Directed evolution of RebH for catalyst-controlled halogenation of indole C-H bonds

Article abstract of DOI:10.1039/c5sc04680g

RebH variants capable of chlorinating substituted indoles ortho-, meta-, and para-to the indole nitrogen were evolved by directly screening for altered selectivity on deuterium-substituted probe substrates using mass spectrometry. This systematic approach allowed for rapid accumulation of beneficial mutations using simple adaptive walks and should prove generally useful for altering and optimizing the selectivity of C-H functionalization catalysts. Analysis of the beneficial mutations showed that structure-guided selection of active site residues for targeted mutagenesis can be complicated either by activity/selectivity tradeoffs that reduce the possibility of detecting such mutations or by epistatic effects that actually eliminate the benefits of a mutation in certain contexts. As a corollary to this finding, the precise manner in which the beneficial mutations identified led to the observed changes in RebH selectivity is not clear. Docking simulations suggest that tryptamine binds to these variants as tryptophan does to native halogenases, but structural studies will be required to confirm these models and shed light on how particular mutations impact tryptamine binding. Similar directed evolution efforts on other enzymes or artificial metalloenzymes could enable a wide range of C-H functionalization reactions.

Full text of DOI:10.1039/c5sc04680g

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Pleas Ce hꢀ d eo mꢀ ni oc ta ꢀ la dS jcu i se t nꢀ mc ea rginsꢀ
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DOI: 10.1039/C5SC04680G
JournalꢀNameꢀ
ARTICLEꢀ
ꢀ
ꢀ
DirectedꢀEvolutionꢀofꢀRebHꢀforꢀCatalyst-
ControlledꢀHalogenationꢀofꢀIndoleꢀC-Hꢀ
Bondsꢀ
Receivedꢀ00thꢀJanuaryꢀ20xx,ꢀ
Acceptedꢀ00thꢀJanuaryꢀ20xxꢀ
Maryꢀ C.ꢀ Andorfer,ꢀ Hyunꢀ Juneꢀ Park,ꢀ Jaylieꢀ Vergara-Coll,ꢀ andꢀ
JaredꢀC.ꢀLewis ꢀ
DOI:ꢀ10.1039/x0xx00000xꢀ
*
www.rsc.org/ꢀ
ABSTRACT:ꢀRebHꢀvariantsꢀcapableꢀofꢀchlorinatingꢀsubstitutedꢀindolesꢀ
ortho-,ꢀmeta-,ꢀandꢀpara-ꢀtoꢀtheꢀindoleꢀnitrogenꢀwereꢀevolvedꢀbyꢀdirectlyꢀ
screeningꢀforꢀalteredꢀselectivityꢀonꢀdeuterium-substitutedꢀprobeꢀ
substratesꢀusingꢀmassꢀspectrometry.ꢀThisꢀsystematicꢀapproachꢀallowedꢀforꢀ
rapidꢀaccumulationꢀofꢀbeneficialꢀmutationsꢀusingꢀsimpleꢀadaptiveꢀwalksꢀ
andꢀshouldꢀproveꢀgenerallyꢀusefulꢀforꢀalteringꢀandꢀoptimizingꢀtheꢀ
selectivityꢀofꢀC-Hꢀfunctionalizationꢀcatalysts.ꢀAnalysisꢀofꢀtheꢀbeneficialꢀ
mutationsꢀshowedꢀthatꢀstructure-guidedꢀselectionꢀofꢀactiveꢀsiteꢀresiduesꢀ
forꢀtargetedꢀmutagenesisꢀcanꢀbeꢀcomplicatedꢀeitherꢀbyꢀactivity/selectivityꢀ
tradeoffsꢀthatꢀreduceꢀtheꢀpossibilityꢀofꢀdetectingꢀsuchꢀmutationsꢀorꢀbyꢀ
epistaticꢀeffectsꢀthatꢀactuallyꢀeliminateꢀtheꢀbenefitsꢀofꢀaꢀmutationꢀinꢀ
certainꢀcontexts.ꢀAsꢀaꢀcorollaryꢀtoꢀthisꢀfinding,ꢀtheꢀpreciseꢀmannerꢀinꢀwhichꢀ
theꢀbeneficialꢀmutationsꢀidentifiedꢀledꢀtoꢀtheꢀobservedꢀchangesꢀinꢀRebHꢀ
selectivityꢀisꢀnotꢀclear.ꢀDockingꢀsimulationsꢀsuggestꢀthatꢀtryptamineꢀbindsꢀ
toꢀtheseꢀvariantsꢀasꢀtryptophanꢀdoesꢀtoꢀnativeꢀhalogenases,ꢀbutꢀstructuralꢀ
studiesꢀwillꢀbeꢀrequiredꢀtoꢀconfirmꢀtheseꢀmodelsꢀandꢀshedꢀlightꢀonꢀhowꢀ
particularꢀmutationsꢀimpactꢀtryptamineꢀbinding.ꢀSimilarꢀdirectedꢀevolutionꢀ
effortsꢀonꢀotherꢀenzymesꢀorꢀartificialꢀmetalloenzymesꢀcouldꢀenableꢀaꢀwideꢀ
rangeꢀofꢀC-Hꢀfunctionalizationꢀreactions.ꢀ
Introductionꢀ
ꢀ
Catalyticꢀ C-Hꢀ bondꢀ functionalizationꢀ hasꢀ theꢀ potentialꢀ toꢀ
reduceꢀ theꢀ needꢀ forꢀ functionalꢀ groupꢀ manipulationꢀ duringꢀ
chemicalꢀ synthesesꢀ byꢀ allowingꢀ conversionꢀ ofꢀ C-Hꢀ bondsꢀ
1
directlyꢀ toꢀ functionalityꢀ presentꢀ inꢀ desiredꢀ targets. ꢀ Thisꢀ
increasesꢀ stepꢀ economy,ꢀ decreasesꢀ waste,ꢀ andꢀ expandsꢀ theꢀ
2
poolꢀ ofꢀ substratesꢀ availableꢀ forꢀ syntheticꢀ efforts. ꢀ
1
Organometallicꢀcatalystsꢀdominateꢀprogressꢀinꢀthisꢀfield, ꢀbutꢀ
mostꢀofꢀtheseꢀrequireꢀsubstratesꢀbearingꢀparticularꢀfunctionalꢀ
groupsꢀ termedꢀ directingꢀ groupsꢀ forꢀ reactivity,ꢀ selectivity,ꢀ orꢀ
3
both. ꢀ Whileꢀ suchꢀ groupsꢀ mayꢀ beꢀ presentꢀ inꢀ aꢀ targetꢀ
-5
6
molecule, ꢀ oftenꢀ theyꢀ areꢀ not,ꢀ orꢀ theirꢀ directingꢀ effectsꢀ areꢀ
mitigatedꢀ byꢀ catalystꢀ bindingꢀ toꢀ otherꢀ functionalityꢀ inꢀ aꢀ
7
substrate .ꢀInꢀsuchꢀcases,ꢀappropriateꢀdirectingꢀgroupsꢀmustꢀbeꢀ
installedꢀ andꢀ removed,ꢀ whichꢀ decreasesꢀ theꢀ benefitsꢀ ofꢀ C-Hꢀ
functionalizationꢀ(Fig.ꢀ1A).ꢀTheꢀsiteꢀselectivityꢀofꢀdirectedꢀC-Hꢀ
functionalizationꢀisꢀalsoꢀintentionallyꢀlimited;ꢀaꢀgivenꢀdirectingꢀ
groupꢀenforcesꢀaꢀparticularꢀselectivityꢀonꢀaꢀsubstrateꢀbyꢀdesignꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
J.ꢀName.,ꢀ2013,ꢀ00,ꢀ1-3ꢀ|ꢀ1ꢀꢀ
ꢀ
Pleaseꢀdoꢀnotꢀadjustꢀmarginsꢀ

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DOI: 10.1039/C5SC04680G
8
29
Fig.ꢀ 1B). ꢀ Inꢀ mostꢀ cases,ꢀ directingꢀ groupsꢀ areꢀ usedꢀ toꢀ RebHꢀ catalyzesꢀ 7-halogenationꢀ ofꢀ tryptophan. ꢀ Thisꢀ processꢀ
(
functionalizeꢀproximalꢀC-Hꢀbonds, ꢀbutꢀdistalꢀC-Hꢀbondsꢀhaveꢀ
3
-5
2 2
involvesꢀtheꢀreactionꢀofꢀO ꢀwithꢀboundꢀFADH ꢀtoꢀformꢀaꢀflavinꢀ
-
9
beenꢀ accessedꢀ usingꢀ elaborateꢀ directingꢀ groups ꢀ orꢀ catalystsꢀ
peroxideꢀ thatꢀ oxidizesꢀ halideꢀ anionꢀ (X ,ꢀ Xꢀ =ꢀ Cl,ꢀ Br)ꢀ toꢀ theꢀ
correspondingꢀ hypohalousꢀ acidꢀ (HOX).ꢀ HOXꢀ isꢀ proposedꢀ toꢀ
travelꢀ throughꢀ aꢀ poreꢀ withinꢀ theꢀ enzymeꢀ toꢀ theꢀ activeꢀ siteꢀ
whereꢀ itꢀ hasꢀ beenꢀ shownꢀ toꢀ reactꢀ withꢀ K79ꢀ toꢀ formꢀ aꢀ
1
0,11
withꢀpendantꢀfunctionalꢀgroupꢀrecognitionꢀelements
someꢀ catalystsꢀ areꢀ capableꢀ ofꢀ functionalizingꢀ C-Hꢀ bondsꢀ
.ꢀWhileꢀ
1
withoutꢀtheꢀneedꢀforꢀdirectingꢀgroups, ꢀsubstrate-dependentꢀ
2
30
haloamineꢀ species. ꢀ Aromaticꢀ halogenationꢀ isꢀ believedꢀ toꢀ
proceedꢀ viaꢀ electrophilicꢀ aromaticꢀ substitutionꢀ ofꢀ enzyme-
1
steric, ꢀ electronic,
3
14-16
17-19
ꢀ orꢀ stereoelectronic ꢀ differentiationꢀ
ofꢀC-Hꢀbondsꢀisꢀrequiredꢀforꢀselectivityꢀinꢀtheseꢀcases.ꢀDespiteꢀ
+ 31
boundꢀsubstrateꢀbyꢀaꢀproximalꢀhaleniumꢀionꢀ(X )ꢀdonor. ꢀThisꢀ
2
,8,20
3
0
theꢀsyntheticꢀutilityꢀofꢀtheseꢀmethods,
catalystꢀcontrolꢀoverꢀtheꢀselectivityꢀofꢀC-Hꢀfunctionalization, ꢀ HOX,
and,ꢀ justꢀ asꢀ importantly,ꢀ theꢀ abilityꢀ toꢀ tuneꢀ thatꢀ selectivity,ꢀ hydrolysisꢀandꢀhydrogenꢀbondedꢀwithinꢀtheꢀactiveꢀsite.ꢀꢀ
remainꢀfundamentalꢀchallenges.ꢀ
WeꢀestablishedꢀthatꢀRebHꢀhalogenatesꢀaꢀrangeꢀofꢀsubstitutedꢀ
Manyꢀ enzymesꢀ catalyzeꢀ selectiveꢀ C-Hꢀ functionalizationꢀ byꢀ indolesꢀ andꢀ electronꢀ richꢀ aromaticꢀ substrates ꢀ andꢀ evolvedꢀ
ꢀtheyꢀhighlightꢀhowꢀ
speciesꢀ isꢀ proposedꢀ toꢀ beꢀ eitherꢀ theꢀ K79ꢀ haloamine ꢀ orꢀ
2
1
31,32
ꢀ theꢀ latterꢀ presumablyꢀ regeneratedꢀ viaꢀ haloamineꢀ
3
3
3
4
bindingꢀsubstratesꢀsuchꢀthatꢀaꢀsingleꢀC-Hꢀbondꢀisꢀpresentedꢀtoꢀ variantsꢀofꢀthisꢀenzymeꢀwithꢀimprovedꢀstability ꢀandꢀexpandedꢀ
2
2
35
activeꢀ siteꢀ residuesꢀ andꢀ cofactorsꢀ involvedꢀ inꢀ C-Hꢀ cleavage. ꢀ substrateꢀ scope. ꢀ Whileꢀ variantsꢀ withꢀ highꢀ selectivityꢀ forꢀ aꢀ
Indeed,ꢀ theꢀ impactꢀ thatꢀ C-Hꢀ bondꢀ functionalizationꢀ canꢀ haveꢀ singleꢀsiteꢀonꢀdifferentꢀsubstratesꢀwereꢀreadilyꢀidentifiedꢀfromꢀ
onꢀ syntheticꢀ efficiencyꢀ isꢀ perhapsꢀ bestꢀ appreciatedꢀ byꢀ theseꢀ efforts,ꢀ variantsꢀ withꢀ differentꢀ selectivitiesꢀ onꢀ aꢀ
comparingꢀ naturalꢀ productꢀ biosynthesisꢀ involvingꢀ suchꢀ particularꢀ substrateꢀ wereꢀ rarelyꢀ observed.ꢀ Relatedꢀ enzymesꢀ
2
3
enzymesꢀ andꢀ totalꢀ synthesesꢀ usingꢀ conventionalꢀ methods. ꢀ thatꢀchlorinateꢀtheꢀ6-ꢀandꢀ5-positionsꢀofꢀtryptophanꢀhaveꢀalsoꢀ
3
6
Theseꢀcatalystsꢀevolvedꢀtoꢀfunctionalizeꢀparticularꢀsubstrates,ꢀ beenꢀ characterized,ꢀ howeverꢀ (Thalꢀ andꢀ PyrH,ꢀ respectively), ꢀ
2
4
butꢀ directedꢀ evolution ꢀ providesꢀ aꢀ systematicꢀ approachꢀ forꢀ suggestingꢀthatꢀitꢀshouldꢀbeꢀpossibleꢀtoꢀalterꢀRebHꢀselectivity.ꢀ
improvingꢀ enzymeꢀ activity,ꢀ selectivity,ꢀ scope,ꢀ andꢀ otherꢀ Furthermore,ꢀ site-directedꢀ mutagenesisꢀ ofꢀ theꢀ 7-halogenaseꢀ
2
5
properties. ꢀ Withꢀ theꢀ notableꢀ exceptionꢀ ofꢀ cytochromesꢀ PrnAꢀledꢀtoꢀaꢀvariantꢀthatꢀprovidedꢀaꢀ1:2ꢀmixtureꢀofꢀ5-ꢀandꢀ7-
2
6
37
P450, ꢀ however,ꢀ fewꢀ enzymesꢀ thatꢀ functionalizeꢀ C-Hꢀ bondsꢀ chlorotryptophans, ꢀandꢀaꢀsimilarꢀapproachꢀwasꢀusedꢀtoꢀalterꢀ
2
2
haveꢀ beenꢀ evolvedꢀ forꢀ biocatalysis. ꢀ Evenꢀ inꢀ casesꢀ whereꢀ theꢀselectivityꢀofꢀPrnAꢀtowardꢀ2-aminobenzoicꢀacidꢀsoꢀthatꢀ5-ꢀ
enzymesꢀ haveꢀ beenꢀ engineeredꢀ forꢀ selectiveꢀ C-Hꢀ chlorinationꢀ wasꢀ favoredꢀ overꢀ 3-chlorinationꢀ (fromꢀ 84:16ꢀ forꢀ
3
8
functionalization,ꢀ noꢀ selectiveꢀ pressureꢀ wasꢀ appliedꢀ toꢀ alterꢀ PrnAꢀtoꢀ38:62). ꢀWhileꢀtheseꢀexamplesꢀshowꢀthatꢀhalogenaseꢀ
theirꢀ selectivity;ꢀ activeꢀ variantsꢀ wereꢀ identified,ꢀ andꢀ theirꢀ selectivityꢀcanꢀbeꢀaltered,ꢀlowꢀselectivitiesꢀwereꢀobserved,ꢀandꢀ
2
6-28
39
selectivityꢀwasꢀdeterminedꢀpostꢀhoc.
Here,ꢀweꢀshowꢀthatꢀtheꢀselectivityꢀofꢀrebeccamycinꢀhalogenaseꢀ substitutedꢀindolesꢀwereꢀchlorinatedꢀonꢀtheꢀpyrroleꢀring.ꢀGivenꢀ
RebH)ꢀ canꢀ beꢀ evolvedꢀ usingꢀ deuterium-substitutedꢀ probeꢀ
ꢀ
anꢀinitialꢀexaminationꢀofꢀPrnAꢀsubstrateꢀscope ꢀindicatedꢀthatꢀ
(
theꢀabilityꢀofꢀRebHꢀvariantsꢀtoꢀhalogenateꢀtheꢀlessꢀreactiveꢀꢀ
3
3
benzeneꢀ ringꢀ ofꢀ indoleꢀ substrates ꢀ andꢀ ourꢀ successꢀ inꢀ
substratesꢀ inꢀ combinationꢀ withꢀ aꢀ massꢀ spectrometryꢀ assay.ꢀ
Ourꢀresultsꢀconstituteꢀaꢀrareꢀexampleꢀofꢀcatalystꢀoptimizationꢀ
toꢀ enableꢀ C-Hꢀ functionalizationꢀ ortho,ꢀ meta,ꢀ andꢀ paraꢀ toꢀ anꢀ
aromaticꢀsubstituentꢀwithꢀhighꢀselectivityꢀ(Fig.ꢀ1C,ꢀFGꢀ=ꢀCl).ꢀThisꢀ
wasꢀ accomplishedꢀ withoutꢀ theꢀ useꢀ ofꢀ metalsꢀ orꢀ theꢀ harshꢀ
conditionsꢀ typicallyꢀ associatedꢀ withꢀ aromaticꢀ halogenation;ꢀ
33,35
engineeringꢀthisꢀenzyme,
ꢀweꢀinitiatedꢀanꢀeffortꢀtoꢀevolveꢀitsꢀ
selectivityꢀ towardꢀ indoles.ꢀ Theꢀ broadꢀ utilityꢀ ofꢀ substitutedꢀ
indolesꢀ hasꢀ ledꢀ toꢀ theꢀ developmentꢀ ofꢀ aꢀ numberꢀ ofꢀ metal-
catalyzedꢀmethodsꢀforꢀfunctionalizingꢀindoleꢀC-Hꢀbonds.ꢀMostꢀ
4
0
ofꢀtheseꢀtargetꢀtheꢀmoreꢀreactiveꢀpyrroleꢀring, ꢀbutꢀdirectingꢀ
andꢀ blockingꢀ groupꢀ strategiesꢀ haveꢀ beenꢀ usedꢀ toꢀ accessꢀ theꢀ
RebHꢀ isꢀ anꢀ FADH
halideꢀ saltsꢀ asꢀ aꢀ halogenꢀ sourceꢀ andꢀ O
2
-dependentꢀ halogenaseꢀ (FDH)ꢀ thatꢀ usesꢀ
ꢀ asꢀ anꢀ oxidant.ꢀ Theꢀ
2
generalityꢀ ofꢀ theꢀ evolutionꢀ strategyꢀ andꢀ theꢀ selectivityꢀ assayꢀ indoleꢀbenzeneꢀring.ꢀꢀ
usedꢀinꢀthisꢀeffortꢀsuggestꢀthatꢀtheꢀselectivityꢀofꢀotherꢀenzymesꢀ Forꢀ example,ꢀ anꢀ N-silylꢀ directingꢀ groupꢀ wasꢀ usedꢀ toꢀ borylateꢀ
couldꢀbeꢀevolvedꢀinꢀaꢀsimilarꢀfashionꢀtoꢀenableꢀaꢀrangeꢀofꢀnon- theꢀ7-positionꢀofꢀ2-unsubstitutedꢀindolesꢀviaꢀaꢀ3-stepꢀsequenceꢀ
directedꢀC-Hꢀfunctionalizationꢀreactions.ꢀ
ꢀ
ꢀ
ꢀ
ResultsꢀandꢀDiscussion
ꢀ
ꢀ
Figureꢀ1.ꢀA)ꢀSelectiveꢀinstallationꢀofꢀfunctionalꢀgroupsꢀ(FG)ꢀonꢀindolesꢀviaꢀC-HꢀbondꢀactivationꢀusingꢀB)ꢀdifferentꢀcatalystꢀdirectingꢀgroupsꢀ(DG)ꢀorꢀC)ꢀnon-directedꢀenzymeꢀcatalysis.ꢀ
2
ꢀ|ꢀJ.ꢀName.,ꢀ2012,ꢀ00,ꢀ1-3ꢀ
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4
(Fig.ꢀ1A/B). ꢀSubstratesꢀbearingꢀaꢀsubstituentꢀatꢀtheꢀindoleꢀ2- tryptophanꢀ(Fig.ꢀS1-S2),ꢀsoꢀrateꢀdifferencesꢀofꢀtheꢀisotopomersꢀ
1
ꢀ
positionꢀhaveꢀbeenꢀalkenylatedꢀatꢀtheꢀ6-positionꢀusingꢀaꢀsimilarꢀ didꢀ notꢀ haveꢀ toꢀ beꢀ takenꢀ intoꢀ account. ꢀ Halogenationꢀ
4
9
4
2
directingꢀ groupꢀ approach ꢀ andꢀ borylatedꢀ atꢀ theꢀ 7-positionꢀ selectivityꢀcouldꢀbeꢀreliablyꢀdeterminedꢀbyꢀMALDI-MSꢀanalysisꢀ
4
3
usingꢀ onlyꢀ theꢀ indoleꢀ nitrogenꢀ asꢀ aꢀ directingꢀ group ꢀ (Fig.ꢀ ofꢀcrudeꢀreactionꢀmixturesꢀarrayedꢀontoꢀaꢀstandardꢀ384-spotꢀ
A/B).ꢀ Veryꢀ recently,ꢀ anꢀ N-silylꢀ blockingꢀ groupꢀ wasꢀ usedꢀ toꢀ sampleꢀ targets,ꢀ whichꢀ allowedꢀ forꢀ rapidꢀ evaluationꢀ ofꢀ
borylateꢀ theꢀ 6-positionꢀ ofꢀ 3-substitutedꢀ indoles,ꢀ althoughꢀ halogenaseꢀlibraries.ꢀ
1
4
4
significantꢀ 5-borylationꢀ wasꢀ alsoꢀ observed. ꢀ RebHꢀ variantsꢀ
capableꢀofꢀhalogenatingꢀtheꢀbenzeneꢀringꢀofꢀ1,2-unsubstitutedꢀ
indolesꢀ wouldꢀ thusꢀ illustrateꢀ theꢀ potentialꢀ forꢀ enzymesꢀ toꢀ
eliminateꢀ theꢀ needꢀ forꢀ directing/blockingꢀ groupsꢀ typicallyꢀ
requiredꢀforꢀselectiveꢀC-Hꢀfunctionalizationꢀ(Fig.ꢀ1C).ꢀHalogenꢀ
substituentsꢀareꢀknownꢀtoꢀgreatlyꢀimpactꢀtheꢀbiologicalꢀactivityꢀ
ꢀ
4
5,46
ofꢀsmallꢀmolecules
couplingꢀ reactionsꢀ toꢀ accessꢀ additionalꢀ functionality,
makingꢀ halogenationꢀ aꢀ particularlyꢀ usefulꢀ process.ꢀ Moreꢀ
ꢀandꢀcanꢀbeꢀusedꢀforꢀsubsequentꢀcross-
4
7,48
ꢀ
ꢀ
broadly,ꢀ successfulꢀ evolutionꢀ ofꢀ RebHꢀ selectivityꢀ wouldꢀ Figureꢀ2.ꢀMassꢀspectrometryꢀassayꢀforꢀhalogenaseꢀselectivityꢀusingꢀprobeꢀsubstrateꢀ1.ꢀ
establishꢀ aꢀ generalꢀ approachꢀ forꢀ evolvingꢀ theꢀ selectivityꢀ ofꢀ
3
6
otherꢀFDHs, ꢀeachꢀofꢀwhichꢀhasꢀitsꢀownꢀuniqueꢀselectivityꢀandꢀ
substrateꢀscope.ꢀThis,ꢀinꢀturn,ꢀwouldꢀprovideꢀaccessꢀtoꢀaꢀrangeꢀ
ofꢀengineeredꢀhalogenasesꢀforꢀlateꢀstageꢀC-Hꢀfunctionalizationꢀ
ofꢀ syntheticꢀ intermediates,ꢀ naturalꢀ products,ꢀ andꢀ otherꢀ
biologicallyꢀactiveꢀcompounds.ꢀ
Roundsꢀ0-6:ꢀAlteringꢀRebHꢀSelectivityꢀUsingꢀProbeꢀ1ꢀ
TryptamineꢀchlorinationꢀbyꢀseveralꢀRebHꢀvariantsꢀdevelopedꢀinꢀ
3
4,35
ourꢀlaboratory
ꢀwasꢀexaminedꢀtoꢀidentifyꢀaꢀsuitableꢀparentꢀ
forꢀourꢀselectivityꢀevolutionꢀeffortꢀ(summarizedꢀinꢀFig.ꢀ3A).ꢀThisꢀ
analysisꢀrevealedꢀthatꢀvariantsꢀcontainingꢀtheꢀmutationꢀN470Sꢀ
providedꢀtheꢀhighestꢀchlorinationꢀyields,ꢀwhichꢀwereꢀrequiredꢀ
toꢀmaintainꢀaꢀMSꢀsignal-to-noiseꢀratioꢀsufficientꢀforꢀanalysisꢀofꢀ
reactionsꢀ conductedꢀ inꢀ cellꢀ lysate.ꢀ Introducingꢀ thisꢀ mutationꢀ
intoꢀ RebHꢀ gaveꢀ variantꢀ 0S,ꢀ whichꢀ providedꢀ theꢀ highestꢀ
chlorinationꢀ yieldsꢀ ofꢀ allꢀ variantsꢀ evaluatedꢀ (2.5-foldꢀ higherꢀ
thanꢀRebH,ꢀ>99%ꢀ7-chlorination,ꢀFigureꢀ3B).ꢀDespiteꢀtheꢀhighꢀ
selectivityꢀofꢀtheseꢀenzymesꢀforꢀ7-chlorination,ꢀaꢀtraceꢀamountꢀ
ofꢀanꢀadditionalꢀchlorinatedꢀspeciesꢀwasꢀalsoꢀdetectedꢀbyꢀLC-
MS,ꢀandꢀauthenticꢀstandardsꢀwereꢀusedꢀtoꢀestablishꢀthatꢀthisꢀ
wasꢀ 5-ꢀ and/orꢀ 6-chlorotryptamineꢀ (chromatographicꢀ
separationꢀ ofꢀ theseꢀ compoundsꢀ wasꢀ notꢀ possible).ꢀ Weꢀ
believedꢀthatꢀthisꢀactivity,ꢀwhileꢀminor,ꢀwouldꢀbeꢀsufficientꢀtoꢀ
enableꢀevolutionꢀofꢀenzymesꢀwithꢀhighꢀselectivityꢀforꢀbothꢀofꢀ
theseꢀ positions.ꢀ Aꢀ libraryꢀ ofꢀ 0Sꢀ variantsꢀ wasꢀ thereforeꢀ
generatedꢀusingꢀerror-proneꢀPCR,ꢀtheꢀlibraryꢀwasꢀexpressedꢀinꢀ
Escherichiaꢀcoli,ꢀandꢀchlorinationꢀofꢀ1ꢀusingꢀlysatesꢀfromꢀ1000ꢀ
clonesꢀwasꢀevaluatedꢀusingꢀanꢀautomatedꢀMALDI-MSꢀmethod.ꢀ
Theꢀratioꢀofꢀ4ꢀ(m/zꢀ=ꢀ196)ꢀtoꢀ3ꢀ(m/zꢀ=ꢀ195)ꢀwasꢀcalculatedꢀforꢀ
eachꢀ reaction,ꢀ andꢀ hitsꢀ wereꢀ definedꢀ asꢀ thoseꢀ withꢀ 196/195ꢀ
ratiosꢀhigherꢀthanꢀthatꢀofꢀparent.ꢀThisꢀledꢀtoꢀtheꢀidentificationꢀ
ofꢀvariantꢀ1Pꢀ(0S-S448P),ꢀwhichꢀprovidedꢀaꢀ4.5-foldꢀincreaseꢀinꢀ
ꢀ
MALDI-MSꢀasꢀaꢀScreenꢀforꢀSelectivityꢀ
EngineeringꢀRebHꢀvariantsꢀwithꢀalteredꢀselectivityꢀrequiresꢀanꢀ
assayꢀcapableꢀofꢀdifferentiatingꢀhalogenatedꢀproductꢀisomersꢀ
regardlessꢀ ofꢀ theꢀ siteꢀ ofꢀ halogenation.ꢀ Liꢀ andꢀ coworkersꢀ
reportedꢀ aꢀ methodꢀ toꢀ determineꢀ theꢀ enantioselectivityꢀ ofꢀ C-
H/Dꢀ hydroxylationꢀ reactionsꢀ conductedꢀ onꢀ deuterium-
substitutedꢀ substratesꢀ usingꢀ massꢀ spectrometryꢀ (GC/MSꢀ orꢀ
4
9
LC/MS). ꢀWeꢀenvisionedꢀthatꢀdeuteratedꢀtryptaminesꢀcouldꢀbeꢀ
usedꢀinꢀaꢀsimilarꢀfashionꢀtoꢀidentifyꢀRebHꢀvariantsꢀwithꢀalteredꢀ
regioselectivity.ꢀ RebHꢀ halogenatesꢀ tryptamineꢀ withꢀ theꢀ sameꢀ
3
3
highꢀ 7-selectivityꢀ thatꢀ itꢀ exhibitsꢀ onꢀ tryptophan, ꢀ butꢀ
deuteratedꢀ tryptaminesꢀ areꢀ moreꢀ readilyꢀ preparedꢀ thanꢀ theꢀ
correspondingꢀ tryptophans.ꢀ Weꢀ thereforeꢀ preparedꢀ 7-
deuterotryptamine,ꢀ1,ꢀasꢀaꢀprobeꢀsubstrateꢀsinceꢀanyꢀalternateꢀ
regioselectivityꢀ wouldꢀ leadꢀ toꢀ productsꢀ (4)ꢀ withꢀ m/zꢀ 1ꢀ unitꢀ
higherꢀthanꢀthatꢀassociatedꢀwithꢀtheꢀnativeꢀregioselectivityꢀ(3,ꢀ
Fig.ꢀ2).ꢀGivenꢀtheꢀhighꢀselectivityꢀofꢀRebHꢀonꢀtryptamine,ꢀweꢀ
believedꢀ thatꢀ thisꢀ unbiasedꢀ probeꢀ wouldꢀ provideꢀ theꢀ bestꢀ
opportunityꢀ forꢀ identifyingꢀ alteredꢀ selectivityꢀ thatꢀ couldꢀ thenꢀ
beꢀ optimizedꢀ usingꢀ site-specificꢀ probesꢀ (e.g.ꢀ 5-
deuterotryptamine,ꢀ2,ꢀvideꢀinfra).ꢀNoꢀsignificantꢀkineticꢀisotopeꢀ
5
/6-chlorinationꢀselectivity,ꢀindicatingꢀthatꢀRebHꢀselectivityꢀꢀ
5
effectꢀ wasꢀ observedꢀ forꢀ RebH-catalyzedꢀ chlorinationꢀ ofꢀ d -
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
J.ꢀName.,ꢀ2013,ꢀ00,ꢀ1-3ꢀ|ꢀ3ꢀꢀ
ꢀ
Pleaseꢀdoꢀnotꢀadjustꢀmarginsꢀ

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ARTICLEꢀ
JournalꢀNameꢀ
ꢀ
ꢀ
View Article Online
DOI: 10.1039/C5SC04680G
ꢀ
ꢀ
Figureꢀ3.ꢀA)ꢀLineageꢀdiagramꢀshowingꢀmutagenesisꢀmethodsꢀandꢀmutationsꢀfoundꢀinꢀselectedꢀvariantsꢀaboveꢀandꢀbelowꢀtheꢀlineageꢀarrows,ꢀrespectively.ꢀB/C)ꢀYieldꢀofꢀ7-ꢀ(leftꢀy-axis)ꢀ
andꢀ6-ꢀandꢀ5-chlorotryptamineꢀ(rightꢀy-axis)ꢀforꢀdifferentꢀvariantsꢀalongꢀtheꢀhalogenaseꢀlineage.ꢀReactionsꢀconductedꢀusingꢀ2.5ꢀμMꢀMBP-RebF,ꢀ9ꢀU/mLꢀGDH,ꢀ100ꢀmMꢀNaCl,ꢀ20ꢀmMꢀ
glucose,ꢀ100ꢀμMꢀNADꢀandꢀFAD,ꢀ0.5ꢀmMꢀphenol,ꢀ0.5%ꢀv/vꢀi-PrOH/25ꢀmMꢀHEPESꢀbuffer,ꢀpHꢀ7.4,ꢀ25ꢀ°C.ꢀꢀSubstrateꢀandꢀenzymeꢀconcentrations:ꢀB)ꢀ1.5ꢀmMꢀ2,ꢀ15ꢀμMꢀRebHꢀvariant.ꢀꢀC)ꢀ
0
.5ꢀmMꢀ2,ꢀ25ꢀμMꢀRebHꢀvariant.ꢀ
couldꢀ beꢀ alteredꢀ viaꢀ randomꢀ mutagenesisꢀ andꢀ screening.ꢀ 1Pꢀ andꢀ5-chlorotryptamineꢀyieldsꢀviaꢀchromatographicꢀseparationꢀ
wasꢀusedꢀasꢀtheꢀparentꢀforꢀaꢀsecondꢀroundꢀofꢀerror-proneꢀPCRꢀ ofꢀ theꢀ 7-ꢀ andꢀ 5/6-isomersꢀ andꢀ massꢀ differentiationꢀ ofꢀ theꢀ 5-ꢀ
andꢀ screeningꢀ asꢀ describedꢀ above.ꢀ Twoꢀ variantsꢀ providedꢀ andꢀ 6-isomersꢀ (Fig.ꢀ S12).ꢀ Thisꢀ procedureꢀ revealedꢀ thatꢀ 6Sꢀ
increasedꢀyieldsꢀofꢀ5/6-chlorotryptamines,ꢀandꢀtheseꢀꢀ
mutationsꢀ wereꢀ combinedꢀ toꢀ giveꢀ variantꢀ 2RFQꢀ (1P-Q494R,ꢀ analysisꢀofꢀ6T,ꢀ6L,ꢀandꢀvariantsꢀresultingꢀfromꢀrecombinationꢀofꢀ
L380F,ꢀR509Q,ꢀFig.ꢀ3B).ꢀ L111S,ꢀ I52T,ꢀ andꢀ F465Lꢀ indicatedꢀ thatꢀ variantꢀ 6TLꢀ (5LS-I52T,ꢀ
providedꢀ 47%ꢀ selectivityꢀ forꢀ 6-chlorotryptamine.ꢀ Similarꢀ
O’Connorꢀandꢀcoworkersꢀpreviouslyꢀshowedꢀthatꢀtheꢀmutationꢀ F465L)ꢀprovidedꢀtheꢀhighestꢀselectivityꢀforꢀ5-chlorotryptamineꢀ
Y455Wꢀ improvedꢀ theꢀ specificityꢀ ofꢀ RebHꢀ forꢀ tryptamineꢀ overꢀ (39%)ꢀofꢀallꢀmutantsꢀscreenedꢀ(Fig.ꢀ3C).ꢀ
5
tryptophanꢀ withoutꢀ changingꢀ selectivityꢀ forꢀ 7-chlorination. ꢀ
0
ꢀ
Introducingꢀ thisꢀ mutationꢀ intoꢀ 2RFQꢀ toꢀ generateꢀ 3W,ꢀ onꢀ theꢀ Evolvingꢀ5-ꢀandꢀ6-HalogenasesꢀUsingꢀProbeꢀ2:ꢀ
otherꢀhand,ꢀbothꢀdecreasedꢀ7-chlorinationꢀandꢀincreasedꢀ5/6-
Despiteꢀ theꢀ significantꢀ improvementꢀ inꢀ aromaticꢀ chlorinationꢀ
chlorinationꢀ (Fig.ꢀ 3B).ꢀ Error-proneꢀ PCRꢀ ofꢀ 3Wꢀ followedꢀ byꢀ
recombinationꢀofꢀbeneficialꢀmutationsꢀledꢀtoꢀvariantꢀ4PLꢀ(3W-
S110P,ꢀ F111L),ꢀ whichꢀ furtherꢀ decreasedꢀ 7-chlorinationꢀ (Fig.ꢀ
selectivityꢀ andꢀ highꢀ tryptamineꢀ conversionꢀ inꢀ reactionsꢀ
catalyzedꢀbyꢀ6S,ꢀisolationꢀofꢀtheꢀproductsꢀfromꢀtheseꢀreactionsꢀ
revealedꢀthatꢀ2-oxotryptamineꢀwasꢀalsoꢀbeingꢀformed.ꢀAnalysisꢀ
ofꢀ theꢀ halogenaseꢀ lineageꢀ indicatedꢀ thatꢀ thisꢀ productꢀ onlyꢀ
formedꢀ toꢀ aꢀ significantꢀ extentꢀ afterꢀ theꢀ F111Sꢀ mutationꢀ wasꢀ
introducedꢀintoꢀ5LSꢀtoꢀgiveꢀ6S.ꢀSinceꢀvariantsꢀ6Tꢀ(5LS-I52T)ꢀandꢀ
3
C).ꢀ Toꢀ improveꢀ overallꢀ conversion,ꢀ aꢀ numberꢀ ofꢀ mutationsꢀ
3
4
previouslyꢀ foundꢀ toꢀ improveꢀ theꢀ stabilityꢀ ofꢀ RebH ꢀ wereꢀ
individuallyꢀ introducedꢀ intoꢀ 4PL.ꢀ Beneficialꢀ mutationsꢀ wereꢀ
againꢀ combinedꢀ toꢀ generateꢀ variantꢀ 5LSꢀ (4PL-S130L,ꢀ N166S),ꢀ
whichꢀwasꢀusedꢀasꢀaꢀparentꢀforꢀanotherꢀroundꢀofꢀerror-proneꢀ
PCRꢀ andꢀ MALDIꢀ screening.ꢀ Threeꢀ variantsꢀ fromꢀ thisꢀ library,ꢀ
eachꢀ containingꢀ anꢀ activeꢀ siteꢀ mutation,ꢀ wereꢀ foundꢀ toꢀ
increaseꢀ 5/6-chlorinationꢀ toꢀ levelsꢀ approachingꢀ orꢀ exceedingꢀ
thatꢀofꢀtheꢀresidualꢀ7-chlorinationꢀ(6S,ꢀ5LS-L111S;ꢀ6T,ꢀ5LS-I52T;ꢀ
6
Lꢀ (5LS-F465L)ꢀ alsoꢀ showedꢀ significantꢀ 5-ꢀ andꢀ 6-chlorinationꢀ
withoutꢀoxotryptamineꢀformation,ꢀindividualꢀrandomizationꢀofꢀ
residuesꢀ52ꢀandꢀ465ꢀinꢀ6TLꢀbyꢀsiteꢀdirectedꢀmutagenesisꢀwithꢀ
NNKꢀ codonsꢀ wasꢀ pursuedꢀ asꢀ aꢀ meansꢀ toꢀ furtherꢀ optimizeꢀ
chlorinationꢀselectivityꢀ(Fig.ꢀ3A).ꢀ
Theꢀ resultingꢀ librariesꢀ wereꢀ screenedꢀ forꢀ activityꢀ onꢀ 2ꢀ byꢀ
6
L,ꢀ 5LS-F465L).ꢀ Toꢀ distinguish,ꢀ andꢀ thusꢀ provideꢀ aꢀ meansꢀ toꢀ
sequentialꢀ MALDI-MS/UPLCꢀ toꢀ determineꢀ 7-,ꢀ 6-,ꢀ andꢀ 5-
chlorotryptamineꢀyieldsꢀ(seeꢀsupportingꢀinformation).ꢀSeveralꢀ
hitsꢀ wereꢀ identified,ꢀ includingꢀ 7Mꢀ (6TL-T52M)ꢀ andꢀ 7Hꢀ (6TL-
individuallyꢀoptimizeꢀ5-ꢀandꢀ6-chlorination,ꢀsite-selectiveꢀprobeꢀ
,ꢀ 5-deuterotryptamine,ꢀ wasꢀ prepared.ꢀ LC-MSꢀ analysisꢀ ofꢀ
reactionsꢀ conductedꢀ singꢀ ꢀ canꢀ beꢀ usedꢀ toꢀ determineꢀ 7-,ꢀ 6-,ꢀ
2
2
4
ꢀ|ꢀJ.ꢀName.,ꢀ2012,ꢀ00,ꢀ1-3ꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
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T52H),ꢀ whichꢀ possessꢀ improvedꢀ selectivityꢀ forꢀ 6-ꢀ andꢀ 5- Theꢀcatalyticꢀefficienciesꢀforꢀ0S,ꢀ10Sꢀandꢀ8Fꢀwereꢀcomparedꢀbyꢀ
chlorination,ꢀrespectively.ꢀDegenerateꢀNNKꢀcodonsꢀwereꢀthenꢀ steadyꢀ stateꢀ kineticꢀ analysis.ꢀ ꢀ Theꢀ enzymeꢀ loadingꢀ
introducedꢀ atꢀ residueꢀ 465ꢀ ofꢀ theseꢀ variants,ꢀ andꢀ whileꢀ noꢀ requirementsꢀ forꢀ theseꢀ reactionsꢀ areꢀ reflectedꢀ inꢀ theꢀ kineticꢀ
m
improvementsꢀ wereꢀ observedꢀ inꢀ theꢀ 7Mꢀ libraryꢀ (Fig.ꢀ 3A),ꢀ parameters,ꢀwithꢀ0Sꢀmaintainingꢀaꢀsignificantlyꢀhigherꢀkcat/K ꢀ
variantꢀ 8Cꢀ (7H-F465C)ꢀ providedꢀ 86%ꢀ selectivityꢀ forꢀ 5- thanꢀ 10Sꢀ andꢀ 8Fꢀ (Tableꢀ 1).ꢀ ꢀ Thisꢀ lossꢀ inꢀ catalyticꢀ efficiencyꢀ isꢀ
chlorinationꢀ (upꢀ fromꢀ 39%ꢀ withꢀ 6TL).ꢀ Despiteꢀ theꢀ improvedꢀ partiallyꢀ dueꢀ toꢀ aꢀ reductionꢀ inꢀ kcat,ꢀ althoughꢀ bothꢀ 5-ꢀ andꢀ 6-
3
3
selectivityꢀofꢀ7Mꢀandꢀ8C,ꢀbothꢀofꢀtheseꢀvariantsꢀprovidedꢀlowꢀ halogenasesꢀdisplayꢀslightlyꢀhigherꢀkcatꢀvaluesꢀthanꢀRebH. ꢀTheꢀ
3
3
productꢀyields.ꢀGivenꢀtheꢀsignificantꢀimpactꢀofꢀresidueꢀ111ꢀonꢀ
M
K ꢀ ofꢀ theꢀ 7-halogenaseꢀ matchedꢀ thatꢀ ofꢀ RebH, ꢀ whileꢀ theꢀ
bothꢀ halogenaseꢀ activityꢀ andꢀ selectivity,ꢀ weꢀ examinedꢀ theꢀ mutantsꢀ withꢀ non-nativeꢀ selectivity,ꢀ 10Sꢀ andꢀ 8F,ꢀ displayedꢀ
effectsꢀofꢀmutatingꢀthisꢀresidueꢀinꢀbothꢀ7Mꢀandꢀ8Cꢀ(Fig.ꢀ3A).ꢀ substantiallyꢀ higherꢀ KMꢀ values,ꢀ suggestingꢀ weakerꢀ substrateꢀ
Siteꢀdirectedꢀmutagenesisꢀofꢀresidueꢀ111ꢀinꢀ7MꢀusingꢀanꢀNNKꢀ binding.ꢀ Whileꢀ substrateꢀ inhibitionꢀ hasꢀ beenꢀ observedꢀ
3
1
codonꢀ wasꢀ usedꢀ toꢀ randomizeꢀ thisꢀ site.ꢀ Remarkably,ꢀ theꢀ previouslyꢀforꢀtryptophanꢀhalogenases, ꢀthisꢀwasꢀnotꢀseenꢀforꢀ
variantꢀwithꢀtheꢀhighestꢀselectivityꢀforꢀ6-chlorinationꢀ(85%)ꢀasꢀ eitherꢀ8Fꢀorꢀ10Sꢀatꢀtheꢀconcentrationsꢀinvestigatedꢀ(upꢀtoꢀ2.5ꢀ
wellꢀ asꢀ theꢀ highestꢀ yieldꢀ ofꢀ 6-chlorotryptamineꢀ (11-foldꢀ andꢀ4.5ꢀmM,ꢀꢀFig.ꢀS16-20).ꢀ
increaseꢀ overꢀ 7M)ꢀ fromꢀ thisꢀ library,ꢀ 8Fꢀ (7M-L111F),ꢀ resultedꢀ
fromꢀreversionꢀofꢀtheꢀF111Lꢀmutationꢀthatꢀoriginallyꢀledꢀtoꢀaꢀ
significantꢀchangeꢀinꢀ7-selectivityꢀofꢀ3W.ꢀGivenꢀthisꢀfinding,ꢀweꢀ
revertedꢀthisꢀsameꢀmutationꢀandꢀseveralꢀadditionalꢀmutationsꢀ
inꢀ8Cꢀtoꢀgenerateꢀ10S,ꢀwhichꢀledꢀtoꢀaꢀ5-foldꢀimprovementꢀinꢀ
yieldꢀ whileꢀ maintainingꢀ itsꢀ highꢀ selectivityꢀ forꢀ 5-chlorinationꢀ
ꢀ
Tableꢀ1.ꢀKineticꢀparametersꢀforꢀRebH,ꢀ0S,ꢀ8F,ꢀandꢀ10S.ꢀ
-
1
-1
Enzymeꢀ
K
m
ꢀ(µM)ꢀ
k
catꢀ(min )ꢀ
m
kcat/K ꢀ(min•µM) ꢀ
b
RebH ꢀ
9ꢀ
0.023ꢀ
2.6E-03ꢀ
2.6E-02ꢀ
2.1E-05ꢀ
1.8E-04ꢀ
0
8
Sꢀ
Fꢀ
10.6ꢀ
1747ꢀ
160ꢀ
0.135ꢀ
0.037ꢀ
(87%).ꢀꢀ
IsolatedꢀYields,ꢀKinetics,ꢀandꢀSubstrateꢀScopeꢀ
1
0Sꢀ
0.028ꢀ
a
2
-4500ꢀ μMꢀ tryptamine,ꢀ 2.5ꢀ μMꢀ MBP-RebF,ꢀ 9ꢀ U/mLꢀ GDH,ꢀ 100ꢀ mMꢀ NaCl,ꢀ 20ꢀ mMꢀ
Optimizationꢀofꢀreactionꢀconditionsꢀtoꢀmaximizeꢀproductꢀyieldsꢀ
usingꢀ 0S,ꢀ 8F,ꢀ andꢀ 10Sꢀ indicatedꢀ thatꢀ 8Fꢀ andꢀ 10Sꢀ gaveꢀ higherꢀ
yieldsꢀatꢀlowerꢀtemperaturesꢀ(16ꢀandꢀ10ꢀ°C,ꢀrespectively)ꢀandꢀ
thatꢀ 100ꢀ mMꢀ NaClꢀ furtherꢀ increasedꢀ yieldsꢀ inꢀ reactionsꢀ
catalyzedꢀbyꢀ10S.ꢀWhileꢀhigherꢀratesꢀwereꢀobservedꢀforꢀ8Fꢀandꢀ
glucose,ꢀ100ꢀμMꢀNADꢀandꢀFAD,ꢀ0.5ꢀmMꢀphenol,ꢀ2.5%ꢀv/vꢀDMSO/25ꢀmMꢀHEPESꢀbufferꢀ
pHꢀ7.4,ꢀ25ꢀ°C.ꢀꢀ0.1ꢀμMꢀ0S,ꢀ25ꢀμMꢀ10S,ꢀ25ꢀμMꢀ8F.ꢀꢀTimeꢀpointsꢀcollectedꢀfromꢀ10-60ꢀ
b
33
minutes.ꢀ Valuesꢀtakenꢀfromꢀaꢀpreviousꢀstudy. ꢀꢀ
GivenꢀthatꢀRebHꢀhalogenatesꢀ(Xꢀ=ꢀCl,ꢀBr)ꢀaꢀnumberꢀofꢀindoleꢀ
3
3
derivativesꢀ withꢀ highꢀ selectivity, ꢀ theꢀ activityꢀ ofꢀ 8Fꢀ andꢀ 10Sꢀ
wasꢀ evaluatedꢀ onꢀ severalꢀ compoundsꢀ (Tableꢀ 2),ꢀ includingꢀ 2-
methyltryptamineꢀ (entryꢀ 3),ꢀ N-methyltryptamineꢀ (entryꢀ 4),ꢀ
tryptopholꢀ(entryꢀ5),ꢀandꢀtryptophan.ꢀ8Fꢀprovidedꢀasꢀgoodꢀorꢀ
betterꢀselectivityꢀforꢀ6-chlorinationꢀofꢀtheseꢀsubstratesꢀthanꢀitꢀ
didꢀ onꢀ tryptamineꢀ whileꢀ providingꢀ reasonableꢀ toꢀ excellentꢀ
yields.ꢀ Onꢀ theꢀ otherꢀ hand,ꢀ 10Sꢀ hadꢀ essentiallyꢀ perfectꢀ
selectivityꢀforꢀ5-chlorinationꢀofꢀN-methyltryptamineꢀandꢀgoodꢀ
selectivityꢀ forꢀ 5-chlorinationꢀ ofꢀ tryptopholꢀ butꢀ lowꢀ selectivityꢀ
onꢀ2-methyltryptamineꢀ(reasonableꢀtoꢀgoodꢀyieldsꢀwereꢀagainꢀ
observed).ꢀꢀ
1
0Sꢀ withꢀ higherꢀ substrateꢀ concentrations,ꢀ 0.5ꢀ mMꢀ substrateꢀ
wasꢀ usedꢀ toꢀ maximizeꢀ conversionꢀ ratherꢀ thanꢀ totalꢀ turnoverꢀ
numbers.ꢀ Theꢀ selectivityꢀ ofꢀ theseꢀ enzymesꢀ remainedꢀ
essentiallyꢀ unchangedꢀ despiteꢀ theseꢀ variations.ꢀ Tryptamineꢀ
chlorinationꢀreactionsꢀ(10ꢀmg)ꢀwereꢀthenꢀconductedꢀusingꢀtheꢀ
optimalꢀconditionsꢀandꢀloadingꢀforꢀeachꢀenzymeꢀ(Fig.ꢀ4).ꢀGoodꢀ
yieldsꢀ (73-98%)ꢀ andꢀ highꢀ selectivitiesꢀ (90-100%)ꢀ wereꢀ
obtained.ꢀꢀ
Neitherꢀ 8Fꢀ norꢀ 10Sꢀ catalyzedꢀ chlorinationꢀ ofꢀ tryptophanꢀ (0.5ꢀ
mM),ꢀ theꢀ nativeꢀ substrateꢀ ofꢀ RebHꢀ (Fig.ꢀ S27).ꢀ Thisꢀ lackꢀ ofꢀ
activityꢀcanꢀbeꢀrationalizedꢀforꢀ8FꢀsinceꢀitꢀincludesꢀtheꢀY455Wꢀ
mutationꢀ knownꢀ toꢀ improveꢀ RebHꢀ specificityꢀ forꢀ tryptamineꢀ
5
0
overꢀ tryptophan, ꢀ butꢀ theꢀ absenceꢀ ofꢀ thisꢀ mutationꢀ inꢀ 10Sꢀ
makesꢀtheꢀoriginꢀofꢀitsꢀalteredꢀꢀsubstrateꢀspecificityꢀlessꢀclear.ꢀ
Inꢀ addition,ꢀ whileꢀ bothꢀ 8Fꢀ andꢀ 10Sꢀ catalyzeꢀ tryptamineꢀ
bromination,ꢀ onlyꢀ modestꢀ selectivityꢀ wasꢀ observedꢀ (Tableꢀ 2,ꢀ
ꢀ
a
Figureꢀ 4.ꢀ Chlorinationꢀ ofꢀ tryptamineꢀ usingꢀ engineeredꢀ halogenases.ꢀ Conversionꢀ ofꢀ entryꢀ 2).ꢀ Theseꢀ findingsꢀ contrastꢀ withꢀ ourꢀ previousꢀ resultsꢀ
b
startingꢀ materialꢀ determinedꢀ byꢀ UPLCꢀ analysisꢀ ofꢀ crudeꢀ reactionꢀ mixtures.ꢀ Isolatedꢀ showingꢀ thatꢀ engineeredꢀ RebHꢀ variantsꢀ typicallyꢀ maintainꢀ
c
yieldꢀofꢀpureꢀproduct.ꢀ SelectivityꢀdeterminedꢀbyꢀNMRꢀanalysisꢀofꢀaꢀpurifiedꢀmixtureꢀofꢀ
35
activityꢀ onꢀ tryptophan ꢀ andꢀ catalyzeꢀ chlorinationꢀ andꢀ
d
isomersꢀ(inseparableꢀbyꢀpreparativeꢀchromatography).ꢀ 10ꢀμMꢀ0S,ꢀ0.5ꢀmMꢀtryptamineꢀ
33
brominationꢀ withꢀ similarꢀ selectivity .ꢀ Onꢀ theꢀ otherꢀ hand,ꢀ anꢀ
(
10ꢀmg),ꢀ2.5ꢀμMꢀMBP-RebF,ꢀ9ꢀU/mLꢀGDH,ꢀ10ꢀmMꢀNaCl,ꢀ20ꢀmMꢀglucose,ꢀ100ꢀμMꢀNADꢀ
e
initialꢀ investigationꢀ ofꢀ 10Sꢀ substrateꢀ scopeꢀ revealedꢀ thatꢀ itꢀ
chlorinatesꢀseveralꢀadditionalꢀsubstratesꢀandꢀprovidesꢀalteredꢀ
productꢀdistributionsꢀonꢀtheseꢀsubstratesꢀrelativeꢀtoꢀRebHꢀ(Fig.ꢀ
andꢀFAD,ꢀ0.5%ꢀv/vꢀi-PrOH/25ꢀmMꢀHEPESꢀbuffer,ꢀpHꢀ7.4,ꢀ25ꢀ°C.ꢀ Asꢀinꢀ(d)ꢀbutꢀ50ꢀμMꢀ8F,ꢀ
f
1
6ꢀ°C.ꢀ Asꢀinꢀ(d)ꢀbutꢀ50ꢀμMꢀ10S,ꢀ100ꢀmMꢀNaCl,ꢀ10ꢀ°C.ꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
J.ꢀName.,ꢀ2013,ꢀ00,ꢀ1-3ꢀ|ꢀ5ꢀ
ꢀ
Pleaseꢀdoꢀnotꢀadjustꢀmarginsꢀ

Pleas Ce hꢀ d eo mꢀ ni oc ta ꢀ la dS jcu i se t nꢀ mc ea rginsꢀ
Page 6 of 11
ARTICLEꢀ
JournalꢀNameꢀ
ꢀ
View Article Online
DOI: 10.1039/C5SC04680G
S28).ꢀꢀTheseꢀdataꢀshowꢀthatꢀ10Sꢀandꢀ8F,ꢀwhichꢀwereꢀevolvedꢀ pointsꢀinꢀtheꢀselectivityꢀlineageꢀ(WT-0S,ꢀ1P-2RFQ,ꢀ8C-10S,ꢀandꢀ
forꢀ alteredꢀ selectivityꢀ onꢀ tryptamine,ꢀ canꢀ halogenateꢀ 7M-8F),ꢀ theꢀ percentꢀ yieldꢀ ofꢀ theꢀ majorꢀ chlorotryptamineꢀ
substratesꢀ otherꢀ thanꢀ tryptamineꢀ withꢀ alteredꢀ selectivity.ꢀ isomerꢀ(7,ꢀ6,ꢀorꢀ5-Cl)ꢀwasꢀsignificantlyꢀimprovedꢀ(1.7-11ꢀfold)ꢀ
Whileꢀdeviationsꢀfromꢀexpectedꢀactivityꢀonꢀindolesꢀcanꢀresultꢀ whileꢀimprovingꢀorꢀnotꢀaffectingꢀselectivityꢀ(Fig.ꢀ3B/C;ꢀFig.ꢀS29).ꢀ
fromꢀ minorꢀ structuralꢀ differences,ꢀ evenꢀ majorꢀ structuralꢀ Thisꢀfindingꢀisꢀconsistentꢀwithꢀourꢀpreviousꢀevolutionꢀeffortsꢀinꢀ
differencesꢀareꢀtoleratedꢀinꢀmanyꢀcases.ꢀꢀ
whichꢀactivityꢀonꢀnon-nativeꢀsubstratesꢀwasꢀimprovedꢀwithoutꢀ
3
5
sacrificingꢀ theꢀ observedꢀ selectivity. ꢀ Sewaldꢀ hasꢀ alsoꢀ shownꢀ
thatꢀcross-linkedꢀRebHꢀcanꢀbeꢀusedꢀtoꢀhalogenateꢀsubstitutedꢀ
Tableꢀ 2.ꢀ Conversionꢀ andꢀ selectivityꢀ forꢀ halogenationꢀ ofꢀ differentꢀ substratesꢀ usingꢀ 8Fꢀ
andꢀ10S.ꢀ
5
3
tryptophansꢀonꢀgramꢀscale. ꢀTheseꢀapproachesꢀtoꢀimprovingꢀ
halogenaseꢀ activityꢀ andꢀ reactionꢀ scale,ꢀ coupledꢀ withꢀ ourꢀ
methodꢀforꢀevolvingꢀhalogenaseꢀselectivity,ꢀprovideꢀaꢀgeneralꢀ
frameworkꢀforꢀimprovingꢀhalogenasesꢀforꢀselectiveꢀcatalysis.ꢀ
ꢀ
ꢀ
1
2
a
b
Entryꢀ R ꢀ
R ꢀ
Xꢀ
8Fꢀ(6-halogenase) ꢀ
10Sꢀ(5-halogenase) ꢀ
c
d
c
d
Conv.ꢀ(%) ꢀ 6-Xꢀ(%) ꢀ Conv.ꢀ(%) ꢀ 5-Xꢀ(%) ꢀ
TryptamineꢀHaleniumꢀAffinityꢀ
e
1
ꢀ
Hꢀ
Hꢀ
NH
NH
2
ꢀ
Clꢀ
74ꢀ
90ꢀ
83ꢀ
95ꢀ
Asꢀ previouslyꢀ noted,ꢀ RebHꢀ catalysisꢀ isꢀ believedꢀ toꢀ involveꢀ
electrophilicꢀaromaticꢀsubstitutionꢀofꢀenzyme-boundꢀsubstrateꢀ
byꢀaꢀproximalꢀhaleniumꢀionꢀdonor,ꢀbelievedꢀtoꢀbeꢀeitherꢀaꢀK79ꢀ
2
ꢀ
2
2
ꢀ
ꢀ
Brꢀ
Clꢀ
84ꢀ
97ꢀ
69ꢀ
99ꢀ
35ꢀ
77ꢀ
59ꢀ
24ꢀ
3
4
5
ꢀ
ꢀ
ꢀ
Meꢀ NH
Hꢀ NHMeꢀ Clꢀ
Hꢀ OHꢀ Clꢀ
54ꢀ
48ꢀ
98ꢀ
89ꢀ
74ꢀ
48ꢀ
>99ꢀ
84ꢀ
3
0-32
haloamineꢀorꢀHOX.
1
ꢀTheꢀobservedꢀselectivityꢀofꢀ0S,ꢀ8F,ꢀandꢀ
0Sꢀ towardꢀ 3-substitutedꢀ indolesꢀ showsꢀ thatꢀ theseꢀ enzymesꢀ
a
5
0ꢀμMꢀ8F,ꢀ0.5ꢀmMꢀsubstrateꢀ(1-2ꢀmg),ꢀ2.5ꢀμMꢀMBP-RebF,ꢀ9ꢀU/mLꢀGDH,ꢀ10ꢀmMꢀ
canꢀ differentiateꢀ similarlyꢀ reactiveꢀ sitesꢀ onꢀ indoleꢀ benzeneꢀ
ringsꢀ bothꢀ fromꢀ oneꢀ anotherꢀ andꢀ fromꢀ theꢀ moreꢀ reactiveꢀ
NaCl,ꢀ 20ꢀ mMꢀ glucose,ꢀ 100ꢀ μMꢀ NADꢀ andꢀ FAD,ꢀ 0.5%ꢀ v/vꢀ i-PrOH/25ꢀ mMꢀ HEPESꢀ
b
bufferꢀ pHꢀ 7.4,ꢀ 16ꢀ °C.ꢀ Sameꢀ asꢀ inꢀ (a)ꢀ butꢀ withꢀ 50ꢀ μMꢀ 10Sꢀ andꢀ 100ꢀ mMꢀ NaCl.ꢀ
3
9,54
c
d
Conversionꢀ determinedꢀ byꢀ UPLCꢀ Selectivityꢀ determinedꢀ byꢀ NMRꢀ analysisꢀ ofꢀ aꢀ indoleꢀ pyrroleꢀ ring.
ꢀ Similarꢀ selectivityꢀ preferencesꢀ areꢀ aꢀ
3
6
purifiedꢀmixtureꢀofꢀinseparableꢀisomersꢀ(X=Cl)ꢀorꢀbyꢀLCMSꢀanalysisꢀofꢀreactionsꢀ hallmarkꢀ ofꢀ tryptophanꢀ halogenationꢀ byꢀ nativeꢀ FDHs, ꢀ butꢀ aꢀ
e
conductedꢀusingꢀprobeꢀ2ꢀ(X=Br).ꢀ Dataꢀfromꢀpreparativeꢀreactionꢀ(Fig.ꢀ4).ꢀ
quantitativeꢀ evaluationꢀ ofꢀ haleniumꢀ ionꢀ reactivityꢀ towardꢀ
differentꢀ substrateꢀ sites,ꢀ andꢀ thusꢀ theꢀ extentꢀ toꢀ whichꢀ FDHsꢀ
Theꢀ novelꢀ scopeꢀ andꢀ selectivityꢀ ofꢀ halogenasesꢀ alongꢀ ourꢀ
overrideꢀ theꢀ chemoselectivityꢀ ofꢀ substratesꢀ towardꢀ haleniumꢀ
selectivityꢀlineageꢀmakeꢀtheseꢀenzymesꢀpromisingꢀcatalystsꢀforꢀꢀ
50,52
5
lateꢀ stageꢀ halogenation ꢀ andꢀ metabolicꢀ engineering
1
ionꢀ donors,ꢀ suchꢀ asꢀ HOXꢀ orꢀ haloamines,ꢀ hasꢀ notꢀ beenꢀ
reported.ꢀCalculatedꢀhaleniumꢀaffinityꢀ(HalA)ꢀhasꢀbeenꢀusedꢀtoꢀ
predictꢀ theꢀ reactivityꢀ ofꢀ aꢀ wideꢀ rangeꢀ ofꢀ substratesꢀ towardꢀ
.ꢀ
Achievingꢀ highꢀ isolatedꢀ yieldsꢀ inꢀ larger-scaleꢀ reactionsꢀ (>10ꢀ
mg),ꢀonꢀtheꢀotherꢀhand,ꢀwillꢀrequireꢀsignificantꢀimprovementsꢀ
inꢀtheꢀactivityꢀofꢀtheseꢀenzymes.ꢀWhileꢀtheꢀfocusꢀofꢀthisꢀstudyꢀ
wasꢀchangingꢀRebHꢀselectivity,ꢀourꢀdataꢀshowꢀthatꢀactivityꢀcanꢀ
alsoꢀbeꢀimprovedꢀwithoutꢀdecreasingꢀselectivity.ꢀAtꢀseveralꢀꢀ
ꢀ
5
5
haleniumꢀionꢀdonors. ꢀTheꢀcalculatedꢀHalAꢀvaluesꢀ(Xꢀ=ꢀCl)ꢀforꢀ
theꢀ2-,ꢀ4-,ꢀ5-,ꢀ6-ꢀandꢀ7-positionsꢀofꢀtryptamineꢀwereꢀ177,ꢀ166,ꢀ
163,ꢀ 166,ꢀ andꢀ 161ꢀ kcal/mol,ꢀ respectivelyꢀ (seeꢀ supportingꢀ
information).ꢀByꢀthisꢀmeasure,ꢀ0Sꢀchlorinatesꢀtheꢀleastꢀreactiveꢀꢀ
ꢀ
Figureꢀ5.ꢀA)ꢀKeyꢀresiduesꢀinꢀtheꢀRebHꢀ(greyꢀcarbons)ꢀandꢀPyrHꢀ(lightꢀblueꢀcarbons)ꢀactiveꢀsites.ꢀInteractionsꢀinvolvedꢀinꢀtryptophanꢀbindingꢀinꢀB)ꢀRebHꢀandꢀC)ꢀPyrH.ꢀArrowꢀdenotesꢀ
chlorinationꢀsite.ꢀ
ꢀ
6
ꢀ|ꢀJ.ꢀName.,ꢀ2012,ꢀ00,ꢀ1-3ꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
ꢀ
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Pleas Ce hꢀ d eo mꢀ ni oc ta ꢀ la dS jcu i se t nꢀ mc ea rginsꢀ
View Article Online
DOI: 10.1039/C5SC04680G
JournalꢀNameꢀ
ARTICLEꢀ
ꢀ
ꢀ
siteꢀonꢀtryptamine,ꢀ8Fꢀandꢀ10Sꢀselectivelyꢀchlorinateꢀsitesꢀthatꢀ halogenation.ꢀNotably,ꢀhowever,ꢀbothꢀRebHꢀandꢀPyrHꢀpossessꢀ
differꢀ inꢀ reactivityꢀ byꢀ onlyꢀ 3ꢀ kcal/mol,ꢀ andꢀ allꢀ threeꢀ enzymesꢀ backboneꢀamidesꢀsuitableꢀforꢀN-Hꢀhydrogenꢀbondingꢀinꢀeitherꢀ
chlorinateꢀsitesꢀsubstantiallyꢀlessꢀreactiveꢀthanꢀtheꢀ2-position.ꢀ orientationꢀ (C
7
-H:ꢀ E357/E354;ꢀ C
5
-H:ꢀ S110/P93).ꢀ Additionalꢀ
Allꢀ ofꢀ theꢀ engineeredꢀ halogenasesꢀ thereforeꢀ overrideꢀ theꢀ interactions,ꢀ includingꢀ
π
-stackingꢀ betweenꢀ tryptophanꢀ andꢀ
expectedꢀ haleniumꢀ ionꢀ chemoselectivityꢀ towardꢀ tryptamine,ꢀ W466ꢀ inꢀ RebHꢀ andꢀ aꢀ secondꢀ indoleꢀ N-Hꢀ hydrogenꢀ bondꢀ toꢀ
butꢀ theꢀ abilityꢀ ofꢀ 8Fꢀ andꢀ 10Sꢀ toꢀ accomplishꢀ thisꢀ featꢀ isꢀ Y454ꢀ inꢀ PyrH,ꢀ haveꢀ beenꢀ proposedꢀ toꢀ favorꢀ theꢀ substrateꢀ
5
7
particularlyꢀ notableꢀ givenꢀ theirꢀ relativelyꢀ weakꢀ substrateꢀ orientationꢀobservedꢀforꢀeachꢀenzyme. ꢀꢀ
bindingꢀ(Tableꢀ1).ꢀAssumingꢀK ꢀcanꢀbeꢀapproximatedꢀbyꢀK ꢀforꢀ Withꢀ theseꢀ aspectsꢀ ofꢀ tryptophan-RebHꢀ andꢀ -PyrHꢀ bindingꢀ inꢀ
Gꢀforꢀtryptamineꢀbindingꢀtoꢀ8Fꢀandꢀ10Sꢀ mind,ꢀ dockingꢀ simulationsꢀ wereꢀ usedꢀ toꢀ identifyꢀ bindingꢀ
d
M
5
6
theseꢀenzymes, ꢀtheꢀ
Δ
isꢀ onlyꢀ 3.8ꢀ andꢀ 5.2ꢀ kcal/mol,ꢀ respectively,ꢀ showingꢀ thatꢀ evenꢀ interactionsꢀ inꢀ tryptamineꢀ posesꢀ consistentꢀ withꢀ theꢀ FDHꢀ
relativelyꢀ weakꢀ bindingꢀ canꢀ overcomeꢀ largeꢀ differencesꢀ (>10ꢀ mechanismꢀandꢀselectivityꢀofꢀ0S,ꢀ8F,ꢀandꢀ10S.ꢀMechanisticallyꢀ
kcal/mol)ꢀinꢀHalA.ꢀ
ꢀ
TryptophanꢀBindingꢀandꢀTryptamineꢀDockingꢀ
relevantꢀposesꢀwereꢀtakenꢀtoꢀbeꢀthoseꢀinꢀwhichꢀtheꢀtryptamineꢀ
indoleꢀbindsꢀinꢀaꢀplanarꢀorientationꢀbetweenꢀH109/F111ꢀwithꢀ
anꢀaromaticꢀC-Hꢀbondꢀatꢀtheꢀsiteꢀoccupiedꢀbyꢀtheꢀtryptophanꢀ
5
7
7
C -Hꢀ bondꢀ inꢀ theꢀ tryptophan-RebHꢀ complexꢀ (Fig.ꢀ 5A/B). ꢀ Toꢀ
Betterꢀunderstandingꢀofꢀhowꢀsubstrateꢀbindingꢀinꢀ0S,ꢀ8F,ꢀandꢀ
5
9
validateꢀ thisꢀ approach,ꢀ AutoDockꢀ Vina ꢀ wasꢀ usedꢀ toꢀ dockꢀ
tryptophanꢀintoꢀanꢀapoꢀRebHꢀstructureꢀminimizedꢀusingꢀtheꢀꢀ
1
0Sꢀ mightꢀ controlꢀ haleniumꢀ selectivityꢀ canꢀ beꢀ gleanedꢀ fromꢀ
previousꢀworkꢀonꢀtheꢀselectivityꢀofꢀRebH-ꢀandꢀPyrH-catalyzedꢀ
6
0
5
tryptophanꢀchlorination. ꢀAligningꢀtheꢀstructuresꢀofꢀtheꢀRebH-
7
GROMOSꢀ 43B1ꢀ forceꢀ fieldꢀ inꢀ Swiss-PDBViewer. ꢀ Theꢀ lowestꢀ
energyꢀposesꢀwereꢀconsistentꢀwithꢀtheꢀbindingꢀobservedꢀinꢀtheꢀ
5
8
57
andꢀ PyrH-tryptophan ꢀ complexesꢀ (Fig.ꢀ 5A)ꢀ showsꢀ thatꢀ theꢀ
ꢀ
5
8
crystalꢀstructureꢀofꢀtheꢀRebH-tryptophanꢀcomplexꢀ(Fig.ꢀS23), ꢀ
althoughꢀ higherꢀ energyꢀ posesꢀ withꢀ tryptophanꢀ boundꢀ inꢀ
7 5
chlorinatedꢀ sitesꢀ (RebH,ꢀ C -H;ꢀ PyrH,ꢀ C -H)ꢀ lieꢀ atꢀ nearlyꢀ theꢀ
sameꢀpointꢀandꢀroughlyꢀwithinꢀaꢀplaneꢀthatꢀbisectsꢀtheꢀspaceꢀ
betweenꢀ conservedꢀ activeꢀ siteꢀ lysineꢀ andꢀ glutamateꢀ residuesꢀ
5
7
flippedꢀ orientationsꢀ (consistentꢀ withꢀ 5-halogenation )ꢀ wereꢀ
alsoꢀobtained.ꢀ
Swiss-PDBViewerꢀwasꢀthenꢀusedꢀtoꢀminimizeꢀtheꢀstructuresꢀofꢀ
(
proposedꢀtoꢀeitherꢀbindꢀandꢀactivateꢀHOX, ꢀorꢀformꢀaꢀreactiveꢀ
RebH,ꢀK79/E357;ꢀPyrH,ꢀK75/E354).ꢀTheseꢀresiduesꢀhaveꢀbeenꢀ
3
2
0
S,ꢀ 8F,ꢀ andꢀ 10S,ꢀ tryptamineꢀ wasꢀ dockedꢀ intoꢀ eachꢀ ofꢀ theꢀ
3
0
chloramineꢀ andꢀ serveꢀ asꢀ aꢀ generalꢀ base,ꢀ respectively, ꢀ toꢀ
enableꢀ electrophilicꢀ aromaticꢀ substitutionꢀ ofꢀ theꢀ tryptophanꢀ
benzeneꢀ ring.ꢀ Regardlessꢀ ofꢀ theꢀ natureꢀ ofꢀ theꢀ halogenꢀ
electrophile,ꢀitsꢀlocationꢀproximalꢀtoꢀtheꢀtryptophanꢀC-Hꢀbondꢀ
structures,ꢀandꢀtheꢀresultingꢀbindingꢀposesꢀwereꢀanalyzed.ꢀForꢀ
eachꢀ variant,ꢀ aꢀ poseꢀ consistentꢀ withꢀ theꢀ observedꢀ selectivityꢀ
wasꢀobtained,ꢀbutꢀposesꢀconsistentꢀwithꢀalternateꢀselectivitiesꢀ
wereꢀagainꢀalsoꢀobtained.ꢀIndeed,ꢀstructuresꢀconsistentꢀwithꢀ5-
halogenatedꢀ byꢀ eachꢀ enzymeꢀ providesꢀ aꢀ rationaleꢀ forꢀ theꢀ ,ꢀ6-,ꢀandꢀ7-halogenationꢀwereꢀobtainedꢀforꢀ0S,ꢀwhichꢀcontainsꢀ
3
observedꢀselectivity. ꢀꢀ
1
onlyꢀ aꢀ singleꢀ pointꢀ mutationꢀ (N470S)ꢀ relativeꢀ toꢀ RebHꢀ (Fig.ꢀ
Theꢀ interactionsꢀ involvedꢀ inꢀ tryptophanꢀ bindingꢀ areꢀ thusꢀ S24).ꢀ Theꢀ posesꢀ consistentꢀ withꢀ 7-ꢀ (theꢀ observedꢀ selectivity;ꢀ
Fig.ꢀ6B)ꢀandꢀ5-halogenationꢀwereꢀessentiallyꢀidenticalꢀtoꢀthoseꢀ
obtainedꢀfromꢀdockingꢀtryptamineꢀinꢀRebHꢀandꢀanalogousꢀtoꢀ
centralꢀtoꢀtheꢀabilityꢀofꢀRebHꢀandꢀPyrHꢀtoꢀorientꢀthisꢀsubstrateꢀ
5
7
toꢀ controlꢀ halogenationꢀ selectivity. ꢀ Inꢀ bothꢀ enzymes,ꢀ
tryptophanꢀ isꢀ boundꢀ viaꢀ extensiveꢀ hydrogenꢀ bondingꢀ
interactionsꢀ toꢀ theꢀ aminoꢀ acidꢀ moietyꢀ (andꢀ ionꢀ pairingꢀ
betweenꢀ theꢀ amineꢀ andꢀ E461ꢀ inꢀ RebH)ꢀ (Fig.ꢀ 5A),ꢀ conservedꢀ
aromaticꢀresiduesꢀthatꢀsandwichꢀtheꢀbenzeneꢀringꢀ(H109/F111ꢀ
inꢀ RebH;ꢀ H92/F94ꢀ inꢀ PyrH),ꢀ andꢀ hydrogenꢀ bondingꢀ betweenꢀ
theꢀindoleꢀN-Hꢀandꢀbackboneꢀamideꢀcarbonylꢀmoietiesꢀ(E357ꢀinꢀ
5
8
57
thoseꢀ foundꢀ inꢀ theꢀ RebH- ꢀ andꢀ PyrH-tryptophan ꢀ structuresꢀ
Fig.ꢀS25).ꢀTheꢀposeꢀconsistentꢀwithꢀ6-halogenationꢀlacksꢀbothꢀ
(
theꢀ indoleꢀ N-Hꢀ hydrogenꢀ bondingꢀ andꢀ amineꢀ ionꢀ pairingꢀ
interactionsꢀ observedꢀ inꢀ allꢀ tryptamineꢀ orꢀ tryptophanꢀ posesꢀ
consistentꢀwithꢀ7-ꢀorꢀ5-halogenation.ꢀItꢀisꢀrotated,ꢀratherꢀthanꢀ
flipped,ꢀ withinꢀ theꢀ 0Sꢀ activeꢀ siteꢀ toꢀ projectꢀ C
K79/E357.ꢀThisꢀrotationꢀisꢀapparentlyꢀenabledꢀbyꢀN470S,ꢀwhichꢀ
6
-Hꢀ towardꢀ
RebH;ꢀ P93ꢀ inꢀ PyrH).ꢀ Becauseꢀ P93ꢀ isꢀ locatedꢀ onꢀ theꢀ oppositeꢀ allowsꢀformationꢀofꢀaꢀhydrogenꢀbondꢀbetweenꢀtheꢀtryptamineꢀ
sideꢀ ofꢀ theꢀ PyrHꢀ activeꢀ siteꢀ relativeꢀ toꢀ E357ꢀ inꢀ RebH,ꢀ P93ꢀ amineꢀ andꢀ theꢀ backboneꢀ carbonylꢀ ofꢀ S110.Interestingly,ꢀ theꢀ
hydrogenꢀbondingꢀleadsꢀtoꢀaꢀflippedꢀorientationꢀofꢀtheꢀindoleꢀ poseꢀ consistentꢀ withꢀ theꢀ selectivityꢀ ofꢀ theꢀ 6-selectiveꢀ
halogenaseꢀ8Fꢀdidꢀnotꢀinvolveꢀthisꢀmodeꢀofꢀamineꢀbindingꢀandꢀ
insteadꢀ appearedꢀ largelyꢀ similarꢀ toꢀ theꢀ 7-halogenationꢀ pose,ꢀ
butꢀrotatedꢀsoꢀthatꢀC -HꢀprojectedꢀtowardꢀK79/E357ꢀ(Fig.ꢀ6E).ꢀ
moietyꢀ inꢀ PyrH.ꢀ Thisꢀ differenceꢀ inꢀ orientationꢀ determinesꢀ
7 5
whetherꢀ C -Hꢀ orꢀ C -Hꢀ bondꢀ projectsꢀ towardꢀ theꢀ conservedꢀ
6
activeꢀ siteꢀ lysineꢀ andꢀ glutamateꢀ residuesꢀ andꢀ undergoesꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
J.ꢀName.,ꢀ2013,ꢀ00,ꢀ1-3ꢀ|ꢀ7ꢀꢀ
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DOI: 10.1039/C5SC04680G
JournalꢀNameꢀ
ꢀ
ARTICLEꢀ
ꢀ
ꢀ
Figureꢀ6.ꢀLocationꢀofꢀmutationsꢀandꢀtryptamineꢀposesꢀforꢀ0Sꢀ(red),ꢀ8Fꢀ(green),ꢀandꢀ10Sꢀ(blue)ꢀmappedꢀontoꢀtheꢀRebHꢀstructureꢀ(grey).ꢀA,ꢀD,ꢀandꢀG)ꢀLocationꢀofꢀmutationsꢀ(spheres)ꢀ
andꢀtryptamineꢀposesꢀ(sticks).ꢀB,ꢀE,ꢀandꢀH)ꢀActiveꢀsiteꢀmutations,ꢀconservedꢀresidues,ꢀtryptamineꢀposesꢀconsistentꢀwithꢀobservedꢀselectivity,ꢀandꢀnativeꢀtryptophanꢀposeꢀ(sticks)ꢀ
6
1
andꢀadditionalꢀtryptamineꢀposesꢀ(lines).ꢀC,ꢀF,ꢀandꢀI)ꢀBindingꢀinteractionsꢀinꢀposesꢀconsistentꢀwithꢀobservedꢀselectivity.ꢀColoredꢀarrowsꢀindicateꢀtheꢀchlorinationꢀsiteꢀ .ꢀ
6 5
AꢀflippedꢀposeꢀinꢀwhichꢀbothꢀC -HꢀandꢀC -Hꢀprojectedꢀtowardꢀ toꢀ tryptophanꢀ bindingꢀ inꢀ theꢀ PyrH-tryptophanꢀ crystalꢀ
5
7
K79/E357ꢀ(Fig.ꢀS26)ꢀwasꢀalsoꢀobtained.ꢀNoꢀcrystalꢀstructureꢀofꢀaꢀ structure. ꢀꢀꢀ
tryptophanꢀ 6-halogenaseꢀ hasꢀ beenꢀ reported,ꢀ soꢀ furtherꢀ DirectedꢀEvolutionꢀStrategyꢀandꢀLibraryꢀMethodsꢀ
structuralꢀ characterizationꢀ ofꢀ 8Fꢀ andꢀ nativeꢀ 6-halogenases,ꢀ
Whileꢀ tryptamineꢀ dockingꢀ providesꢀ bindingꢀ posesꢀ consistentꢀ
3
6
includingꢀThal, ꢀwillꢀbeꢀrequiredꢀtoꢀdetermineꢀtheꢀrelevanceꢀofꢀ
aꢀ flippedꢀ orꢀ rotatedꢀ orientationꢀ toꢀ 6-selectiveꢀ halogenation.ꢀ
Onlyꢀoneꢀmechanisticallyꢀrelevantꢀposeꢀwasꢀgeneratedꢀforꢀtheꢀ
withꢀ theꢀ selectivitiesꢀ ofꢀ 0S,ꢀ 8L,ꢀ andꢀ 10S,ꢀ inꢀ noꢀ caseꢀ isꢀ theꢀ
preciseꢀmechanismꢀbyꢀwhichꢀmutationsꢀinꢀtheseꢀvariantsꢀfavorꢀ
theꢀrelevantꢀposeꢀclear.ꢀItꢀisꢀpossibleꢀthatꢀI52MꢀandꢀI52Hꢀinꢀ8Fꢀ
andꢀ10SꢀdisruptꢀtryptamineꢀN-HꢀhydrogenꢀbondingꢀtoꢀE357ꢀdueꢀ
toꢀ theirꢀ proximityꢀ toꢀ thisꢀ residue,ꢀ allowingꢀ additionalꢀ
mutationsꢀ (e.g.ꢀ P110/L465ꢀ inꢀ 8Fꢀ andꢀ C465ꢀ inꢀ 10S)ꢀ toꢀ alterꢀ
binding.ꢀ Characterizationꢀ ofꢀ theseꢀ variantsꢀ andꢀ theirꢀ
tryptamineꢀcomplexesꢀbyꢀX-rayꢀcrystallographyꢀandꢀmolecularꢀ
5
-halogenaseꢀ10S,ꢀandꢀthisꢀwasꢀconsistentꢀwithꢀ5-halogenationꢀ
Fig.ꢀ6H).ꢀThisꢀposeꢀwasꢀsimilarꢀtoꢀtheꢀflippedꢀtryptamineꢀposesꢀ
(
describedꢀforꢀRebHꢀandꢀ0Sꢀdockingꢀ(Fig.ꢀS24),ꢀinvolvingꢀindoleꢀ
N-HꢀhydrogenꢀbondingꢀtoꢀS110ꢀandꢀionꢀpairingꢀwithꢀE461,ꢀandꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
J.ꢀName.,ꢀ2013,ꢀ00,ꢀ1-3ꢀ|ꢀ8ꢀꢀ
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dynamicsꢀ simulationsꢀ isꢀ underwayꢀ toꢀ betterꢀ understandꢀ ꢀꢀ
structuralꢀperturbationsꢀresultingꢀfromꢀmutagenesis.ꢀTheꢀlackꢀ
ofꢀ aꢀ clearꢀ mechanismꢀ byꢀ whichꢀ randomꢀ mutationsꢀ improveꢀ
fitnessꢀ isꢀ aꢀ commonꢀ themeꢀ inꢀ directedꢀ evolution,ꢀ however,ꢀ
Tableꢀ3.ꢀEffectsꢀofꢀmutationsꢀatꢀresiduesꢀ52ꢀandꢀ465ꢀonꢀRebHꢀandꢀ5LSꢀonꢀtheꢀselectivityꢀ
ofꢀaromaticꢀchlorinationꢀ(SD,ꢀn=2).ꢀ
6
2
ꢀ
ꢀ
RebHꢀvariantꢀ
I52Tꢀ
5LSꢀvariantꢀ
I52Tꢀ
evenꢀwhenꢀstructuralꢀdataꢀforꢀevolvedꢀvariantsꢀareꢀavailable. ꢀ
Theꢀsameꢀsubtletiesꢀthatꢀcomplicateꢀsuchꢀanalysisꢀleadꢀtoꢀtheꢀ
difficultyꢀ ofꢀ rationallyꢀ introducingꢀ specificꢀ mutationsꢀ toꢀ
-ꢀ
F465Lꢀ
96.9ꢀ
(0.24)ꢀ
1.5ꢀ
-ꢀ
F465Lꢀ
32.3ꢀ
9
9.0ꢀꢀ
99.3ꢀ
86.8ꢀ
(0.12)ꢀ
7.4ꢀ
73.6ꢀ
%ꢀ7Clꢀ
(
(
(
0.10)ꢀ (0.05)ꢀ
.7ꢀ 0.5ꢀ
0.05)ꢀ (0.04)ꢀ
.3ꢀ 0.2ꢀ
0.05)ꢀ (0.09)ꢀ
(0.20)ꢀ
16.6ꢀ
(0.98)ꢀ
49.2ꢀ
2
4
improveꢀenzymeꢀfunction. ꢀIndeed,ꢀearlierꢀattemptsꢀtoꢀmodifyꢀ
0
3
theꢀ selectivityꢀ ofꢀ PrnAꢀ towardꢀ tryptophan ꢀ orꢀ substitutedꢀ
7
%
%
ꢀ6Clꢀ
ꢀ5Clꢀ
(0.12)ꢀ
1.6ꢀ
(0.09)ꢀ
5.8ꢀ
(0.34)ꢀ
9.8ꢀ
(2.12)ꢀ
18.5ꢀ
3
8
benzenes ꢀ byꢀ mutatingꢀ activeꢀ siteꢀ residuesꢀ ledꢀ toꢀ enzymesꢀ
withꢀpoorꢀselectivity,ꢀandꢀactiveꢀsiteꢀmutationsꢀintroducedꢀintoꢀ
RebHꢀtoꢀalterꢀitsꢀspecificityꢀfromꢀtryptophanꢀtoꢀtryptamineꢀdidꢀ
0
(0.12)ꢀ
(0.03)ꢀ
(0.14)ꢀ
(1.14)ꢀ
5
0
notꢀchangeꢀitsꢀselectivity. ꢀꢀ
Onꢀ theꢀ otherꢀ hand,ꢀ halogenasesꢀ withꢀ highꢀ non-nativeꢀ Conclusionsꢀ
selectivityꢀ wereꢀ obtainedꢀ viaꢀ randomꢀ mutagenesisꢀ andꢀ
RebHꢀ variantsꢀ 0S,ꢀ 8F,ꢀ andꢀ 10S,ꢀ whichꢀ chlorinateꢀ substitutedꢀ
indolesꢀortho-,ꢀmeta-,ꢀandꢀpara-ꢀtoꢀtheꢀindoleꢀnitrogen,ꢀwereꢀ
evolvedꢀ byꢀ directlyꢀ screeningꢀ forꢀ alteredꢀ selectivityꢀ onꢀ
deuterium-substitutedꢀ probeꢀ substratesꢀ usingꢀ massꢀ
spectrometry.ꢀ Thisꢀ systematicꢀ approachꢀ allowedꢀ forꢀ rapidꢀ
accumulationꢀ ofꢀ beneficialꢀ mutationsꢀ usingꢀ simpleꢀ adaptiveꢀ
walksꢀ andꢀ shouldꢀ proveꢀ generallyꢀ usefulꢀ forꢀ alteringꢀ andꢀ
optimizingꢀ theꢀ selectivityꢀ ofꢀ C-Hꢀ functionalizationꢀ catalysts.ꢀ
Analysisꢀ ofꢀ theꢀ selectivityꢀ lineageꢀ showedꢀ howꢀ “rationally”ꢀ
selectingꢀ activeꢀ siteꢀ residuesꢀ forꢀ targetedꢀ mutagenesisꢀ couldꢀ
beꢀ complicatedꢀ eitherꢀ byꢀ activity/selectivityꢀ tradeoffsꢀ thatꢀ
reduceꢀ theꢀ possibilityꢀ ofꢀ detectingꢀ suchꢀ mutationsꢀ orꢀ byꢀ
epistaticꢀeffectsꢀthatꢀactuallyꢀeliminateꢀtheirꢀbenefitsꢀinꢀcertainꢀ
screeningꢀ usingꢀ anꢀ assayꢀ forꢀ alteredꢀ selectivity,ꢀ followedꢀ byꢀ
saturationꢀmutagenesisꢀofꢀresiduesꢀthatꢀsignificantlyꢀimpactedꢀ
selectivityꢀ orꢀ activityꢀ (Fig.ꢀ 3A).ꢀ Dockingꢀ simulationsꢀ suggestꢀ
thatꢀtryptamineꢀbindingꢀinꢀ0Sꢀandꢀ10Sꢀisꢀsimilarꢀtoꢀtryptophanꢀ
5
7
bindingꢀ inꢀ RebHꢀ andꢀ PyrH,ꢀ respectivelyꢀ (Fig.ꢀ 5). ꢀ Whileꢀ
evolvingꢀ RebHꢀ selectivityꢀ tookꢀ onlyꢀ aꢀ handfulꢀ ofꢀ mutations,ꢀ
however,ꢀPyrHꢀandꢀtheꢀ6-halogenaseꢀThalꢀdifferꢀfromꢀRebHꢀbyꢀ
2
05ꢀ andꢀ 335ꢀ residues,ꢀ respectively,ꢀ highlightingꢀ howꢀ
dramaticallyꢀdifferentꢀsolutionsꢀtoꢀsimilarꢀselectivityꢀproblemsꢀ
e.g.ꢀ 5-ꢀ orꢀ 6-chlorinationꢀ ofꢀ indoles)ꢀ canꢀ ariseꢀ fromꢀ
(
homologousꢀenzymes.ꢀ
Despiteꢀtheꢀrelativelyꢀsmallꢀnumberꢀofꢀmutationsꢀrequiredꢀtoꢀ
convertꢀ RebHꢀ toꢀ aꢀ 5-ꢀ orꢀ 6-halogenase,ꢀ identifyingꢀ theseꢀ
mutationsꢀ requiredꢀ upꢀ toꢀ tenꢀ roundsꢀ ofꢀ mutagenesisꢀ andꢀ
screening.ꢀSeveralꢀofꢀtheꢀmutationsꢀidentifiedꢀareꢀinꢀtheꢀRebHꢀ
activeꢀ siteꢀ (Fig.ꢀ 6),ꢀ andꢀ mutationꢀ ofꢀ residuesꢀ 52ꢀ andꢀ 465ꢀ inꢀ
particularꢀledꢀtoꢀmajorꢀbranchꢀpointsꢀinꢀtheꢀselectivityꢀlineageꢀ
62
contexts. ꢀAsꢀaꢀcorollaryꢀtoꢀthisꢀfinding,ꢀtheꢀpreciseꢀmannerꢀinꢀ
whichꢀ theꢀ beneficialꢀ mutationsꢀ improvedꢀ RebHꢀ selectivityꢀ isꢀ
notꢀ clear.ꢀ Dockingꢀ simulationsꢀ suggestꢀ thatꢀ tryptamineꢀ bindsꢀ
toꢀ theseꢀ variantsꢀ asꢀ tryptophanꢀ doesꢀ toꢀ nativeꢀ halogenases,ꢀ
butꢀstructuralꢀstudiesꢀwillꢀbeꢀrequiredꢀtoꢀconfirmꢀtheseꢀmodelsꢀ
andꢀ shedꢀ lightꢀ onꢀ howꢀ theꢀ mutationsꢀ identifiedꢀ impactꢀ
tryptamineꢀbinding.ꢀꢀ
(
atꢀ5LSꢀandꢀ6TL,ꢀFig.ꢀ3A),ꢀsuggestingꢀthatꢀtargetedꢀmutagenesisꢀ
ofꢀtheseꢀsitesꢀcouldꢀhaveꢀdecreasedꢀtheꢀeffortꢀrequiredꢀtoꢀalterꢀ
RebHꢀ selectivity.ꢀ Weꢀ thereforeꢀ comparedꢀ theꢀ effectsꢀ ofꢀ
introducingꢀI52TꢀandꢀF465LꢀintoꢀbothꢀRebHꢀandꢀ5LSꢀ(Tableꢀ3).ꢀ
Theꢀfirstꢀofꢀtheseꢀmutations,ꢀI52T,ꢀdecreasesꢀtheꢀ5/6-selectivityꢀ
ofꢀRebHꢀ(1.4-1.5ꢀfold),ꢀbut,ꢀasꢀshownꢀinꢀFigureꢀ3,ꢀitꢀsignificantlyꢀ
improvesꢀ 5/6-selectivityꢀ inꢀ 5LSꢀ (1.7-2.2ꢀ fold).ꢀ Whileꢀ F465Lꢀ
increasesꢀ 5/6-selectivityꢀ inꢀ bothꢀ RebHꢀ andꢀ 5LS,ꢀ theꢀ foldꢀ
improvementꢀforꢀ5-ꢀversusꢀ6-selectivityꢀisꢀoppositeꢀinꢀtheꢀtwoꢀ
casesꢀ(favoringꢀ5ClꢀinꢀRebHꢀandꢀ6Clꢀinꢀ5LS)ꢀandꢀnoꢀgreaterꢀthanꢀ
theꢀimprovementꢀaffordedꢀbyꢀtheꢀS448Pꢀmutationꢀ(variantꢀ1P)ꢀ
inꢀ ourꢀ selectivityꢀ lineageꢀ (Fig.ꢀ 3).ꢀ Weꢀ nextꢀ introducedꢀ theꢀ
mutationsꢀatꢀresiduesꢀ52ꢀandꢀ465ꢀthatꢀledꢀtoꢀoptimalꢀ5-ꢀandꢀ6-
selectivityꢀ (I52H/F465Cꢀ andꢀ I52M/F465L,ꢀ respectively)ꢀ intoꢀ
RebH.ꢀ Noꢀ measureableꢀ activityꢀ wasꢀ observedꢀ forꢀ RebH-I52H/ꢀ
F465C,ꢀ andꢀ whileꢀ RebH-I52M/F465Lꢀ didꢀ haveꢀ alteredꢀ
selectivityꢀ (Fig.ꢀ S22),ꢀ extremelyꢀ lowꢀ conversionꢀ (<0.5%ꢀ atꢀ 5ꢀ
mol%ꢀenzymeꢀloading)ꢀwasꢀobserved.ꢀTogether,ꢀtheseꢀresultsꢀ
showꢀ thatꢀ severalꢀ ofꢀ theꢀ keyꢀ mutationsꢀ responsibleꢀ forꢀ theꢀ
selectivityꢀ ofꢀ 8Fꢀ andꢀ 10Sꢀ haveꢀ aꢀ minimalꢀ orꢀ evenꢀ negativeꢀ
impactꢀonꢀtheꢀ5/6-selectivityꢀofꢀRebHꢀitself.ꢀ
Interestingly,ꢀ 8Fꢀ andꢀ 10Sꢀ bindꢀ tryptamineꢀ ratherꢀ poorly,ꢀ butꢀ
stillꢀchlorinateꢀthisꢀsubstrateꢀwithꢀalmostꢀexclusiveꢀselectivityꢀ
forꢀtheꢀ6-ꢀandꢀ5-positions,ꢀrespectively,ꢀratherꢀthanꢀtheꢀRebH-
preferredꢀ 7-positionꢀ orꢀ theꢀ moreꢀ reactiveꢀ 2-position.ꢀ Similarꢀ
selectivityꢀ wasꢀ observedꢀ forꢀ chlorinationꢀ ofꢀ 2-
methyltryptamine,ꢀN-methyltryptamine,ꢀandꢀtryptopholꢀbyꢀ8Fꢀ
andꢀ 10S.ꢀ Theseꢀ resultsꢀ indicateꢀ thatꢀ evenꢀ weakꢀ substrateꢀ
bindingꢀ canꢀ beꢀ sufficientꢀ toꢀ enableꢀ highlyꢀ selectiveꢀ C-Hꢀ
63-65
functionalizationꢀ inꢀ anꢀ enzymeꢀ activeꢀ site,ꢀ
ꢀ andꢀ directedꢀ
evolutionꢀprovidesꢀaꢀmeansꢀtoꢀsystematicallyꢀtuneꢀthisꢀbindingꢀ
toꢀ functionalizeꢀ differentꢀ C-Hꢀ bondsꢀ onꢀ aꢀ givenꢀ substrate.ꢀ
22
Similarꢀ effortsꢀ onꢀ otherꢀ enzymes ꢀ orꢀ artificialꢀ
66
metalloenzymes ꢀ thatꢀ catalyzeꢀ C-Hꢀ functionalizationꢀ couldꢀ
thereforeꢀenableꢀaꢀwideꢀrangeꢀofꢀtransformations.ꢀ
Acknowledgementsꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ20xxꢀ
J.ꢀName.,ꢀ2013,ꢀ00,ꢀ1-3ꢀ|ꢀ9ꢀ
ꢀ
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1
(
1
7 Gormisky,ꢀP.ꢀE.;ꢀWhite,ꢀM.ꢀC.ꢀJ.ꢀAm.ꢀChem.ꢀSoc.ꢀ2013,ꢀ135ꢀ
38),ꢀ14052.ꢀ
8 Chen,ꢀK.;ꢀEschenmoser,ꢀA.;ꢀBaran,ꢀP.ꢀS.ꢀAngew.ꢀChem.ꢀInt.ꢀ
Ed.ꢀ2009,ꢀ48ꢀ(51),ꢀ9705.ꢀ
9 Davies,ꢀH.ꢀM.ꢀL.;ꢀMorton,ꢀD.ꢀChem.ꢀSoc.ꢀRev.ꢀ2011,ꢀ40ꢀ(4),ꢀ
1857.ꢀ
0 Michaudel,ꢀ Q.;ꢀ Journot,ꢀ G.;ꢀ Regueiro-Ren,ꢀ A.;ꢀ Goswami,ꢀ
Thisꢀ workꢀ wasꢀ supportedꢀ byꢀ theꢀ NIHꢀ (5R00GM087551ꢀ andꢀ
1
R01GM115665),ꢀ theꢀ Searleꢀ Scholarsꢀ Program,ꢀ Theꢀ Novartisꢀ
InstitutesꢀforꢀBiomedicalꢀResearch,ꢀandꢀtheꢀNSFꢀunderꢀtheꢀCCIꢀ
Centerꢀ forꢀ Selectiveꢀ C–Hꢀ Functionalizationꢀ (CHE-1205646).ꢀ
MCAꢀwasꢀsupportedꢀbyꢀanꢀNIHꢀChemistryꢀandꢀBiologyꢀInterfaceꢀ
trainingꢀgrantꢀ(T32ꢀGM008720),ꢀanꢀNSFꢀpredoctoralꢀfellowshipꢀ
1
2
A.;ꢀGuo,ꢀZ.;ꢀTully,ꢀT.ꢀP.;ꢀZou,ꢀL.;ꢀRamabhadran,ꢀR.ꢀO.;ꢀHouk,ꢀK.ꢀ
N.;ꢀBaran,ꢀP.ꢀS.ꢀAngew.ꢀChem.ꢀInt.ꢀEd.ꢀ2014,ꢀ53ꢀ(45),ꢀ12091.ꢀ
(
DGE-1144082),ꢀ aꢀ Universityꢀ ofꢀ Chicagoꢀ Departmentꢀ ofꢀ
Chemistryꢀ Helenꢀ Sellei-Beretvasꢀ Fellowship,ꢀ andꢀ anꢀ ARCSꢀ
ScholarꢀAward.ꢀJVCꢀwasꢀsupportedꢀbyꢀtheꢀUniversityꢀofꢀChicagoꢀ
Departmentꢀ ofꢀ Chemistryꢀ undergraduateꢀ summerꢀ researchꢀ
fund.ꢀThisꢀworkꢀwasꢀcompletedꢀinꢀpartꢀwithꢀresourcesꢀprovidedꢀ
byꢀtheꢀUniversityꢀofꢀChicagoꢀResearchꢀComputingꢀCenter,ꢀandꢀ
MSꢀ dataꢀ wereꢀ acquiredꢀ onꢀ instrumentsꢀ purchasedꢀ usingꢀ anꢀ
NSFꢀ instrumentationꢀ grantꢀ (CHE-1048528).ꢀ Weꢀ wouldꢀ likeꢀ toꢀ
thankꢀJamesꢀPayneꢀforꢀconductingꢀtheꢀtryptamine/tryptophanꢀ
competitionꢀreactions,ꢀProf.ꢀBenoitꢀRouxꢀandꢀDr.ꢀYilinꢀMengꢀforꢀ
assistanceꢀwithꢀdockingꢀsimulations,ꢀandꢀmembersꢀofꢀtheꢀNSFꢀ
CCHFꢀforꢀhelpfulꢀdiscussions.ꢀꢀ
2
1 Mahatthananchai,ꢀJ.;ꢀDumas,ꢀA.ꢀM.;ꢀBode,ꢀJ.ꢀW.ꢀAngew.ꢀ
Chem.ꢀInt.ꢀEd.ꢀ2012,ꢀ51ꢀ(44),ꢀ10954.ꢀ
22 Lewis,ꢀ J.ꢀ C.;ꢀ Coelho,ꢀ P.ꢀ S.;ꢀ Arnold,ꢀ F.ꢀ H.ꢀ Chem.ꢀ Soc.ꢀ Rev.ꢀ
2
2
1
2
2
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Notesꢀandꢀreferencesꢀ
2
ꢀ
ABBREVIATIONS:ꢀ FDH,ꢀ flavinꢀ dependentꢀ halogenase;ꢀ MBP,ꢀ
maltoseꢀ bindingꢀ protein;ꢀ GDH,ꢀ glucoseꢀ dehydrogenase;ꢀ NAD,ꢀ
nicotinamideꢀ adenineꢀ dinucleotide;ꢀ FAD,ꢀ flavinꢀ adenineꢀ
dinucleotideꢀ
KEYWORDS:ꢀ Directedꢀ Evolution,ꢀ Halogenase,ꢀ RebH,ꢀ C-Hꢀ
Functionalizationꢀ
ꢀ
3
3
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