prepared in 96-well plates, containing 0.2 mM racemic substrate, purified photoenzyme (10 mol%)”. For a reaction in a 96-well plate with 0.2 mM substrate, how was 10% pure enzyme extracted?\nResponse: Thank the reviewer for raising this important clarification. We apologize for any ambiguity in our original description. The corrected procedure is as follows:\nA reaction mixture (200 μL) containing purified photoenzyme (10 mol%) and racemic substrate (0.2 mM) in PBS buffer (pH 7.4) with (5%–10% v/v) DMSO in a 96-well plate was illuminated using a 365 nm LED for 15–30 min. The mixture was continuously agitated at 200 rpm on an orbital shaker within an ice bath.\nResponse: We thank the reviewer for this valuable suggestion. As requested, a scale-up reaction was carried out using 10 mg of rac-3b with purified PEA2.0. Purification by column chromatography afforded (S)-3b in 85% yield with 90% e.e. The isolated products were characterized by 1H NMR. These results have been included in the revised manuscript and the Supplementary Information.\nQ10. In Fig.5 and the main text, it should be “1,2-diradical intermediate” rather than “1,3-biradical intermediate” or “1,3-diradical intermediate”.\nResponse: We sincerely thank the reviewer for identifying this important terminological inaccuracy. The text and Figure 5 have been revised to consistently use \"1,2-diradical intermediate\" throughout the manuscript.\nQ11. Figure 4d, DMSO:PBS = 1:19 rather than 19:1.\nResponse: We appreciate the reviewer's careful reading. The ratio is now correctly shown as DMSO:PBS = 1:19.\nQ12. Reviews on photobiocatalysis, like Chin. J. Chem. 2024, 42, 3553-3558, and recent biocatalytic allenes synthesis (10.1021/jacs.5c08146) should be cited.\nResponse: We thank the reviewer for suggesting these important references. Both recommended works have now been cited in the Introduction section.\nQ13. The HPLC traces of sustrate1c and 1f: the signal-to-noise ratio is too low. Please increase the concentration of the reaction substrate and repeat the test.\nResponse: We thank the reviewer for identifying the issue with the signal-to-noise ratio in the HPLC traces for substrates 1c and 1f. As suggested, we repeated the HPLC analysis using increased substrate concentrations. The new HPLC traces have been included in the Supplementary Information.\nQ14. On line 368, the citation page number needs to be corrected.\nQ15. On lines 375, 390, and 406, the journal volume number should be bolded.\nResponse: We appreciate the reviewer’s attention to detail. The requested bolding of volume numbers at lines 375, 390, and 406 has been implemented in the revised version.\nQ16. On line 447, it should be “Ed.” rather than “Edit.”.\nResponse: We appreciate the reviewer’s meticulous reading. \"Edit.\" on line 447 has been corrected to \"Ed.\" in the revised manuscript.\nQ17. Fig. S30, the peaks in 13C NMR spectrum shouldn’t be integrated.\nResponse: We thank the reviewer for highlighting this issue. The 13C NMR spectrum in Figure S30 has been appropriately revised by removing the peak integrations.\nTo Reviewer #3\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We sincerely thank both reviewers for their valuable comments. We have carefully noted all feedback and have thoroughly addressed every point raised in the revision.\nTo Reviewer #4\nThis manuscript describes the rational engineering of a photoenzyme to produce axially chiral allenic acids and amides. The three design strategies are logically conceived and yield promising results. The crystal structures are well refined, and the electron density for the bound ligands is clearly defined. I have several suggestions for minor revision:\nResponse: We appreciate the reviewer for their positive assessment of our rational enzyme design strategies and structural analyses. All your comments have been carefully addressed in the revised manuscript.\nResponse: We sincerely thank the reviewer for this valuable observation. We acknowledge that the initial e.e. values of 16-20% do not justify the description of \"high enantioenrichment.\" To address this limitation, a protein engineering strategy was implemented to enhance enzyme–substrate interactions, following optimization of the reaction conditions. The engineered mutant PEC4.0-Q72R-M86K exhibited substantially improved stereoselectivity toward allenic esters, affording (S)-2a, (S)-2b, and (S)-2c with e.e. values of 52%, 47%, and 48%, respectively. These results and relevant experimental details have been included in the main text and the Supplementary Information. The description in the main text was as follows:\nSubsequently, catalytic activity was observed for allenic esters when PEC4.0 was used, albeit with lower stereoselectivity compared to the corresponding allenic carboxylic acids ((S)-2a, 20% e.e.; (S)-2b, 15% e.e.; (S)-2c, 8% e.e.), likely attributable to disruption of the essential hydrogen-bonding network by the ester group. Following cosolvent optimization (Supplementary Table 15), we engineered the enzyme to enhance enzyme–substrate interactions. Guided by the molecular docking of (R)-2a with PEC4.0 (Supplementary Fig. 9), site-directed mutagenesis was conducted at residues surrounding the ester group and the phenyl ring, specifically I15, Y52, Q72, M86, and K117 (Supplementary Table 16). The engineered variant PEC4.0-Q72R-M86K demonstrated significantly enhanced stereoselectivity, affording (S)-2a with 52% e.e., along with improved enantiocontrol for substrates 2b and 2c (47% and 48% e.e., respectively).\nQ19. Electron density maps: All maps shown are 2Fo–Fc. Are these omit maps? If not, the authors should provide composite omit 2Fo–Fc maps to confirm ligand placement.\nResponse: We thank the reviewer for this important clarification. The original 2Fo–Fc maps were not omit maps. As suggested, composite omit 2Fo–Fc maps have now been included in the Supplementary Information (Fig. R2).\nQ20. PDB codes: Please include the PDB accession codes for all deposited structures in the main text.\nResponse: We thank the reviewer for this suggestion. The PDB accession codes for all deposited structures have now been included in the main text as suggested.\nQ21. Line 168: Strategy 2 is intended to bring H111 closer to the substrate, but the authors did not demonstrate this with the crystal structure of PEC4.0-(R)-1e. This should be clarified or supported with structural evidence.\nResponse: We thank the reviewer for highlighting this point. To directly demonstrate that Strategy 2 brings H111 closer to the substrate, we conducted molecular docking of (R)-1e with the modeled structure of PEC2.0. The distance between the centroids of the benzene ring of H111 and that of (R)-1e decreased from 4.5 Å in PEC2.0 to 4.2 Å in the PEC4.0-(R)-1e complex (Fig. R3). This reduction in distance further supports the rationale of Strategy 2. The corresponding results have been incorporated into the main text and the Supplementary Information.\nQ22. Line 189: The authors suggest that the ester group may disrupt the hydrogen-bonding network. Should they also examine whether the reduced stereoselectivity could arise from steric clashes introduced by the ethyl group?\nResponse: We thank the reviewer for this insightful suggestion. Following the comment, we performed site-directed mutagenesis based on the molecular docking model of (R)-2a with PEC4.0. Specifically, two residues near the ethyl group, I15 and V17, were mutated to alanine to examine potential steric effects on enantioselectivity (Table R4). Compared to PEC4.0, both mutants exhibited a significant decrease in e.e., suggesting that steric hindrance is likely not the major factor influencing the stereoselectivity in this case.\nTable R4. Effects of steric hindrance on the deracemization of (R)-2a.\nEntry CTB10-based photoenzymes e.e. (%)\nQ23. Line 229: Please elaborate on how the M86I mutation restricts the flexibility of BpA40. Additionally, could altered substrate–enzyme–substrate interactions also contribute to enhanced enantioselectivity?\nThe corresponding resluts have been added to the main text and the Supporting Information.\nFig. R4. Comparative analysis of enzyme-BpA and enzyme-substrate interaction networks in PEA1.0 versus PEA2.0.\nQ24. Lines 253–257: The authors should clarify whether the analysis presented here is consistent with the high e.e. value of 93%.\nResponse: We thank the reviewer for raising this important point regarding the consistency between the structural data and the high enantioselectivity. We added the following sentence in the manuscript:\nQ25. Supporting information line 270-280: the concentrations of the ligands as well as the incubation or soaking time are not provided. Please include these details to ensure reproducibility.\nResponse: We thank the reviewer for highlighting this omission. The ligand concentrations and soaking/incubation times have now been added in the Supporting Information to ensure experimental reproducibility.\nThe authors have well addressed all concerns raised by reviewers. This MS is now suitable for publication in NC.\nResponse: We thank the reviewer for your vaulable comments again.\nTo Reviewer #2:\nThe authors have addressed all of my concerns. I recommend the manuscript for acceptance. Congratulations on this excellent work on artificial photoenzymes.\nResponse: We thank the reviewer for your vaulable comments again.\nTo Reviewer #3:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank the reviewer for your vaulable comments again.\nTo Reviewer #4\nThe authors have addressed all of my previous comments. However, in the main text, the ligand maps are still shown as 2Fo–Fc electron density maps rather than omit maps. I note that this adjustment has been made only in Supplementary Fig. 12.\nResponse: We thank the reviewer for this comment. In response to this suggestion, we have modified all the electron density maps in the main text to omit maps."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have well addressed all concerns raised by reviewers. This MS is now suitable for publication in NC.", "reviewer_2": "The authors have addressed all of my concerns. I recommend the manuscript for acceptance. Congratulations on this excellent work on artificial photoenzymes.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The authors have addressed all of my previous comments. However, in the main text, the ligand maps are still shown as 2Fo–Fc electron density maps rather than omit maps. I note that this adjustment has been made only in Supplementary Fig. 12."}, "author_response": "To Reviewer #1\nThe authors have well addressed all concerns raised by reviewers. This MS is now suitable for publication in NC.\nResponse: We thank the reviewer for your vaulable comments again.\nTo Reviewer #2:\nThe authors have addressed all of my concerns. I recommend the manuscript for acceptance. Congratulations on this excellent work on artificial photoenzymes.\nResponse: We thank the reviewer for your vaulable comments again.\nTo Reviewer #3:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank the reviewer for your vaulable comments again.\nTo Reviewer #4\nThe authors have addressed all of my previous comments. However, in the main text, the ligand maps are still shown as 2Fo–Fc electron density maps rather than omit maps. I note that this adjustment has been made only in Supplementary Fig. 12.\nResponse: We thank the reviewer for this comment. In response to this suggestion, we have modified all the electron density maps in the main text to omit maps."}]

Decision letter