Biocatalysis with the milk protein β-lactoglobulin: Promoting retroaldol cleavage of α,β-unsaturated aldehydes

Article abstract of DOI:10.1039/c8ob00139a

Enzymes with a hydrophobic binding site and an active site lysine have been suggested to be promiscuous in their catalytic activity. β-Lactoglobulin (BLG), the principle whey protein found in milk, possesses a central calyx that binds non-polar molecules. Here, we report that BLG can catalyze the retro-aldol cleavage of α,β-unsaturated aldehydes making it a naturally occurring protein capable of catalyzing retro-aldol reactions on hydrophobic substrates. Retroaldolase activity was seen to be most effective on substrates with phenyl or naphthyl side-chains. Use of a brominated substrate analogue inhibitor increases the product yield by a factor of three. BLG's catalytic activity and its ready availability make it a prime candidate for the development of commercial biocatalysts.

Full text of DOI:10.1039/c8ob00139a

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Biomol ec ular
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Foley, J. Du, M. Esteb and C. Watanabe, Org. Biomol. Chem., 2018, DOI: 10.1039/C8OB00139A.
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January 2016 Pages 1–372
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COMMUNICATION
Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
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O rP gl ea na is ce &ꢀ d Bo i ꢀon mo to ꢀ lae dc juu l sa tr ꢀ Cm h ae r mg i ins ts rꢀ y
View Article Online
DOI: 10.1039/C8OB00139A
JournalꢀNameꢀ
COMMUNICATIONꢀ
ꢀ
BiocatalysisꢀwithꢀtheꢀMilkꢀProteinꢀβ-Lactoglobulin:ꢀPromotingꢀ
RetroaldolꢀCleavageꢀofꢀα,β-UnsaturatedꢀAldehydesꢀ
Receivedꢀ00thꢀJanuaryꢀ20xx,ꢀ
Acceptedꢀ00thꢀJanuaryꢀ20xxꢀ
VishruthꢀGowda,ꢀBrendanꢀFoley,ꢀJasmineꢀDu,ꢀMeganꢀEsteb,ꢀCoranꢀM.ꢀH.ꢀWatanabe*ꢀ
DOI:ꢀ10.1039/x0xx00000xꢀ
www.rsc.org/ꢀ
9
10
Enzymesꢀwithꢀaꢀhydrophobicꢀbindingꢀsiteꢀandꢀanꢀactiveꢀsiteꢀlysineꢀ Michaelꢀadditions ꢀandꢀKnoevenagelꢀcondensations ꢀindicateꢀ
haveꢀbeenꢀsuggestedꢀtoꢀbeꢀpromiscuousꢀinꢀtheirꢀcatalyticꢀactivity.ꢀ thatꢀtheꢀminimumꢀrequirementꢀforꢀpromiscuityꢀinꢀcatalyzingꢀC-
β-lactoglobulinꢀ (BLG),ꢀ theꢀ principleꢀ wheyꢀ proteinꢀ foundꢀ inꢀ milk,ꢀ Cꢀbondꢀgeneratingꢀreactionsꢀisꢀanꢀactiveꢀsiteꢀcapableꢀofꢀbindingꢀ
possessesꢀaꢀcentralꢀcalyxꢀthatꢀbindsꢀnon-polarꢀmolecules.ꢀHere,ꢀweꢀ hydrophobicꢀ moleculesꢀ andꢀ anꢀ activeꢀ siteꢀ lysineꢀ toꢀ formꢀ
reportꢀ thatꢀ BLGꢀ canꢀ catalyzeꢀ theꢀ retro-aldolꢀ cleavageꢀ ofꢀ α, βꢀ iminiumꢀionsꢀorꢀenamines.ꢀꢀꢀ
unsaturatedꢀ aldehydesꢀ makingꢀ itꢀ aꢀ naturallyꢀ occurringꢀ proteinꢀ
ꢀ
Bovineꢀ β-lactoglobulinꢀ (BLG)ꢀ constitutesꢀ betweenꢀ 12–
capableꢀ ofꢀ catalyzingꢀ retro-aldolꢀ reactionsꢀ onꢀ hydrophobicꢀ 15%ꢀ ofꢀ theꢀ proteinꢀ contentꢀ ofꢀ milk.ꢀ Despiteꢀ extensiveꢀ
substrates.ꢀ Retroaldolaseꢀ activityꢀ wasꢀ seenꢀ toꢀ beꢀ mostꢀ effectiveꢀ investigationsꢀintoꢀitsꢀphysicalꢀandꢀbiochemicalꢀpropertiesꢀforꢀ
onꢀ substratesꢀ withꢀ phenylꢀ orꢀ naphthylꢀ side-chains.ꢀ Useꢀ ofꢀ aꢀ approximatelyꢀ 80ꢀ years,ꢀ theꢀ protein’sꢀ functionꢀ remainsꢀ
11
brominatedꢀ substrateꢀ analogueꢀ inhibitorꢀ increasesꢀ theꢀ productꢀ unassigned. ꢀꢀItsꢀsize,ꢀstability,ꢀbioavailabilityꢀandꢀcapacityꢀtoꢀ
yieldꢀ byꢀ aꢀ factorꢀ ofꢀ three.ꢀ ꢀ BLG’sꢀ catalyticꢀ activityꢀ andꢀ itsꢀ readyꢀ bindꢀhydrophobicꢀmoleculesꢀinꢀitsꢀcentralꢀcalyxꢀhaveꢀresultedꢀ
availabilityꢀ makeꢀ itꢀ aꢀ primeꢀ candidateꢀ forꢀ theꢀ developmentꢀ ofꢀ inꢀ applicationsꢀ includingꢀ encapsulationꢀ andꢀ nutraceuticalꢀ
12
commercialꢀbiocatalysts.ꢀ
delivery. ꢀ ꢀ Theꢀ presenceꢀ ofꢀ anꢀ activeꢀ siteꢀ lysineꢀ capableꢀ ofꢀ
activatingꢀ aldehydesꢀ byꢀ imineꢀ formationꢀ andꢀ aꢀ hydrophobicꢀ
cavityꢀ ledꢀ usꢀ toꢀ proposeꢀ thatꢀ BLG,ꢀ likeꢀ RA95.5-8,ꢀ couldꢀ alsoꢀ
serveꢀasꢀanꢀeffectiveꢀretroaldolase.ꢀPreviously,ꢀweꢀhaveꢀshownꢀ
thatꢀ BLGꢀ canꢀ promoteꢀ condensationꢀ ofꢀ α,ꢀ β-unsaturatedꢀ
ꢀ
Enzymesꢀ thatꢀ areꢀ versatileꢀ inꢀ theirꢀ activityꢀ andꢀ substrateꢀ
toleranceꢀ canꢀ beꢀ commerciallyꢀ appliedꢀ toꢀ theꢀ productionꢀ ofꢀ
chemicalsꢀandꢀpharmaceuticalsꢀtherebyꢀincreasingꢀefficiencyꢀofꢀ
manufacturingꢀplantsꢀwhileꢀreducingꢀenvironmentallyꢀharmfulꢀ
aldehydes,ꢀ toꢀ theirꢀ respectiveꢀ cyclodimersꢀ throughꢀ C-Cꢀ bondꢀ
13-15
formation.
ꢀForꢀexample,ꢀcondensationꢀofꢀall-transꢀretinalꢀ1ꢀ
yieldsꢀ theꢀ formationꢀ ofꢀ all-transꢀ retinalꢀ dimer,ꢀ cycloretinalꢀ 2ꢀ
1
waste. ꢀ Naturalꢀ enzymes,ꢀ whileꢀ highlyꢀ proficientꢀ inꢀ theꢀ
(
Figureꢀ1).ꢀ
catalysisꢀ ofꢀ theirꢀ nativeꢀ substratesꢀ areꢀ notꢀ veryꢀ versatile.ꢀ
Aldolasesꢀ areꢀ aꢀ characteristicꢀ exampleꢀ ofꢀ suchꢀ enzymesꢀ withꢀ
highꢀactivityꢀtowardsꢀtheirꢀnativeꢀpolarꢀsubstratesꢀbutꢀveryꢀlowꢀ
2
,ꢀ 3
toleranceꢀ towardsꢀ hydrophobicꢀ ones. ꢀ Variousꢀ strategiesꢀ
4
includingꢀ directedꢀ evolution, ꢀ catalyticꢀ antibodies, ꢀ andꢀ
5,ꢀ 6
7
computationalꢀ design ꢀ haveꢀ beenꢀ exploredꢀ toꢀ designꢀ anꢀ
O
O
BLG
aldolaseꢀwithꢀhighꢀactivityꢀtowardsꢀnon-polarꢀmolecules.ꢀꢀTheꢀ
de-novoꢀ designꢀ andꢀ directedꢀ evolutionꢀ ofꢀ aꢀ retro-aldolase,ꢀ
RA95.5-8ꢀ withꢀ activityꢀ approachingꢀ thatꢀ ofꢀ naturalꢀ classꢀ Iꢀ
1
all-trans retinal
2
cycloretinal
8
aldolasesꢀ hasꢀ madeꢀ theꢀ mostꢀ progressꢀ towardꢀ thisꢀ goal. ꢀꢀ
ꢀ
FurtherꢀinvestigationsꢀofꢀRA95.5-8ꢀindicateꢀthatꢀthisꢀenzymeꢀisꢀ
notꢀ onlyꢀ promiscuousꢀ inꢀ substrateꢀ selectivity,ꢀ butꢀ alsoꢀ inꢀ
catalyticꢀactivity.ꢀStudiesꢀintoꢀtheꢀenzyme’sꢀabilityꢀtoꢀcatalyzeꢀ
Figureꢀ1.ꢀBLGꢀcatalyzedꢀcyclodimerizationꢀofꢀall-transꢀretinalꢀ1ꢀ
toꢀcycloretinalꢀ2ꢀ
ꢀ
Here,ꢀ weꢀ reportꢀ onꢀ theꢀ resultsꢀ ofꢀ ourꢀ investigations,ꢀ whichꢀ
suggestꢀthatꢀBLGꢀisꢀableꢀtoꢀcatalyzeꢀtheꢀretro-aldolꢀbreakdownꢀ
ofꢀ α,ꢀ β-unsaturatedꢀ aldehydes.ꢀ ꢀ Explorationꢀ ofꢀ theꢀ substrateꢀ
scopeꢀofꢀBLG’sꢀretroaldolaseꢀactivityꢀrevealedꢀthatꢀsubstratesꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
J.ꢀName.,ꢀ2013,ꢀ00,ꢀ1-3ꢀ|ꢀ1ꢀꢀ
Pleaseꢀdoꢀnotꢀadjustꢀmarginsꢀ

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COMMUNICATIONꢀ
JournalꢀNameꢀ
withꢀaromatic,ꢀnon-polarꢀsideꢀchainsꢀareꢀpreferredꢀtoꢀaliphaticꢀ Dueꢀ toꢀ theꢀ similarityꢀ betweenꢀ compoundꢀ 5ꢀ andꢀ methodol,ꢀ aꢀ
View Article Online
7,ꢀ 18
1
DOI: 10.1039/C8OB00139A
orꢀpolarꢀheadꢀgroups.ꢀꢀꢀ
compoundꢀusedꢀtoꢀstudyꢀretroaldolꢀreactions,
ꢀweꢀinitiallyꢀ
choseꢀthisꢀcompoundꢀtoꢀevaluateꢀtheꢀcleavageꢀproductsꢀofꢀtheꢀ
reaction.ꢀꢀCompoundꢀ5ꢀwasꢀincubatedꢀwithꢀBLGꢀinꢀPBSꢀbufferꢀ
atꢀ 37ꢀ °Cꢀ andꢀ theꢀ organicsꢀ subsequentlyꢀ extractedꢀ withꢀ ethylꢀ
acetate.ꢀ ꢀ Analysisꢀ byꢀ HPLC,ꢀ revealedꢀ theꢀ formationꢀ ofꢀ fourꢀ
peaksꢀ (Figureꢀ 3A).ꢀ Eachꢀ peakꢀ wasꢀ collectedꢀ andꢀ analyzedꢀ byꢀ
NMRꢀspectroscopy.ꢀTheꢀexpectedꢀhomodimerꢀ6ꢀwasꢀobservedꢀ
inꢀ peakꢀ a.ꢀ Peakꢀ bꢀ wasꢀ structurallyꢀ characterizedꢀ asꢀ
acetonaphthoneꢀ 3,ꢀ whichꢀ wasꢀ alsoꢀ validatedꢀ byꢀ comparisonꢀ
withꢀ acetonaphthoneꢀ 3ꢀ standardꢀ (Figureꢀ 3C).ꢀ Peakꢀ dꢀ wasꢀ
identifiedꢀ asꢀ startingꢀ materialꢀ 5ꢀ byꢀ H-NMRꢀ spectroscopyꢀ andꢀ
Peakꢀ cꢀ wasꢀ shownꢀ toꢀ beꢀ aꢀ compoundꢀ closelyꢀ relatedꢀ toꢀ theꢀ
startingꢀ material;ꢀ however,ꢀ itꢀ wasn’tꢀ isolatedꢀ inꢀ sufficientꢀ
quantitiesꢀtoꢀfullyꢀcharacterizeꢀit.ꢀAꢀcontrolꢀreactionꢀwhereꢀ5ꢀ
wasꢀincubatedꢀwithꢀPBSꢀoverꢀ4ꢀdaysꢀresultedꢀinꢀtheꢀretrievalꢀofꢀ
onlyꢀstartingꢀmaterialꢀ(SIꢀpageꢀ7).ꢀ
Figureꢀ 2.ꢀ ꢀ Oxidativeꢀ cleavageꢀ asꢀ mediatedꢀ byꢀ BLG:ꢀ [A]ꢀ BLGꢀ
catalyzedꢀoxidativeꢀcleavageꢀofꢀβ-methylꢀnaphtheneꢀaldehydeꢀ
3
;ꢀ [B]ꢀ Synthesisꢀ ofꢀ β-methylꢀ naphtheneꢀ aldehydeꢀ 3:ꢀ iꢀ NaH,ꢀ
DCM;ꢀiiꢀDIBAL-H,ꢀ-60ꢀ°C;ꢀiiiꢀH
ꢀ
2
Oꢀ
ꢀ Theꢀscopeꢀofꢀtheꢀreactionꢀwasꢀevaluatedꢀwithꢀaꢀseriesꢀofꢀ
α,β-unsaturatedꢀaldehydeꢀsubstrates,ꢀTableꢀ1.ꢀꢀTheꢀretro-aldolꢀ
Synthesisꢀ ofꢀ theꢀ α,β-unsaturatedꢀ substratesꢀ wasꢀ achievedꢀ byꢀ
invokingꢀaꢀHorner-Wadsworth-EmmonsꢀreactionꢀtoꢀgiveꢀtheꢀE-
alkene,ꢀfollowedꢀbyꢀDIBALꢀreductionꢀofꢀtheꢀnitrile,ꢀasꢀshownꢀforꢀ
breakdownꢀ productꢀ forꢀ eachꢀ wasꢀ characterizedꢀ byꢀ HPLCꢀ co-
injectionꢀ withꢀ syntheticꢀ standardsꢀ andꢀ theꢀ totalꢀ turnoverꢀ
16
theꢀformationꢀofꢀβ-methylꢀnaphtheneꢀaldehydeꢀ5,ꢀFigureꢀ2A. ꢀꢀ
numberꢀ (TTN)ꢀ forꢀ eachꢀ substrateꢀ wasꢀ evaluatedꢀ asꢀ shownꢀ inꢀ
Tableꢀ1.ꢀꢀBLGꢀwasꢀshownꢀtoꢀbeꢀmostꢀeffectiveꢀinꢀtheꢀretroaldolꢀ
cleavageꢀofꢀcompoundsꢀwithꢀaromatic,ꢀphenylꢀ10ꢀorꢀnaphthylꢀ5ꢀ
sideꢀchains.ꢀꢀExtendingꢀtheꢀsideꢀchainꢀtoꢀthatꢀofꢀaꢀbiphenyl-ꢀ11ꢀ
orꢀfluoreneꢀ12ꢀgroupꢀresultedꢀinꢀaꢀ3-ꢀtoꢀ10-foldꢀreductionꢀinꢀ
retroaldolaseꢀ activity,ꢀ respectively.ꢀ ꢀ Likewise,ꢀ theꢀ moreꢀ polarꢀ
aromaticꢀ substrate,ꢀ 2-methylꢀ furanylꢀ 9ꢀ moietyꢀ andꢀ theꢀ
aliphaticꢀ substrateꢀ 8ꢀ showedꢀ veryꢀ littleꢀ toꢀ noꢀ retroaldolaseꢀ
activity.ꢀꢀThisꢀisꢀthusꢀsuggestiveꢀthatꢀtheꢀretroaldolaseꢀactivityꢀ
favorsꢀ thatꢀ ofꢀ aꢀ hydrophobicꢀ bindingꢀ cavityꢀ thatꢀ mostꢀ
effectivelyꢀaccommodatesꢀphenylꢀandꢀnaphthylꢀsidechains.ꢀꢀ
ꢀ
Tableꢀ1ꢀ
ꢀ
Figureꢀ3.ꢀEvaluationꢀofꢀBLGꢀreactionꢀbyꢀHPLCꢀanalysis:ꢀA]ꢀBLGꢀ
reactionꢀwithꢀcompoundꢀ5;ꢀa:ꢀcompoundꢀ6,ꢀb:ꢀacetonaphthoneꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3
reactionꢀ withꢀ compoundꢀ 1;ꢀ e:ꢀ compoundꢀ 13,ꢀ f:ꢀ
,ꢀc:ꢀrelatedꢀtoꢀstartingꢀmaterial,ꢀd:ꢀstartingꢀmaterialꢀꢀꢀB]ꢀBLGꢀ
acetonaphthoneꢀ 3,ꢀ g:ꢀ notꢀ characterized,ꢀ h:ꢀ compoundꢀ 5ꢀ ꢀ C]ꢀ
acetonaphthoneꢀ3ꢀsyntheticꢀstandardꢀꢀ
ꢀ
Schemeꢀ1ꢀProposedꢀmechanismꢀforꢀBLGꢀcatalyzedꢀretroaldolꢀ
cleavageꢀ
BLG
BLG
O
N
H
3
O
5
H
H
B:
BLG
BLG
N
H
N
H
OH
H
O
H
B:
B
O
O
+
H2O
3
4
7
7
2
ꢀ|ꢀJ.ꢀName.,ꢀ2012,ꢀ00,ꢀ1-3ꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
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O rP gl ea na is ce &ꢀ d Bo i ꢀon mo to ꢀ lae dc juu l sa tr ꢀ Cm h ae r mg i ins ts rꢀ y
JournalꢀNameꢀ
Schemeꢀ2.ꢀCovalentꢀlabellingꢀofꢀBLGꢀwithꢀInhibitorꢀ13ꢀ
ꢀCOMMUNICATIONꢀ
ꢀ
View Article Online
DOI: 10.1039/C8OB00139A
forꢀtheꢀretroaldolꢀcleavage.ꢀꢀHence,ꢀinhibitorꢀ13ꢀisꢀexpectedꢀtoꢀ
haveꢀaꢀmoreꢀsubstantiveꢀaffectꢀonꢀtheꢀdimerizationꢀreactionꢀasꢀ
opposedꢀtoꢀthatꢀofꢀtheꢀoxidativeꢀcleavage.ꢀꢀꢀCompoundꢀ13ꢀwasꢀ
synthesizedꢀfromꢀacetophenoneꢀ15ꢀusingꢀtheꢀVilsmeier-Haackꢀ
Br
Br
BLG
BLG
O
N
H
8
1
3
2
H N
19
reactionꢀaccordingꢀtoꢀFigureꢀ4B. ꢀꢀBLGꢀwasꢀincubatedꢀwithꢀaꢀ
:1.5ꢀ ratioꢀ ofꢀ 5ꢀ toꢀ 13ꢀ inꢀ PBSꢀ bufferꢀ atꢀ 37ꢀ °C.ꢀ Theꢀ reactionꢀ
BLG
1
Br
BLG
BLG
mixtureꢀwasꢀsubsequentlyꢀquenchedꢀandꢀextractedꢀwithꢀethylꢀ
acetate.ꢀHPLCꢀanalysisꢀshowedꢀ4ꢀpeaksꢀ(Figureꢀ3B)ꢀthatꢀwereꢀ
collectedꢀ andꢀ characterizedꢀ byꢀ H-NMRꢀ spectroscopy.ꢀ Peakꢀ fꢀ
wasꢀ confirmedꢀ asꢀ acetonaphthoneꢀ 3.ꢀ Cyclodimerꢀ 6ꢀ wasꢀ notꢀ
observedꢀ indicatingꢀ thatꢀ treatmentꢀ withꢀ 13ꢀ eliminatedꢀ
cyclodimerizationꢀactivityꢀofꢀBLG.ꢀWhileꢀpeakꢀeꢀelutedꢀatꢀaboutꢀ
theꢀ sameꢀ retentionꢀ timeꢀ asꢀ theꢀ homodimerꢀ 6,ꢀ itꢀ wasꢀ
structurallyꢀ characterizedꢀ asꢀ 13.ꢀ Peakꢀ hꢀ wasꢀ confirmedꢀ asꢀ 5ꢀ
andꢀ peakꢀ gꢀ wasꢀ alsoꢀ notꢀ isolatedꢀ inꢀ sufficientꢀ quantitiesꢀ forꢀ
accurateꢀcharacterization.ꢀ
HN
N
H
BLG
N
NH
H
BLG
BLG
HN
Br
O
O
11
14
2
H O
ꢀ
Theꢀlowꢀturnoverꢀnumbersꢀareꢀpotentiallyꢀdueꢀtoꢀnon-covalentꢀ
binding/interactionꢀ ofꢀ BLGꢀ andꢀ startingꢀ materialꢀ thatꢀ eitherꢀ
doesꢀ notꢀ resultꢀ inꢀ anyꢀ productꢀ formationꢀ orꢀ inꢀ productꢀ notꢀ
beingꢀreleasedꢀfromꢀtheꢀprotein.ꢀꢀWhenꢀweꢀextractꢀwithꢀethylꢀ
acetate,ꢀtheꢀaqueousꢀlayerꢀretainsꢀaꢀcolorꢀindicativeꢀofꢀstartingꢀ
material-proteinꢀSchiffꢀbase,ꢀevenꢀafterꢀmultipleꢀextractions.ꢀꢀꢀꢀ
ꢀ
Theꢀ amountꢀ ofꢀ acetonaphthoneꢀ 3ꢀ formedꢀ inꢀ eachꢀ
experimentꢀ wasꢀ quantifiedꢀ byꢀ HPLCꢀ analysisꢀ utilizingꢀ aꢀ
standardꢀcurveꢀ(seeꢀSIꢀpageꢀ17).ꢀAdditionꢀofꢀcyclodimerizationꢀ
inhibitorꢀ13ꢀmoreꢀthanꢀdoubledꢀtheꢀyieldꢀofꢀacetonaphthoneꢀ
(
0.45ꢀ mg/L)ꢀ comparedꢀ toꢀ incubationꢀ ofꢀ BLGꢀ withꢀ 5ꢀ alone,ꢀ
whichꢀyieldedꢀonlyꢀ0.18ꢀmg/Lꢀofꢀtheꢀacetonaphthoneꢀcleavageꢀ
productꢀ(SIꢀpageꢀ18).ꢀꢀTheꢀenhancementꢀinꢀyieldꢀobservedꢀwithꢀ
inhibitorꢀ13ꢀcoupledꢀwithꢀtheꢀsubstrateꢀspecificityꢀseenꢀonꢀtheꢀ
retroaldolꢀ cleavageꢀ reactionꢀ (Tableꢀ 1),ꢀ isꢀ suggestiveꢀ ofꢀ theꢀ
involvementꢀ ofꢀ specificꢀ lysinesꢀ onꢀ theꢀ proteinꢀ catalyzingꢀ theꢀ
reaction.ꢀ
ꢀ
ꢀ
Theꢀ proposedꢀ routeꢀ forꢀ cleavageꢀ ofꢀ theꢀ α,ꢀ β-
unsaturatedꢀdoubleꢀbondꢀofꢀ5ꢀtoꢀgenerateꢀacetonaphthoneꢀ3ꢀisꢀ
depictedꢀ inꢀ Schemeꢀ 1.ꢀ Imineꢀ formationꢀ viaꢀ aꢀ lysineꢀ residueꢀ
withinꢀtheꢀBLGꢀbindingꢀpocketꢀinitiatesꢀtheꢀprocess.ꢀꢀThisꢀsetsꢀ
theꢀ stageꢀ forꢀ theꢀ additionꢀ ofꢀ waterꢀ toꢀ theꢀ imineꢀ activatedꢀ
doubleꢀ bondꢀ andꢀ subsequentꢀ retro-aldolꢀ likeꢀ cleavageꢀ ofꢀ theꢀ
C—Cꢀ singleꢀ bond.ꢀ ꢀ Itꢀ isꢀ suggestiveꢀ thatꢀ theꢀ preferenceꢀ forꢀ
aromaticꢀgroupsꢀoverꢀaliphaticꢀside-chainsꢀisꢀdueꢀtoꢀresonanceꢀ
stabilizationꢀ ofꢀ aꢀ transitionꢀ stateꢀ C-3ꢀ carbocationꢀ duringꢀ C-Cꢀ
bondꢀ cleavage,ꢀ withꢀ non-aromaticꢀ orꢀ polarꢀ moleculesꢀ beingꢀ
lessꢀableꢀtoꢀstabilizeꢀtheꢀcarbocation.ꢀꢀꢀ
ꢀ
Controlꢀexperimentsꢀwithꢀ5ꢀorꢀ14,ꢀwhichꢀdifferꢀonlyꢀbyꢀ
theꢀpresenceꢀofꢀaꢀbromo-ꢀorꢀmethyl-groupꢀatꢀC-3,ꢀwasꢀcarriedꢀ
out.ꢀSynthesisꢀofꢀ14ꢀwasꢀachievedꢀinꢀtheꢀsameꢀmannerꢀasꢀtheꢀ
synthesisꢀ ofꢀ 13ꢀ (SIꢀ pageꢀ 6).ꢀ ESI-MSꢀ analysisꢀ showedꢀ lysineꢀ
labelingꢀwithꢀ14ꢀwhileꢀnoꢀlabelingꢀwasꢀobservedꢀwithꢀ5,ꢀwhichꢀ
furtherꢀ substantiatesꢀ thatꢀ theꢀ bromo-groupꢀ isꢀ criticalꢀ forꢀ
covalentꢀlabellingꢀ(SIꢀpageꢀ21).ꢀꢀ
ꢀ Inꢀ lightꢀ ofꢀ thisꢀ mechanism,ꢀ weꢀ evaluatedꢀ whetherꢀ aꢀ
cyclodimerizationꢀ inhibitor,ꢀ 3-bromocinnamaldehydeꢀ 13ꢀ
15
(Figureꢀ 4A), ꢀ couldꢀ effectꢀ anꢀ enhancementꢀ inꢀ retro-aldolꢀ
activity.ꢀ ꢀ Brominatedꢀ 13ꢀ hasꢀ beenꢀ previouslyꢀ shownꢀ toꢀ
inactivateꢀ keyꢀ lysineꢀ residuesꢀ (Schemeꢀ 2)ꢀ asꢀ validatedꢀ byꢀ
Conclusionꢀ
15
trypsinꢀdigestionꢀandꢀESI/MSꢀanalysis. ꢀꢀ
HereꢀweꢀhaveꢀshownꢀthatꢀBLGꢀisꢀaꢀversatileꢀproteinꢀwithꢀtheꢀ
ꢀ
involvementꢀofꢀtwoꢀBLGꢀlysineꢀresidues ꢀasꢀopposedꢀtoꢀoneꢀꢀ
Theꢀ cyclodimerizationꢀ processꢀ necessitatesꢀ theꢀ abilityꢀ toꢀ notꢀ onlyꢀ promoteꢀ condensationꢀ reactions,ꢀ butꢀ alsoꢀ
15
retro-aldolꢀcleavageꢀofꢀα, β-unsaturatedꢀaldehydes.ꢀCatalysisꢀisꢀ
proposedꢀ toꢀ proceedꢀ throughꢀ initialꢀ hydrolysisꢀ ofꢀ theꢀ α,ꢀ β-
unsaturatedꢀ imineꢀ followedꢀ byꢀ aꢀ retro-aldolꢀ cleavageꢀ ofꢀ theꢀ
alcoholꢀ (Schemeꢀ 1).ꢀ Useꢀ ofꢀ aꢀ cyclodimerizationꢀ inhibitor,ꢀ
disablesꢀthisꢀcompetingꢀreactionꢀandꢀenhancesꢀtheꢀretroaldolꢀ
process.ꢀ Whileꢀ theꢀ otherꢀ well-studiedꢀ milkꢀ protein,ꢀ bovineꢀ
serumꢀalbuminꢀ(BSA)ꢀhasꢀbeenꢀreportedꢀtoꢀcatalyzeꢀmoreꢀthanꢀ
A
Br
O
O
O
+
BLG
3
8
4
5
13
3
B
20
O
Br
tenꢀ reactionsꢀ ,ꢀ thisꢀ isꢀ onlyꢀ theꢀ secondꢀ catalyticꢀ activityꢀ
PBr , 0 °C
O
discoveredꢀ forꢀ BLG.ꢀ Althoughꢀ theꢀ TTNꢀ forꢀ BLGꢀ catalyzedꢀ
retroaldolꢀ cleavageꢀ isꢀ muchꢀ lowerꢀ thanꢀ de-novoꢀ designedꢀ
proteins,ꢀtheꢀreadyꢀavailabilityꢀofꢀBLGꢀmakesꢀitꢀaꢀviableꢀchoiceꢀ
forꢀ biocatalyticꢀ investigations.ꢀ Daviscoꢀ Foodsꢀ Internationalꢀ
aloneꢀ producesꢀ overꢀ 10ꢀ millionꢀ poundsꢀ ofꢀ wheyꢀ proteinꢀ
isolatesꢀ annually.ꢀ Evolutionꢀ ofꢀ theseꢀ enzymesꢀ and/orꢀ furtherꢀ
understandingꢀofꢀtheꢀmechanismꢀofꢀtheseꢀreactionsꢀmightꢀleadꢀ
toꢀ theꢀ developmentꢀ ofꢀ BLGꢀ variantsꢀ withꢀ higherꢀ catalyticꢀ
efficiencyꢀ and/orꢀ adaptableꢀ biocatalystsꢀ forꢀ commercialꢀ use.ꢀꢀ
3
+
N
O
9
10
8
1
55
1613
13
3
ꢀ
Figureꢀ4.ꢀEffectꢀofꢀsubstrateꢀanalogꢀ13ꢀonꢀretroaldolꢀcleavage:ꢀ
A]ꢀ BLGꢀ catalyzedꢀ oxidativeꢀ cleavageꢀ ofꢀ β-methylꢀ naphtheneꢀ
[
aldehydeꢀ 5ꢀ inꢀ theꢀ presenceꢀ ofꢀ inhibitorꢀ 13;ꢀ [B]ꢀ Synthesisꢀ ofꢀ
substrateꢀanalogꢀ13ꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
J.ꢀName.,ꢀ2013,ꢀ00,ꢀ1-3ꢀ|ꢀ3ꢀ
Pleaseꢀdoꢀnotꢀadjustꢀmarginsꢀ

O rP gl ea na is ce &ꢀ d Bo i ꢀon mo to ꢀ lae dc juu l sa tr ꢀ Cm h ae r mg i ins ts rꢀ y
Page 4 of 5
COMMUNICATIONꢀ
JournalꢀNameꢀ
AsꢀBSAꢀandꢀotherꢀproteinsꢀwithꢀaꢀhydrophobicꢀactiveꢀsiteꢀandꢀ 15.ꢀV.ꢀGowda,ꢀJ.ꢀFoulke-Abel,ꢀH.ꢀAgbo,ꢀB.ꢀJ.ꢀBench,ꢀJV.ieꢀCwhAarteic,leꢀWOn.lꢀinKe.ꢀ
DOI: 10.1039/C8OB00139A
Russellꢀ andꢀ C.ꢀ M.ꢀ H.ꢀ Watanabe,ꢀ Biochemistry,ꢀ 2017,ꢀ 56,ꢀ
anꢀ activeꢀ siteꢀ lysineꢀ haveꢀ beenꢀ shownꢀ toꢀ catalyzeꢀ Michaelꢀ
addition,ꢀKnoevenagelꢀcondensationꢀandꢀKempꢀelimination,ꢀitꢀ
mightꢀ beꢀ predictedꢀ thatꢀ BLGꢀ couldꢀ potentiallyꢀ catalyzeꢀ suchꢀ 16.ꢀC.ꢀJellimann,ꢀM.ꢀMathé-Allainmat,ꢀJ.ꢀAndrieux,ꢀS.ꢀKloubert,ꢀJ.ꢀ
5715-5719.ꢀ
reactionsꢀasꢀwell.ꢀꢀꢀꢀ
A.ꢀBoutin,ꢀJ.-P.ꢀNicolas,ꢀC.ꢀBennejean,ꢀP.ꢀDelagrangeꢀandꢀM.ꢀ
Langlois,ꢀ Journalꢀ ofꢀ Medicinalꢀ Chemistry,ꢀ 2000,ꢀ 43,ꢀ 4051-
4
062.ꢀ
Conflictsꢀofꢀinterestꢀ
Theꢀauthorsꢀdeclareꢀnoꢀcompetingꢀfinancialꢀinterest.ꢀ
1
1
7.ꢀB.ꢀ List,ꢀ C.ꢀ F.ꢀ Barbasꢀ andꢀ R.ꢀ A.ꢀ Lerner,ꢀ Proceedingsꢀ ofꢀ theꢀ
NationalꢀAcademyꢀofꢀSciences,ꢀ1998,ꢀ95,ꢀ15351-15355.ꢀ
8.ꢀJ.ꢀ Schmidt,ꢀ C.ꢀ Ehasz,ꢀ M.ꢀ Epperson,ꢀ K.ꢀ Klas,ꢀ J.ꢀ Wyatt,ꢀ M.ꢀ
Hennigꢀ andꢀ M.ꢀ Forconi,ꢀ Organicꢀ &ꢀ Biomolecularꢀ Chemistry,ꢀ
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Acknowledgmentꢀ
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2
9.ꢀG.ꢀWang,ꢀX.ꢀChen,ꢀG.ꢀMiao,ꢀW.ꢀYaoꢀandꢀC.ꢀMa,ꢀTheꢀJournalꢀofꢀ
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Weꢀ areꢀ gratefulꢀ toꢀ theꢀ Welchꢀ Foundationꢀ (A-1828)ꢀ andꢀ theꢀ
Nationalꢀ Scienceꢀ Foundationꢀ (CHE-1608580)ꢀ forꢀ financialꢀ
support.ꢀꢀTheꢀauthorsꢀwouldꢀlikeꢀtoꢀthankꢀDr.ꢀHowardꢀWilliamsꢀ
andꢀ Dr.ꢀ Yohannesꢀ Rezenomꢀ inꢀ theꢀ NMRꢀ andꢀ massꢀ specꢀ
facilities.ꢀꢀ
1
ꢀ
0588-10598.ꢀ
ꢀ
Notesꢀandꢀreferencesꢀ
‡
ꢀFootnotesꢀrelatingꢀtoꢀtheꢀmainꢀtextꢀshouldꢀappearꢀhere.ꢀTheseꢀ
mightꢀ includeꢀ commentsꢀ relevantꢀ toꢀ butꢀ notꢀ centralꢀ toꢀ theꢀ
matterꢀunderꢀdiscussion,ꢀlimitedꢀexperimentalꢀandꢀspectralꢀdata,ꢀ
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Organic & Biomolecular Chemistry
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DOI: 10.1039/C8OB00139A
TOC Entry
The milk protein BLG catalyzes retro-aldol cleavage of α, β-unsaturated aldehydes. Use of a
cyclodimerization inhibitor enhances the reaction rate.