University of Groningen

Efficient protein engineering by combining computational design and directed evolutionFloor, Robert

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Chapter4. ComputationalLibraryDesignforIncreasingHaloalkaneDehalogenaseStability

Robert J. Floor, Hein J.Wijma, Dana I. Colpa, Aline Ramos‐Silva, Peter A. Jekel,

WiktorSzymański,BenL.Feringa,SiewertJ.MarrinkandDickB.Janssen

Weexploredtheuseofacomputationaldesignframeworkforthestabilizationof

the haloalkane dehalogenase LinB. Energy calculations, disulfide bond design,

molecular dynamics simulations, and rational inspection of mutant structures

predicted many stabilizing mutations. Screening of these in small mutant

libraries led to the discovery of seventeen point mutations and one disulfide

bondthatenhancedthermostability.Mutations located inorcontacting flexible

regionsoftheproteinhadalargerstabilizingeffectthanmutationsoutsidesuch

regions.Thecombinedintroductionoftwelvestabilizingmutationsresultedina

LinB mutant with a 23°C increase in apparent melting temperature (Tm,app,

72.5°C) and an over 200‐fold longer half‐life at 60°C. The most stable LinB

variants also displayed increased compatibilitywith co‐solvents, thus allowing

substrateconversionandkineticresolutionatmuchhigherconcentrationsthan

withthewild‐typeenzyme.

Thischapterhasbeenpublished:

ChemBioChem2014,(15),1660‐1672

doi:10.1002/cbic.201402128

Chapter4

92

IntroductionAlthough enzymes are very proficient catalysts and can combine high

acceleration of chemical reactions with good selectivity[1], their application in

industrialprocessescanbehamperedbytheirlimitedstability[2].Forexample,at

hightemperaturesorinthepresenceoforganicco‐solvents,enzymesoftenlose

activity due to unfolding and aggregation. Performing enzyme reactions at

elevated temperatures has several benefits, such as improved solubility of

reactants, higher conversion rates, and reduced risk of microbial

contamination[1‐3].Improvingintrinsicenzymestabilitythereforecontributesto

the applicability of enzymes under realistic process conditions. Furthermore,

thermostableenzymesarebettersuitedthanmesostableenzymesforimproving

catalyticactivitybymutagenesis[2c,4].

Effective methods for improving intrinsic enzyme stability include

directedevolution(repeatedroundsofrandommutagenesisandscreening)[2b,5]

and the introduction of consensus amino acids detected bymultiple sequence

alignments[5b,6].Replacementofaminoacidsbasedonstructuralinspectionand

reconstructionofancestralsequenceshasalsobeenused[2b,5].Directedevolution

usually requires the screening of thousands of variants, and thus depends on

high‐throughputexpressionandassaymethods,whicharenotalwaysavailable.

Rationallypredicted stabilizingmutations canbe foundwith less experimental

screening effort and have also been used[7]. However, the design of individual

stabilizing mutations is often complicated and time‐consuming[5c].

Computational design has evolved from rational design and can predict many

potentially stabilizing mutations based on free energy calculations, and can

replace or complement chemical intuition. Computational methods led to an

improvement in theapparentmelting temperature (Tm,app)of smallproteinsof

morethan15°C[8],whilereducingtheamountofexperimentalscreeningneeded

toidentifystabilizedvariants.

Manyapproachesforthecomputationaldesignofproteinswithenhanced

thermostability have been explored[8c]. One method is calculation of the

electrostatic contributions of surface charges to the overall stability of the

protein, and subsequent removal of charges that have destabilizing

contributions.ThismethodwasusedtoimprovetheTm,appofanacylphosphatase

and GTPase by 9°C[9]. Another approach uses semi‐empirical force fields to

compute the differences in folding energy (ΔΔGfold) between the wild‐type

enzyme structure and the predicted mutant structures. Algorithms that can

calculate such folding‐energy differences include PoPMuSiC[8e], FoldX[8d, 10],

SCADES[8a] andRosetta[11].RosettaDesignhasbeenused to improvepacking in

ComputationalLibraryDesignforIncreasingHaloalkaneDehalogenaseStability

93

the hydrophobic core of a protein, and this resulted in an 18°C increase in

melting temperature of a domain of methionine aminopeptidase[8b].

Furthermore, the design of stabilizing disulfide bonds has been reported for

several proteins[12]. However, computational methods to design chemically

differenttypesofstabilizingmutationinasinglecomputationalapproachremain

a challenge[8c], It is also difficult to accurately predict the ΔΔGfold required to

achieve a large increase in unfolding temperature[8c,13] and to retain catalytic

activityatlowtemperatureinmutantswithincreasedthermostability[2c,8a].

Werecentlyproposedacomputationalstrategy forenzymestabilization

based on the calculation of folding energies for all possible substitutions, the

integrationofconformationalsamplingindisulfide‐bonddesigns,andtheuseof

molecular dynamics as a fast screening/ranking tool (FRESCO, framework for

rapidenzymestabilizationbycomputation)[8f].TheFRESCOstrategyisnotbased

on finding just a single or a few effective mutations, but on discovering and

combiningasmanystabilizingmutationsaspossibleeachofwhichmighthave

onlya small effect. Furthermore,asmostof the screening isdone in silico, the

number of variants that needs to be produced and tested experimentally is

significantly reduced. By using the FRESCO approach, the Tm,app of limonene

epoxide hydrolasewas improved by 32°C,with a total screening effort of less

than 80 variants. The stabilization arose from both computationally designed

point mutations and introduced disulfide bonds. The constructed enzymes,

whichcontained10‐12mutations,remainedactiveat30°C[8f].

In this study,we examined the use of FRESCO for the stabilization of a

larger monomeric protein that catalyzes both the degradation of recalcitrant

environmental pollutants and performs stereoselective biocatalysis[14], that is,

haloalkane dehalogenase (LinB, EC 3.8.1.5) from Sphingomonas paucimobilis

UT26[14b, 15]. LinB is responsible for the hydrolytic dehalogenation of 1,3,4,6‐

tetrachloro‐1,4‐cyclohexadiene, which is an intermediate in the degradation

pathway for the insecticide hexachlorocyclohexane (HCH, common name

lindane)[15].Itisalsocapableofhydrolysisofthehighlyrecalcitrantβ‐isomerof

HCH[16]andisaversatiledehalogenasethatconvertsmanychloro‐,bromo‐,and

iodoalkanes,haloamides,andhaloesters[14a].Otherenvironmentalchemicalsthat

aresubstrates forLinB include1,2‐dibromoethane,1‐bromo‐2‐chloroethane,3‐

chloro‐2‐methylpropene,and1‐bromopropane[14a,14b].Furthermore,theenzyme

can be used for the enantioselective preparation α‐bromoamides[14c] and

haloalcohols[14d]. Its catalytic mechanism has been studied in depth by

structural[17],mutational[17b,18] and quantummechanicalmethods[19]. However,

thepossibilities to engineer and apply the enzymeare limitedby itsmediocre

Chapter4

94

stability, as was found when performing mutagenesis experiments aimed to

improvetheenzymeselectivityfortheenantioselectiveconversionofN‐benzyl‐

2‐bromohexanamide(unpublishedresults).Thelimitedstabilityresultsinalow

tolerance of organic co‐solvents[20], thus restricting the use of co‐solvents to

solubilizehydrophobic substrates[14c]. In thispaper,wedemonstrate that, by a

combination of computational design and experimental screening, the FRESCO

approachcandrasticallyimproveboththethermostabilityoftheenzymeandits

toleranceofco‐solvents.

Results

ComputationaldesignofpointmutationsPreviously, the FRESCO computational frameworkwas applied to improve the

Tm,appofthesmalldimericproteinlimoneneepoxidehydrolaseby32°C[8f].Here

we investigated whether this framework, which aims to reduce experimental

screening to a minimum, is also applicable for the stabilization of the larger

monomerichaloalkanedehalogenaseLinB.Wecomparedtheeffectsonstability

and activity bymutations close to the active site to those bymutations away

fromtheactivesite.Inapreviousstudy,severalmutationsclosetotheactivesite

were found to stabilize the haloalkane dehalogenase DhaA[21], but such

mutationsoftenresultinreducedcatalyticactivity.ForthestabilizationofLinB,

mutationswerefirst introducedmorethan9Åfromthedockedsubstratetert‐

butyl‐2‐(2‐bromopropanamido)acetate. This afforded the substitution of 233

residues; 63 other residues were excluded because of their proximity to the

active site (21% of the protein; Figure 4‐1, regions marked in red). Of the

excludedresidues,35wereinsixloops,and28wereinthreehelicesaroundthe

activesite.

Subsequently,weusedRosetta[11b]andFoldX[10]topredictΔΔGfoldinorder

to identify mutations which should stabilize LinB. Such point mutations can

stabilize proteins by improving hydrophobic interactions in the interior, by

removing unsatisfied hydrogen bonds, by improving surface electrostatic

interactions, or by reducing the flexibility of areas that are rate limiting for

kinetic unfolding. FoldX predicted 150 possible stabilizing mutations, and

Rosettapredicted314mutationswithΔΔGfoldhigher than3.5kJmol‐1.Of these,

57 were predicted by both methods, so a total of 407 unique stabilizing

substitutions were expected. Previous work suggests that although FoldX and

Rosettacanbeusedtofindstabilizingmutations,theseprogramsalsogivefalse‐

positive predictions[8f,13,22]. Therefore, the predicted structures of themutants

were visually inspected to eliminate mutants that harbored unconvincing

ComputationalLibraryDesignforIncreasingHaloalkaneDehalogenaseStability

95

substitutions(resultingfromknownproblemsincomputationaldesign),suchas

hydrophobic residues exposed at the surface, internal cavities, and missing

hydrogen‐bond interactions. The criteria for this elimination process are

described in the Experimental Section. By this visual inspection, 219 variants

wereeliminated.

To reduce the required experimental screening effort, a further

computational screening step by molecular dynamics simulations was

performed. This procedure identifies unfavorable interactions that are not

evidentintheinitialstructurepredictedbyFoldX,butoccurdynamicallyduring

MD simulations. Previous MD studies showed that several short parallel MD

simulationscansimulateanenzymeaswellasonelongsimulation[23].Therefore,

five parallel MD simulations of 100 ps were performed for eachmutant with

varying initial atom velocities to obtain different samplings of conformational

space.TheMDscreeningeliminated79ofthe188variants.Themajorityofthe

mutations that were eliminated showed changes in the conformation of the

introduced and surrounding residues during the simulation, compared to the

structurespredictedbyFoldX.Thisresultedinunsatisfiedhydrogenbonds, the

removalofα‐helixcappinginteractions,orthebreakingofasaltbridge.Finally,

109 designed mutations that passed the MD screening were selected for

experimentalanalysis.

Mutantswere constructed byQuikChange reactions inmicrotiter plates

(MTPs).Subsequently,cellsproducingtheproteinofinterestweregrowninMTP

plates and, after lysis, the enzymeswere purified by affinity chromatography.

Withthisprocedure,solubleproteinwasobtainedfromEscherichiacolicellsfor

99 of the 109 mutants. To verify the predicted stabilizing effects of these

mutations, mutantTm,app values were experimentally determined by using the

thermofluormethod[24].Tm,appdoesnotcorrelatedirectlywithΔΔGfold,whichwas

usedforthecomputationalprediction,butearlierresearchsuggestedthatTm,app

correlateswellwithenzymehalf‐lifeandactivityathighertemperature[8f,25].The

useofTm,appwaspreferredinsteadofthemorecommonlyusedquantificationof

theresidualactivityafterheatingtheenzyme,asthelattercanbeinfluencedby

variationsinproteinrefolding,insteadofimprovementsinintrinsicstability[26].

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96

Figure4‐1.Correlationbetweenflexibilityandstabilizingeffectofmutationsin

LinB. A) Sites of stabilizing mutations shown on the X‐ray structure of LinB.

Residueswithin9Åofthesubstratetert‐butyl‐2‐(2‐bromopropanamido)acetate

bound in the active site are shown in red. The thickness of the backbone

represents the protein flexibility as determined by B‐factor. Colored spheres

indicate the effect of the mutation on Tm,app. The introduced disulfide bond

(yellow) is between twopurple spheres. Structure drawn from1MJ5,with the

docked substrate shown in sticks. B) Values for ΔTm,app of the introduced

mutations (○) and B‐factors (‐) against residue number. C) ΔTm,app of the

introducedmutations(○)andRMSF(‐)againstresiduenumber.D)ΔTm,appofthe

introduced mutations (○) and Cα distances to the chloride ion bound in the

activesite(‐)againstresiduenumber.

TheobtainedTm,appvalueswerecomparedtothemeltingtemperatureof

the wild type to obtain ΔTm,app. Mutations that gave a ΔTm,app at least twofold

higher than the standarddeviationof the assay (±0.96°C,n=9)were scored as

stabilizing.Thisresultedinthediscoveryoftenstabilizingmutations(Table4‐1)

a success rate of 10% (number of mutations experimentally found to be

stabilizing divided by the number of predicted mutations). Different types of

stabilizingmutationswereexperimentallyobserved(Table4‐1andFigure4‐2):

introductionofaproline intoa loop(A197P), removalofnegativecharge from

thesurface(E15T,D166N,andD255A),introductionofapositivesurfacecharge

ComputationalLibraryDesignforIncreasingHaloalkaneDehalogenaseStability

97

(A81K, D166K, E192K, and E192R), improving hydrophobic packing in the

interioroftheprotein(A53L),andreplacingaglycineinaloop(G229Q).

Figure4‐2.StructuresofstabilizedmutantsaspredictedbyFoldX.A)Mutation

A53L(ΔTm,app=+1°C);structuralinspectionrevealedthatthismutationimproves

van der Waals interactions. B) Mutation E192K (ΔTm=+3°C); the introduced

lysine resulted in a positive charge at the surface. In the predicted structure,

K192hasonlylong‐rangeelectrostaticinteractions(>7.5Å).C)Modelbasedon

thestructure1MJ5highlightingresidueD147,whichislocatedinaflexibleloop

neartheactivesite.MutationofD147toH,Y,M,orL(ΔTm=+3.0‐5.5°C)improves

stability but reduces activity. The docked substrate tert‐butyl‐2‐(2‐

bromopropanamido)‐acetate is shown in sticks to highlight its proximity to

D147. D) Predicted structure with the disulfide bond introduced between

residues 185 (protein core) and 5 (close to the N terminus); ΔTm=+5°C. The

flexibilityof thewild‐typeprotein,asobservedbycrystallographicB‐factors, is

representedbythethicknessofthechain(3‐35Å2)inpanelsCandD.

To determine if the discovered stabilizing mutations affected

dehalogenase activity, specific activities of the purified enzymes were

determinedwith1‐bromopropane.Nearlyallvariantswereasactiveasthewild‐

type enzyme (Table 4‐1), thus supporting the expectation that mutations

stabilizingLinBawayfromtheactivesitedonotreduceitsactivity.Themajority

of the tested mutations had neither a significantly positive nor a significantly

Chapter4

98

negativeeffectonstability(56%ofthemutations).Thissuggeststhatmostofthe

ΔΔGfold differences of themutations do not significantly influence the overall

stabilityoftheprotein.Possibly,alabileregionoftheprotein(onenotinfluenced

bytheexpectedstabilizingmutations)determinestheoverallunfolding[7b,27].

DesignofdisulfidebondsTheDDDalgorithm[8f]wasusedtopredictsuitablelocationsfortheintroduction

ofdisulfidebonds,basedonboththegeometryoftheS‐Sbondandthemolecular

mechanics energy. Disulfide bonds are known to stabilize proteins both

thermodynamically(reducingtheentropyoftheunfoldedstate)andkinetically

(slowingimportantstepsinproteinunfoldingpathways,suchaslocalunfolding

of flexibleareas) [12a,12b,28]. Sevensuitableresiduepairswerepredicted for the

introductionofdisulfidebonds,basedontheX‐raystructure(PDBID:1MJ5).To

examinewhetheralargernumberofdisulfidebondsispossible,notonlytheX‐

raystructurebutalsosnapshotsofMDsimulationswereusedtopredictpairsof

positions where disulfide‐forming cysteines could be introduced. Previous

experimentssuggestedthatsmallvariationsinthepositionsofbackboneatoms

can have a large effect on the distances, angles, and dihedrals of the designed

disulfidebonds,and thereforeon thepredictedmolecular‐mechanicsenergyof

disulfide bonds[8f]. By using a set ofMD‐generated template structures for the

predictions, an additional 25 pairs of cysteine positionswere discovered, thus

raisingthetotalnumberofpredictedpairsto32.

Aftervisualinspectionofthemutants(basedoncriteriadescribedinthe

Experimental Section) thedynamicbehaviorofmutants carrying thepredicted

disulfidebondswereanalyzedbyMDsimulations, inorder to identifymutants

withdestabilizingfeatures,suchassurface‐exposedaromaticresiduesorbroken

salt bridges. This analysis revealed that 19 of the 32 predicted disulfide bond

mutationslikelyweredestabilizing,andthereforetheywereeliminated.

The remaining 13 disulfide bond designs were characterized

experimentally,withenzymesproducedinbothE.coliTOP10andE.coliSHuffle

(Table 4‐2). The latter is optimized for the cytosolic formation of disulfide

bonds[29].Thisanalysisrevealedthatonlyoneofthe13testedmutantswasmore

stable.Itcarriedadisulfidebondbetweenresidues5and185,andthisstabilized

the protein by 5°C (Table 4‐1, Figure 4‐1 and 4.2). The mutation was only

stabilizing in its oxidized state (Table 4‐2), thus indicating that the observed

increase in Tm,app can be attributed to the introduced disulfide bond. The

dehalogenation activity of the stabilized variant A5C/A185C with the model

substrate1‐bromopropanewassimilartothewild‐typeactivity(Table4‐1),thus

ComputationalLibraryDesignforIncreasingHaloalkaneDehalogenaseStability

99

indicating that the stabilizing disulfide bond does not diminish activity. This

stabilizing disulfide bond was predicted by methods based on both the X‐ray

structureandstructuresgeneratedbyMDsimulations.

Table 4-1. Stabilizing mutations discovered in LinB.

Parent Origin GFOLD

(kJ/mol)

Mutation(s)

wild-type +

TM,app

(°C)

TM,app

(°C)

Relative

activity[b]

LinB WT variants

wild-type - 51.0 0.0 ±

wild-type Rosetta -4.2 E15T 52.0 1.0 ±

wild-type Rosetta -4.0 A53L 52.0 1.0 -

wild-type Rosetta -4.0 A81K 52.0 1.0 ±

wild-type Rosetta -9.2 D166K 53.5 2.5 +

wild-type Rosetta -4.3 D166N 53.0 2.0 ND

wild-type FoldX -3.7 E192K 54.0 3.0 ±

wild-type FoldX -4.6 E192R 53.5 2.5 ND

wild-type FoldX -4.0 A197P 52.5 1.5 +

wild-type - - G229Q + E192R[a] 54.0 3.9 +

wild-type Rosetta -5.0 D255A 51.5 0.5 ±

wild-type DDD /MD - A5C/A185C 56.0 5.0 ±

Wild-type Variant G1

A5C+A185C+G229Q+ 62.0 11 ±

D166K+E192K

LinB G1 variants

G1 Rosetta -15.9 D147H 65.0 3.0 --

G1 Rosetta -25.4 D147Y 67.5 5.5 ---

G1 Rosetta -9.2 D147M 65.0 3.0 ---

G1 Rosetta -15.8 D147L 66.0 4.0 ----

G1 Rosetta -5.8 F169V 64.0 2.0 +

G1 FoldX -5.8 T249L 62.5 0.5 ±

G1 FoldX -4.0 A247F 67.5 5.5 ---

G1 Variant G3 - E15T+A53L+A81K+F169V

+A197P+D255A+A247F 74.0 12/23[c] --

[a] TM,app determined for the double mutant. [b] Symbols: -, activity with 1-bromopropane reduced by 10-

25%; --, reduced by 25-50%; ---, reduced by 50-75%; ---- reduced by 75-100%, ±, similar to the wild-type; +,

improved by 10-25%; ND, not determined. [c] As compared to the wild-type.

Previously, a similar approach aimed at enhancing the stabilization of

limoneneepoxidehydrolaseyieldedthirteenstabilizingdisulfidebonds[8f].Other

computational design studies have found one[12b‐d] or two stabilizing disulfide

bonds[12a]. For LinB, only one disulfide bond was found, and this yielded

moderatestabilization.Possibly,localkineticstabilizationarisingfromtheother

examined disulfide bonds did not contribute to the overall stability of the

protein.ThestabilizingA5C/A185CdisulfidebondwasintheflexibleNterminus

of the protein (Figure 4‐1 and 4.2); no stabilizing disulfide bonds were

Chapter4

100

discovered at more rigid parts of the protein. The observed stabilization in a

flexibleareaisconsistentwithresultsforotherproteins[12a,30].

Table 4-2. The TM,app of all disulfide bond mutants constructed and analyzed in both the

oxidized and reduced state, in both E. coli TOP10 and shuffle strains.

E. coli TOP10 E. coli shuffle

Mutation TM,oxidized (°C) TM,oxidized (°C) TM,reduced (°C)A

WT 51 49.0 49.0

S2C/N47C 48 - -

A5C/A185C 56 55.5 49.0

R20C/D70C 50 - 45.5

A22C/D68C 50 - 49.5

S42C/A60C - - 47.5

I48C/G283C 49 - -

H51C/G283C 48.5 - 46.0

S75C/E199C 48.5 - -

V106C/G114C - 50.0 50.0

V268C/S278C 48.5 - 46.0

S75C/R79C 45.5 - 44.0

S183C/E186C 47 - -

A196C/A200C 48.5 46.0 48.5

A Reduced by the addition of 10 mM DTT

Toanalyzewhytheotherdisulfidebondswerenotstabilizing,themelting

temperatures of all 13 disulfide‐bond variants were determined in both the

reducedandoxidizedstates.Sixofthesedidnotshowaclearmeltingtransition

in their oxidized state (suggestingmisfolding), whereas such a transition was

seenwhenreduced(Table4‐2).Thisreductionresultedinproteinswithclearly

defined melting temperatures, thus indicating homogenous structures. These

results suggest that unintended disulfide bonds were formed between the

introduced and endogenous cysteine residues, as six of the 13 constructed

disulfidebondsresultedinmisfoldedprotein(Table4‐2).

CombiningstabilizingmutationsTo investigate whether a highly thermostable variant could be obtained by

combining confirmedstabilizingmutations,we constructedmutantLinB‐G1, in

which three stabilizing point mutations (G229Q, D166K, and E192K) and one

disulfidebond(A5C/A185C)werecombined.TheTm,appofthisvariantwas11°C

higher than that of the wild‐type enzyme (Table 4‐1, Figure 4‐3A and B). Its

activity was preserved, with slightly higher kcat and higher Km for the

ComputationalLibraryDesignforIncreasingHaloalkaneDehalogenaseStability

101

dehalogenationof1‐bromopropane(Table4‐3).Thedehalogenaseactivitiesfor

arangeofotherhaloalkanesandhalocycloalkaneswerealsofoundtobealmost

the same as for the wild type (Table 4‐4). This supports the hypothesis that

stabilizing mutations away from the active site do not affect activity, and

indicatesthatthemutationsdidnotindirectlydisturbtheactivesiteorpartsof

theenzymethatneedtobeflexibleforsubstrateaccessorproductrelease.The

observed increase inKmsuggests[14c] that,duetothemutations,eitheranearly

stepinthecatalyticmechanismhadbecomeslowerthanthelaterreactionssteps

orthatthemutationsslightlyreducedsubstratebinding.

Figure 4‐3. Thermostabilities of wild‐type LinB and variants G1 and G3

determinedby fourdifferentmethods:wild type(black line),G1(dashed line),

G3(grayline).A)Tm,appmeasuredbythethermofluormethod(intheabsenceof

cosolvent). B) Tm,app measured by DSC. C) Thermal inactivation constants at

different temperatures. D) Specific activities for dehalogenation of 1‐

bromopropane at different temperatures. Because of the experimental setup,

activitiescouldonlybemeasuredupto55°C.

The correlation between increased Tm,app and protein unfolding and

activity at elevated temperatures was investigated by measuring inactivation

rates and enzyme activity at higher temperatures. The rate of unfolding of

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variantG1wassignificantlylowerthanthatofthewildtype(increasedhalf‐lives

atelevatedtemperatures:40‐foldat50°Cand20‐foldat54°C,Figure4‐3C).The

activitiesofbothwildtypeandG1increasedwithtemperature,butthewild‐type

enzyme reached optimum activity at 40°C, whereas G1 showed its highest

activityat50°C.Attheoptimumtemperatures,G1was1.5‐foldmoreactivethan

wildtype(Figure4‐3D).Thus,the11°CimprovementintheTm,appofvariantG1

(Figure4‐3AandB) reflects significantly improvedhalf‐life andactivityat and

above44°C.

Table 4-3. Kinetic parameters for the dehalogenation of 1-bromopropane by wild-type LinB and

evolved variants G1 and G3 at 30°C and at their optimum temperatures of 40, 50 and 50°C.

30°C Optimum temperature

Variant kcat (s-1) KM (mM)

kcat/KM

(s-1mM-1) kcat (s-1) KM (mM)

kcat/KM

(s-1mM-1)

T

(°C)

Wild-type 4.5 ± 0.2 1.9 ± 0.1 2.3 6.1 ± 0.3 3.2 ± 0.3 1.9 40

G1 4.7 ± 0.4 3.0 ± 0.2 1.6 6.7 ± 0.4 3.7 ± 0.1 1.8 50

G3 2.4 ± 0.1 1.3 ± 0.1 1.9 4.3 ± 0.6 2.4 ± 0.2 1.8 50

MutationsclosetotheactivesiteIthasbeensuggestedthatstabilizationofflexibleareasofaproteincontributes

more to the overall stability than does stabilization of rigid areas[7b,27,31]. For

LinB, the region surrounding the active site is relatively flexible (Figure 4‐1).

Structural inspection and analysis of theB‐factors of the protein revealed that

themost flexiblepartsof twohelixes (α4, residues144‐148, andα5,169‐173)

flanktheactivesite(Figure4‐1B).ThecrystallographicB‐factorsofthisareaare

1.5‐fold higher than the average for the protein (9.1 and 5.6 Å2, respectively).

Furthermore, the root‐mean square fluctuation (RMSF) calculated from MD

simulations of thewild‐type enzymewas 1.5‐fold higher in helix α4 (residues

144‐148).Indeed,withtheexceptionoftheNandCtermini,helixα4isthemost

flexiblepartoftheprotein,basedonB‐factorandRMSF(Figure4‐1BandC).The

active site of LinB is buried in the protein, and substrates reach it through an

accesstunnel.Itwasreportedthatflexibilityofresiduesliningthistunnel(169‐

173) is required for substrate access to the active site[32]. Therefore, this area

could be an interesting region for the introduction of stabilizing mutations,

althoughthisbearstheriskofincurringanegativeeffectoncatalyticactivity.

To investigatewhethermutationsclosetotheactivesite(<9Åfromthe

bound chloride) have a larger effect on overall stability, potentially stabilizing

mutations were computationally designed by the above approach, including

folding‐energy predictions, structural inspection, and MD simulations. This

ComputationalLibraryDesignforIncreasingHaloalkaneDehalogenaseStability

103

predicted 27 stabilizing mutations. These were constructed by QuikChange

mutagenesis inaLinB‐G1background;24mutantsweresuccessfullyproduced,

andtheirTm,appvaluesweremeasuredbythethermofluorassay.Thisresultedin

thediscoveryofsevenadditionalstabilizingmutations(Table4‐1).Thesuccess

rate formutations introduced close to the active site (29%)wasmuch higher

than for mutations distant from the active site (10%). Five of the seven

stabilizingmutations improved packing of hydrophobic residues in the apolar

interior of the protein close to the active site (D147M, D147L, F169V, A247F,

T249L,Figure4‐2C).Residues247and249areinaloopcoveringtheactivesite

and interact with a flexible helix (residues 139‐156). Residue 147 is at the

beginningofaveryflexiblestretchofthishelix.Allmutationsthatreplacedthis

residuewith a lesspolar amino acid stabilized the enzyme significantly (Table

4‐1).Likely,theresidueatthispositioninfluencestheflexibilityofthetwomost

flexibleresiduesoftheactivesite,E145andN146.

Table 4-4. Specific activities for the dehalogenation of several haloalkanes by wild-type

LinB and variants G1 and G3

Bromoalkanes A LinB wild-type (U/mg) LinB-G1 (U/mg) LinB-G3 (U/mg)

1-bromohexane 1.2 ± 0.1 1.3 ± 0.2 0.8 ± 0.1

1-bromocyclohexane 1.5 ± 0.1 1.3 ± 0.1 1.5 ± 0.1

1-bromopropanen 6.3 ± 0.1 6.3 ± 0.1 2.7 ± 0.1

1,2-dibromomethane 7.1 ± 0.6 8.1 ± 0.6 0.5 ± 0.1

chloroalkanes[a]

1-chloropropane 14 ± 0.3 12.2 ± 0.4 1.3 ± 0.1

1-chloroheptane 1.7 ± 0.1 1.8 ± 0.1 0.1 ± 0.05

1-chloropentane 1.5 ± 0.2 1.4 ± 0.1 0.9 ± 0.1

1-chlorobutane 1.3 ± 0.1 1.1 ± 0.1 1.2 ± 0.1

1,3-dichloropropane 0.9 ± 0.1 0.7 ± 0.1 0.3 ± 0.1

1-chlorocyclohexane 0.2 ± 0.1 0.1 ± 0.05 0.2 ± 0.1

iodoalkanes[a]

1-iodopropane 4.7 ± 0.1 4.0 ± 0.1 2.7 ± 0.3

A Specific activities in U/mg protein, determined at 30°C.

Stabilizing mutations close to the active site had a more pronounced

effectonoverallthermostability.Formutationsneartheactivesite,66%(16of

24) affected overall stability, whereas only 44% of distalmutations (44 of 99

variantscharacterized) influencedstability,eventhoughthedesignprocedures

were very similar. Mutations that improved local folding interactions (e.g., by

optimizing packing in the hydrophobic interior) were much more stabilizing

whenclosetotheactivesite(4of13,comparedto2of34).Awayfromtheactive

Chapter4

104

site,sevenoutof tenmutationsstabilizedtheenzymebyimproving long‐range

electrostatics at the surface. This suggests that mutations that improve local

folding interactions close to the active site contribute more to the overall

stabilityofLinB.

Assays were carried out with 1‐bromopropane to analyze whether

stabilizingmutationsclosetotheactivesite influenceactivity: fiveoftheseven

stabilizing mutations significantly reduced the activity of the enzyme; only

mutations F169V andT249L increased stabilitywhile retaining activity (Table

4‐1). Thus, although 29% of the mutations surrounding the active site were

stabilizing (compared with 10% for those outside this area), most (71%)

diminishedactivity,whereasallbutoneofthemoredistantmutationsretained

catalyticactivity.

CombiningmutationstoobtainafurtherstabilizedvariantItwasinvestigatedwhetherit ispossibletoobtainahyperstabilizedvariantby

combiningallthemoderatelyandhighlystabilizingmutations.Thus,stabilizing

mutations E15T/A53L/A81K/A197P/D255A (distant from the active site) and

F169V/A247F (close to the active site) were introduced into variant G1. The

resulting variant (LinB‐G3) contained ten stabilizing point mutations and one

disulfidebond.ItsTm,appwasmeasuredbybothdifferentialscanningcalorimetry

(DSC) and thermofluor assays:Tm,appwas (74±0.5)°C, an increaseof 23°C over

wild type (Figure 4‐3A and B). This resulted in improvements to other

parameters relevant for biocatalysis, such as half‐life and activity at higher

temperatures:half‐lifewas200minat60°C,comparedtojust1.0minat55°Cfor

thewildtype(Figure4‐3C).

To determine activity at 30°C, variant G3 was assayed with 1‐

bromopropaneandseveralotherchloroalkanes,iodoalkanes,andbromoalkanes

(Table 4‐4). Most of the G3 mutations did not reduce enzymatic activity

individually (Table 4‐1). However, activity at 30°C was reduced when the

mutationswerecombined:kcatfor1‐bromopropanewasreduced(46%),Kmwas

also weaker (1.9 vs. 1.3 mM), and there was a moderate reduction (19%) in

catalyticefficiency(kcat/Km,Table4‐3).Theactivityforseveralotherhaloalkanes

was also lower (Table 4‐4). However, the improved thermal stability allowed

variantG3toperformdehalogenationreactionsat55°C,whereasthewild‐type

enzyme starts to unfold above 40°C. Variant G3 is as active at its optimum

temperatureof55°Casthewildtypeatitsoptimumtemperatureof40°C(Figure

4‐3D).

ComputationalLibraryDesignforIncreasingHaloalkaneDehalogenaseStability

105

Improveddegradationofβ‐hexachlorocyclohexaneinDMSO/waterThermostableenzymesoftentoleratehigherconcentrationsofco‐solventbetter

than mesostable enzymes[6c,21,33]. This is advantageous for the conversion of

poorlysolublesubstrates inwater/co‐solventmixtures.To investigatewhether

thehigherstabilitiesoftheLinBvariantswereaccompaniedbyhighertolerance

of co‐solvent, catalytic activitywas determined in buffer containing 50% (v/v)

DMSOor1,4‐dioxane(Figure4‐4AandB).Tm,appvaluesinthepresenceofDMSO

or 1,4‐dioxanewere also determined. G1 and G3were inactivatedmuchmore

slowly,whiletheirTm,appvaluesinthepresenceofco‐solventsweresignificantly

increased over that of wild type (Figure 4‐4C and D). G3 displayed some

heterogeneity: a fraction of the enzyme initially unfolded but the rest of the

protein retained activity over time (Figure 4‐4B). These results clearly

demonstrate that these thermostablevariants areable to function significantly

betterinthepresenceoftheseco‐solvents.

A unique property of LinB is its activity with the highly recalcitrant β‐

isomer of HCH[16]. However, efficient conversion by LinB is hampered by the

poor solubility of this compound[21]. In water β‐HCH is soluble up to only 17

μM[16,21], but higher concentrations canbe obtained in aDMSO/watermixture.

Tofurtherimprovethesolubilityofandactivitytowardsβ‐HCH,thetemperature

wasraisedto45°C.Activityassayswithatenfoldhigherconcentrationofβ‐HCH

(180 μM in a DMSO/buffer mixture) at 45°C showed superior conversion

comparedtothatinbufferalone(Figure4‐5).Degradationofβ‐HCHbythewild‐

type enzyme and variant G1 in 25% DMSO at 45°C was compared: G1 fully

converted180μMβ‐HCHwithin 8h (99% conversion),whereas thewild‐type

enzymeconvertedonly26%andwasinactivatedwithin30min(Figure4‐5).The

concurrent increase in thermostabilityandsolvent tolerance suggests that it is

possible to enhance the performance of enzymes in organic solvents by

engineeringtheirstability[6c,21,33].

To investigate the cause of the improved tolerance to co‐solvent,Tm,app

was investigated in the presence of solvent. These experiments showed that

reducedTm,app as a function of co‐solvent concentrationwas rather similar for

wild‐type and G1 and G3 enzymes (Figure 4‐4C and D). However, the higher

meltingtemperaturesofthethermostablevariantswithoutco‐solventsresulted

in higherTm,app values in the presence of co‐solvent, even though themelting

temperatures were lower for both the wild‐type and mutant enzymes in the

presence of co‐solvent (Figure 4‐4C and D). When the addition of co‐solvent

resulted inmelting temperatures close to ambient temperature, LinBunfolded

rapidly, aswas the case for thewild‐type enzyme in 25%1,4‐dioxane (Figure

Chapter4

106

4‐3C and Figure 4‐4B). This demonstrates that the higher overall stability

resultedinahigherTm,appandslowerunfoldinginthepresenceofco‐solvent.

Figure4‐4.Comparisonofactivitiesandapparentmeltingtemperaturesofwild‐

typeLinB(blackline)andvariantsG1(dashedline)andG3(grayline)inbuffer

withorganic co‐solvent.A)Effectof the incubation timeon theactivity for the

dehalogenation of 1‐bromopropane in the presence of 50% DMSO. The initial

activitywithoutco‐solventwasset to100%.B)Asabovewith25%co‐solvent

1,4‐dioxane. C) Observed Tm,app of the different variants in the presence of

differentamountsofDMSO.D)Asabovewith1,4‐dioxane.

KineticresolutionInorder to investigatewhether theFRESCOmethodcould improve thesolvent

toleranceofLinBwhileretainingenantioselectivity,variantG1wasusedforthe

kineticresolutionoftert‐butyl‐2‐(2‐bromopropanamido)‐acetateinthepresence

of co‐solvent. LinB is highly enantioselective in this conversion, but the

compoundissolubleinwateruptoonly1500μM[14c].VariantG1wasusedasit

was themostpromisingmutant. Its toleranceoforganicsolventwas improved

whileitscatalyticactivitywassimilartothatofthewild‐typeenzyme.

ComputationalLibraryDesignforIncreasingHaloalkaneDehalogenaseStability

107

Figure 4‐5. Conversion of 180 μM β‐HCH by wild‐type LinB (black line) and

variantG1(dashedline)at45°Cinthepresenceof25%DMSO(co‐solvent).A)β‐

HCH concentration against time. B) Concentration of the product

pentachlorocyclohexanolagainsttime,asdeterminedbyrelativeGCpeakareas.

Table 4-5. Kinetic resolution of t-butyl-2-(2-bromopropanamido)-acetate by LinB wild-type

and G1 in the presence of 10% THF at 30°C.

wild type G1 mutant

time

(h)

ee

substrate

(%)

ee

product

(%)

conversion

(%)

E

ee

substrate

(%)

ee

product

(%)

conversion

(%)

E

0 0 < 1 0 0 0

5 2 < 1 < 3 -[a] 12 < 1 < 3 -[a]

24 0.3 < 1 < 3 -[a] 56 89 39 30

48 0.2 < 1 < 3 -[a] 80 84 49 28

72 0.6 < 1 < 3 -[a] 91 81 53 29

A cannot be calculated due to the low conversion.

To allow higher substrate concentrations, the kinetic resolution was

examinedinabuffercontaining10%(v/v)THFat30°C.Undertheseconditions,

athree‐foldhighersubstrateconcentrationofcouldbetested(5mM,Table4‐5);

THF can easily be removed from the reaction mixture after extraction of the

substrates and products. Chiral HPLC showed that mutant G1 was active for

morethan72handgaveasubstrateconversionof53%undertheseconditions,

whereas the wild‐type enzyme was inactivated within 5h and its substrate

conversion was less than 3% (Table 4‐5). The enzyme remained moderately

enantioselective in this reaction (E value 28‐30; 67 for wild type without co‐

solvent)[14c]. This demonstrates that it was possible to enhance the solvent

tolerance of the enzyme while retaining most of the catalytic properties.

However, the organic co‐solventmight reduce enantioselectivity. Although the

amountofsolubilizedsubstratecouldbeincreasedbothforβ‐HCHandtert‐butyl

Chapter4

108

2‐(2‐bromopropanamido)acetate,thesubstratetocatalystratioremainedrather

low.Tomakethisconversionmoreefficient,futureenzymeengineeringstudies

needtobeperformedtoincreasetheenzymaticratesforthesereactions.

DiscussionWe describe here the use of a rapid computational design protocol for the

stabilization of the LinB haloalkane dehalogenase. Most of the laboratory

construction of libraries and thermostability screening (typical for directed‐

evolutionprojects)werereplacedby insilicomethods.Only150mutantswere

experimentally tested: 18 stabilizing mutations were found, as well as a

combined variant that showed a 23°C increase in apparent unfolding

temperatureandamorethan200‐foldlowerrateofinactivation.Lessthan3%

ofthemutationsthatwereanalyzedinsilicowereexaminedexperimentally.As

computationalscreeningisfasterandcheaperthanexperimentalscreening,this

FRESCO protocol[8f] provides an attractive route for rapid and cost‐effective

proteinstabilization.Thermostabilizationmethodsthatrelyontheexperimental

evaluation of large libraries of mutations usually require the screening of

thousandsofvariants,asmostrandommutationsareneutralordetrimentalto

stability[2c],evenwhenmore advancedmethods such as B‐fitter[34] are used to

identifytargetpositionsformutagenesis.TheFRESCOmethod,asdemonstrated

here for the stabilization of LinB and previously for limonene epoxide

hydrolase[8f], aims toeliminatemutations thatdonot contribute to thedesired

phenotype,byinsilicomethodsinsteadoflaboratorytesting.

TheincreaseinthermostabilityofthebestLinBvariantthatcarriesthree

point mutations and a disulfide bond was accompanied by an increase in co‐

solvent tolerance. Similar correlations between thermostability and solvent

tolerancewereobservedforhaloalkanedehalogenase[21],anaminopeptidase[33a],

analdolase[33b],anene‐reductase[33c]andanesterase[6c]andotherenzymes[33d‐g]

thus suggesting a common biophysical mechanism. For example, the

introductionofdisulfidebonds,whichinthecaseofLinBcanpreventunfolding

oftheNterminusandreduceheatinactivation[35],mightalsoreduceunfoldingin

thepresenceofco‐solvents,as,inbothcases,thegaininentropyassociatedwith

(local)unfoldingwouldbereduced.Polarsolvents,suchasDMSO,caninactivate

enzymes by breaking hydrogen bonds and by removing or replacing essential

water molecules in the solvent shell surrounding the protein[33d, 35]. Apolar

solvents, such as THF, can disrupt hydrophobic interactions in the protein

interior; these are important for protein folding.There are several biophysical

effects that can increase both thermo‐ and solvent stability, such as

ComputationalLibraryDesignforIncreasingHaloalkaneDehalogenaseStability

109

improvementstohydrophobicpackingintheproteininteriorortheintroduction

of stabilizing interactions in flexible parts. The observed relationship between

foldingenergy,thermostability,andsolventtolerancecanbeusedtoimprovethe

co‐solvent compatibility of enzymes more easily, as demonstrated here. The

ability to improveenzymestability in thepresenceofhydrophobic co‐solvents

(e.g.,THF)canprovidegreatbenefitsfordownstreamprocessinginbiocatalytic

conversion,suchasfacileproductseparationandrecovery.Furthermore,solvent

tolerance is often required for the use of hydrolytic enzymes in synthetic

reactions[36],andcontributestoapplicabilityinpharmaceuticalsynthesis[37]and

polymermodification[38].

Although the computational methods used in this work identified

eighteen mutations that enhanced stability, a significant number of the

mutations that were predicted to enhance stability appeared to lower LinB

thermostability when tested experimentally. Incorrect predictions frequently

involved the introduction of surface‐exposed hydrophobic residues. These

mutations can be discarded by visual inspection because they lack sufficient

interactionwithotherhydrophobicgroups.TheenergycalculationsofFoldXand

Rosettafavorintroductionofsuchresidues;thisis(partially)inagreementwith

observations for small model proteins, where such mutations can be

stabilizing[10,11b]. However, the introduction of hydrophobic side chains at the

protein surface canbedisadvantageous for theTm,app of largerproteins,which

moreeasilyaggregate[22].ArecentstudybyJacaketal.showedthatadaptations

to theRosettaenergy functioncanprevent the formationof thesehydrophobic

patches on the protein surface; these can be applied in future computational

enzymestabilization[22].

Partial unfolding of flexible areas of a protein can cause irreversible

inactivationbyaggregation[13].Mutationsthatimprovethestabilityofsuchareas

mightcontribute toenhancingoverallproteinstabilitymore thansubstitutions

in less‐flexible regions[7b, 27, 31]. The highly flexible regions that can serve as

targets for stabilization can be detected by their high B‐factors in crystal

structures[39] or by a high RMSF during MD simulations[40]. During the

engineeringofLinB,twostronglystabilizingpointmutations(ΔTm,app>4°C)were

discovered at residue 147,which is in a flexible helix (residues 138‐150) that

covers the active site (Table 4‐1, Figure 4‐1). Cα atoms in the loop between

residues138‐150arepredictedtoberelativelyflexible,aswasevidentfromthe

fivefoldhigherB‐factors(comparedtotheaverage)anda1.5‐foldhigherRMSF

during anMD simulation. The latter is the highest RMSF of thewhole protein

(Figure4‐1C).Anotherprominentstabilizingmutation(A247F)isnotitselfina

Chapter4

110

flexiblearea,buthasinteractionswiththisflexibleloop(Figure4‐1Aand4.2D).

Theonlystronglystabilizingmutationfoundinadifferentregionwasadisulfide

bond incorporated near the flexibleN terminus of the protein. Therefore, it is

likelythatthemosteffectivemutationsenhanceoverallstabilitybyreducingthe

unfolding of the weakest parts of the protein and thereby reduce protein

inactivationrates(Figure4‐3C).

By identifying flexibleregionswheremutationsaffect stability, thesizes

of libraries targeting enhanced stability can be significantly decreased[6b].

However, simply introducing themutations at highly flexible residues (the B‐

fitter approach) might not always be the best option. For the stabilization of

limoneneepoxidehydrolasebyFRESCO,themosteffectivepointmutationswere

nearflexibleareasratherthanintheflexibleregionsthemselves[8f]. InLinBthe

mutations with the largest stabilizing effects were in flexible regions, but the

majority of the moderately stabilizing mutations were not (Figure 4‐1B); one

highlystabilizingmutationwasnotinaflexibleareabuthadinteractionswithit.

These mutations would have been missed if only highly flexible residues had

beentargetedformutagenesis.

Inmanyenzymes, theactive site isoneof themost flexiblepartsof the

protein,and introducingstabilizingmutations into thisregionbears theriskof

reducing enzymatic activity, as has been observed in studies on lysozyme[41],

barnase[42], ribonuclease[43], beta‐lactamase[44], protease[45], enolase[46] and

citrate synthase[47]. However, other studies have shown that stabilizing

mutations near the active site do not necessarily affect catalytic activity.

Mutations at the entrance of the tunnel to the active site of haloalkane

dehalogenase fromRhodococcusrhodochrous stabilized theenzymebutdidnot

decreasecatalyticactivity;[21]similarly,theactivityandstabilityofalipasewere

improvedconcurrently[48].TheLinBactivesite isburiedintheproteinandcan

only be accessed after movements of the loops covering it[49]. Therefore,

flexibility of these loops is important for substrate entrance or product

release[49b,50].We indeedobserveda significantreduction inenzymaticactivity

of LinB bymost stabilizingmutations introduced close to the active site. Four

stabilizing mutations introduced at position D147 (Table 4‐1, Figure 4‐2C)

caused a reduction in activity, likely due to stronger interactions between a

flexible helix (residues 145‐155) and a loop and helix covering the active site

(residues166‐178and244‐250); these increasedstabilitybut reducedactivity

with1‐bromopropane(Table4‐1).

The effect of stabilizing mutations on catalytic activity differed

significantlydependingonsubstrate(Table4‐3andTable4‐4)andtheparticular

ComputationalLibraryDesignforIncreasingHaloalkaneDehalogenaseStability

111

mutant enzyme. For variant G1 (no mutations near the active site), the

conversionrateswerenearly identical for11different substrates,even though

theinactivationratedecreased30‐fold(Figure4‐3C)andTm,appandthecatalytic

activityathighertemperaturesincreased.However,withvariantG3,threeofthe

elevensubstrateswereconvertedtentimesmoreslowlythanwiththewild‐type

enzyme,whereas the other eight substrateswere converted only two to three

timesmoreslowly(oratasimilarrate;Table4‐4).

In conclusion, the work reported here demonstrates that the

thermostability and solvent tolerance of haloalkane dehalogenase can be

simultaneously increased by using the FRESCO method for computational

enzyme stabilization. Mutations introduced close to the active site were most

stabilizing, but these should be chosen with care because they have a high

probabilityofresulting ina lossofactivityby influencingfunctionalmovement

aroundtheactivesite.Byintroducingmutationsonlyatmoredistantpositions,it

was possible to engineer a variant that displayed both improved stability and

fullypreservedcatalyticactivityforalltestedsubstrates.Theincreaseinstability

wasaccompaniedbya large improvement inorganicco‐solvent tolerance, thus

enhancingthebiocatalyticpotentialofthisenzyme.

MaterialsandmethodsComputationaldesignofstabilizingmutationsbyFRESCO:TheFRESCOapproachisbasedon

thecombineduseofseveralcomputationalalgorithmstodiscoverstabilizingmutations.Tofind

stabilizingpointmutationsinLinB,allpositionsoftheproteinsequenceweremutatedinsilicoto

allproteinogenicaminoacidsexceptcysteine.Foreachmutant,a3Dstructurewaspredictedby

FoldX[10], with the crystal structure of wild‐type LinB[49a] as the template. The free energy

difference between the folded and unfolded structures (ΔΔGfold) of these point mutants was

predictedbybothFoldX[10]andRosetta[11b]andwascomparedtothatofwildtype.ΔΔGfoldwas

usedasameasureofenzymestability.Settingsdescribedpreviously(Table1,row3inref[11b])

wereusedforRosettacalculations.ForFoldX,thestandardparametersettings,whichhavebeen

testedonalargedatasetofpointmutants,wereused[10].

Themodeledthree‐dimensionalstructuresofthemutantspredictedtobestabilizingby

FoldXorRosettawereinspectedvisuallyinYASARA(www.yasara.org).Mutationsthatresulted

in one of the following types of unfavorable interactions were excluded: 1) steric clashes, 2)

internalcavities,3)solvent‐exposedaromaticresidues,4)solvent‐exposedmethionines[51],5)a

hydrogen bond donor or acceptor that has fewer hydrogen bonding interactions than inwild

type,6)a largehydrophobicpatchontheproteinsurface,7)uncompensatedremovalofasalt

bridge, and 8) destabilization of an α‐helix by removal of α‐helix capping[52]. In the field of

computational design, visual inspection to eliminate designed variants with such defects is a

standardstepandisrequiredbecauseof insufficientconformationalsamplingandinaccuracies

in the energy functions[53]. Mutations were treated in two categories for stability/activity

investigations: those within 9 Å of any atom of the substrate (tert‐butyl‐2‐(2‐

Chapter4

112

bromopropanamido)acetate), and those outside this area. To determine these distances, the

substratewasmodeledintheactivesitebyusingAutoDock4[54].

Positions suitable for the introduction of disulfide‐bond forming cysteines were

predicted by the Dynamic Disulfide Discovery (DDD) algorithm[8f], for both the wild‐type

structureandsnapshotsofMDsimulationsforconformationalsampling.Predictionofdisulfide

bonds is highly dependent on backbone conformation, and can be improved by the use of an

ensembleoftemplatestructuresgeneratedbyMDsimulations.FortheDDDprocedure,thewild‐

typestructurewasplacedinawaterboxandsimulatedat298KwiththeYamber3forcefield[55].

FiveindependentMDsimulationswereperformed.Foreachsimulation,thefirst250pswasused

for equilibration, andafter entering theproductionphase, snapshotswere takenevery250ps

between500and2500ps,thusyielding45snapshotsintotal.Fromthisensembleofstructures

asa template, theDDDalgorithmpredictsdisulfidebondsbasedongeometricconstraints that

are derived from the angles, dihedrals, and bond lengths of disulfide bonds in high‐resolution

structures in the protein database[56] The structures of all predicted disulfide bonds were

analyzedvisuallyaccordingtothescreeningcriteriadescribedintheprevioussection.

Toeliminatemutationsfalselypredictedtobestabilizing,predictedpointmutationsand

disulfide‐bondmutantswerescreenedbyMDsimulation.Thesimulationswerebasedonthe3D

structureofthemutantaspredictedbyFoldX,andusedtheYamber3forcefield[55].Five100ps

MD simulations were performed for each mutant. Each simulation started with a different

random set of initial atomvelocities, assigned according to aMaxwell‐Boltzmann distribution.

Thetemperaturewasincreasedfrom5to298Kover30ps,followedbyequilibration(20ps)and

production (50 ps). Snapshots were taken every 5 ps. For each variant, five independentMD

simulations were performed. During each simulation, ten snapshots were recorded, and the

averagecoordinatesofthesetensnapshotswerecalculated,toobtainoneaveragestructureper

trajectory.Ifoneofthetrajectoriesdisplayedlargelocalconformationaldifferencescomparedto

theothersimulations, itwasconsideredtobeanoutlierandwasnotusedfor furtheranalysis.

Theresultingtrajectorieswerevisuallyscreenedbythecriteriamentionedpreviously.Mutations

were selected for experimental verification if they did not exhibit clearly unfavorable

interactionsduringMDsimulation.

Construction of mutants: A pBAD plasmid containing the linB gene from Sphingomonas

paucimobilisUT26(UniProtaccessionnumberP51698)attachedtoanN‐terminalhexahistidine

sequence was used throughout this study[14c]. Mutations to the linB gene were introduced by

QuikChangemutagenesis (AgilentTechnologies)with thePfuUltraHotstart PCRMasterMix as

recommendedbythemanufacturer.QuikChangePCRproductsweretransformedintochemically

competentE.coliTOP10(LifeTechnologies). Incorporationof themutationswasconfirmedby

DNA sequencing. Successive rounds of mutagenesis were performed to construct variants

containingmultiplemutations.AsE.colioftenhasdifficultywithcytosolicproductionofproteins

containing disulfide bonds, the production of variants containing these bonds was also

performedinE.coliSHuffle(NewEnglandBiolabs).Thisstrainisoptimizedfortheproductionof

cytosolicproteinscontainingdisulfidebonds[29].

Protein expression: Small‐scale production (1.2 mL) of LinB was performed in deep‐well

microtiter plates (MTPs). Large‐scale protein expression (1 L)wasperformed in terrific broth

(TB:yeastextract(24gL‐1),tryptone(12gL‐1),glycerol(4mLL‐1),KH2PO4(17mM),K2HPO4(72

mM), pH 7.0). Themediumwas inoculatedwith 1%of an overnight culture and subsequently

incubated at 37°Cwith shaking at 200pm.AtOD600=0.6 expressionwas induced by adding L‐

ComputationalLibraryDesignforIncreasingHaloalkaneDehalogenaseStability

113

arabinose (0.02%, w/v) and the culture was grown for a further 16h at 24°C. Cells were

harvestedbycentrifugation(2000g,15min,4°C)andsuspendedinPNbuffer(450μLforsmall‐

scaleexperimentsand30mLforlarge‐scaleexperiments:potassiumphosphate(50mM,pH7.5)

withNaCl(150mM)).

Small scale protein purification: Cells from a 1.2 mL culture were lysed by addition of

FastBreak cell lysis reagent (50 μL, Promega) followedby incubation for 15min at 30°Cwith

shakingat200rpm.Topreparecell‐freeextract,thelysatewascentrifuged(2000g,45min,4°C)

andthesupernatantwastransferredtoacleanMTPandusedforproteinpurification.Topurify

proteins on a small scale, the MagneHis protein purification system (Promega) was used

according to the manufacturer’s instructions. Briefly, cell‐free extract was incubated with

magneticbeadssuspension(10μL),thebeadswerecollectedwithamagnet,thesupernatantwas

removed,andthebeadswerewashedthreetimesinPNbuffer.LinBproteinwaselutedinelution

buffer(200μL;PNcontainingimidazole(500mM))andthemagneticbeadswereremovedwith

themagnet.Subsequently,fourroundsofdilutionoftheLinBsolutioninproteinstoragebuffer

(Na‐HEPES(50mM,pH7.5))andconcentrationinanAmiconUltra‐0.5centrifugalfilter(10kDa

cut‐off,MerckMillipore)wereperformedtodesalt theprotein.The finalenzymesolution(100

μL)was obtainedwith a protein concentrationof~0.2mgmL‐1. SDS‐PAGE showed that these

samplescontained>90%targetprotein.

Large‐scaleproteinpurification:Thecellswerelysedbysonication(10son,30soff,10min;

65W,20kHz,4°C).Celldebriswasremovedbycentrifugation(31000g,45min,4°C),thenthe

supernatantwas loadedonaNi‐NTAcolumn(GEHealthcare)andwashedwithPNbuffer (ten

columnvolumes)andelutedbya lineargradientofelutionbuffer.Fractionsthatcontainedthe

desiredproteinwerepooledanddesaltedintoproteinstoragebufferbyusingaHiTrapdesalting

column(GEHealthcare).Forallvariants,theyieldwas50mgofproteinperliterofculture.The

samplewasanalyzedonaSDS‐PAGEgel:asinglebandsuggestedthatproteinpuritywas>95%.

Enzyme assays: Catalytic activity by LinB variants was quantified by dehalogenation of the

modelsubstrate1‐bromopropane.Proteinsample(20μL,0.2mgmL‐1inproteinstoragebuffer)

wasmixedwith1‐bromopropane (285μL,30mM inTrisSO4 (50mM,pH8.2)) inamicrotiter

plate.Themixturewasincubatedat30°C.Samples(40μL)wereremovedatregularintervalsand

mixedwith halide quantification reagent (160 μL; NH4Fe(SO4)2 (16 μL, 0.25 M in HNO3 (9M)),

saturatedHg(SCN)2(16μLinethanol),anddeionizedwater(128μL)).Thisreagentquenchesthe

dehalogenation reaction and allows quantitative determination of halide concentration[57]. The

sampleswere incubated for 5min at 20°C, and absorbance at 460nmwasmeasuredwith an

SynergyMxplatereader(BioTek,Winooski,VT).AcalibrationcurvewasmadewithNaBr,and

the free bromide concentration was calculated. Where necessary, a calibration curve of free

bromideinthepresenceofco‐solventwasconstructedtocorrectfortheeffectofco‐solventon

thehalideassay.A linearfitofthisdatawasusedtoobtaintheinitialrate.Specificactivitiesat

different substrate concentrations were determined and fitted with the Michaelis‐Menten

equationtoobtainkineticsparameters.Specificactivitiesathighertemperaturesweremeasured

byincubatingtheproteinsamplefor5minatthespecifiedtemperatureandmeasuringspecific

activity at this temperature.A unit of catalytic activity (U)wasdefined as the conversionof 1

μmolofsubstrateinoneminute.

The halide release assay was used to determine catalytic activities of dehalogenase

variantswithseveralchloro‐,bromo‐,andiodoalkanes,andhalocycloalkanes.Alinearcalibration

curvewasusedforbromideandiodinequantification;apolynomialcalibrationcurvewasused

Chapter4

114

forchlorideanalysis.Thesubstrateconcentrationswerechosensuchthattheywerebelowthe

solubilitylimitofthiscompoundinwaterand(ifpossible)abovethereportedKmvaluesforthe

dehalogenationreaction[14a].

Proteinconcentrationsweredeterminedbyabsorbanceat280nm(ε280=5.6×104M‐1cm‐

1). For protein samples below 1.0 mL, concentration was determined with a NanoDrop 1000

spectrophotometer(ThermoScientific).

Dehalogenationofβ‐HCH:β‐HCH(2.88mg;Sigma‐Aldrich)wasdissolvedinDMSO(15mL)to

obtain a 660μM solution.An aliquot (3.75mL) of thiswasmixed in a 15mLPyrex tubewith

buffer(11.3mL,potassiumphosphate(50mM,pH7.5)withglycerol(10%))andLinB(6.4mg,

190nmol;wildtypeorvariantG1).Thetubewasincubatedat45°Cwithshakingat200rpm,and

samples (1 mL) were removed at regular intervals. β‐HCH, pentachlorocyclohexanol, and

tetrachlorocyclohexanediol were extracted with ethyl acetate (0.5 mL) containing 1,2,4‐

trichlorobenzene(4μM;internalstandard).ThisextractwasdriedwithMgSO4andanalyzedona

model7890Agaschromatograph(Agilent)withanRtx‐1column(Restek,Bellefonte,PA)andan

electroncapturedetector,asdescribedbyItoetal.[16]Thebaseline‐separatedsignalsofsubstrate

β‐HCH (retention time (tR) 10.7min) and products (tetrachlorocyclohexanediol (tR=11.6min)

andpentachlorocyclohexanol (tR=11.8min))weremeasured.Toquantifysubstrateconversion,

the peak area of β‐HCHwas divided by the peak area of the internal standard. This response

valuewasconvertedtoabsoluteconcentrationbyusingacalibrationcurve.

Kineticresolution:Thesubstratetert‐butyl‐2‐(2‐bromopropanamido)‐acetatewassynthesized

as reported by Westerbeek et al.[14c] To follow its kinetic resolution, tert‐butyl‐2‐(2‐

bromopropanamido)‐acetate(50mMinTHF)wasdiluted(1:10)inTris‐SO4(50mM,pH8.2)to

obtain a 5mM solution in a buffer/co‐solventmixture.An aliquot (15mL)was incubated in a

Pyrextubeinawaterbathat30°Cwithwild‐typeLinBorLinB‐G1(0.5mgmL‐1).Samples(1mL)

were removedat regular intervals, and the substrates andproductswere extractedwith ethyl

acetate(1mL).ThisextractwasdriedwithMgSO4and filtered.Volatileswereevaporated,and

theenantiomericexcessofα‐bromoamideandα‐hydroxyamidewasdeterminedbychiralHPLC

withanAD‐Hcolumn(Chiralcel,Illkirch,France;4.6×256mm,5μm,heptane/iPrOH(95:5),flow

rate0.5mLmin‐1).Retentiontimesforsubstrate:22.4(R)and24.1min(S);forproduct:30.4(R)

and36.1min(S).Theenantioselectivity(E)andconversionswerecalculatedbysubstitutingthe

measuredenantiomericexcessofsubstrateandproductintopublishedequations[58].

Determination of apparent melting temperatures: The thermofluor assay was used to

determineTm,app[24].SyproOrange(μL,100‐folddiluted;LifeTechnologies)wasaddedtopurified

enzyme(20μL,0.2‐1.5mgmL‐1).ThissamplewastransferredtoaiQPCR96‐wellplate(Bio‐rad,

Hercules,CA)and sealedwith iQ96‐wellPCRPlate seal (Bio‐rad).Fluorescence (RFU;λex=490

nm,λem=575nm)wasmonitoredwhilethesamplewasheatedfrom20to99°C(1.1°Cmin‐1)ina

CFX96Q‐PCR device (Bio‐rad).Tm,appwas defined as themaximumof dRFU/dT. To determine

Tm,appafterreductionofdisulfidebonds,dithiothreitol(DTT,10mM)wasadded,andthemixture

wasincubatedfor15minat20°Cbeforemeasurement.

Differential scanning calorimetry: Apparent melting temperatures were measured by DSC.

Proteinsamplesweredialyzedfor16hagainstNa‐HEPES(50mM,pH7.5)toremovesalts.The

Tm,app was determined by measuring excess heat capacity of a protein sample (20 μm) while

heatingfrom20to110°C(1°Cmin‐1)inaVP‐DSCMicroCalsystem(GEHealthcare).Areference

signalwasmeasuredwithbufferinthesamewayandsubtractedfromtheproteinsamplesignal

ThetemperatureatwhichtheexcessheatcapacitywasmaximalwastakenastheTm,app.

ComputationalLibraryDesignforIncreasingHaloalkaneDehalogenaseStability

115

Thermal inactivationrates:Thermal inactivation ratesweremeasuredby incubatingprotein

samples(0.2mgmL‐1inproteinstoragebuffer)forvaryingtimesatthespecifiedtemperatures,

andsubsequentlyincubatingtheenzymesat4°Cfor5min.Theresidualspecificactivitywasthen

determinedbyusingthestandardactivityassay.Aplotofresidualactivityversusincubationtime

was fittedwith a single exponential decay to obtain the irreversible thermal inactivation rate

(kinact).Itwasexperimentallyconfirmedthattheenzymeunfoldedirreversiblybymeasuringthe

activityofthermallyunfoldedenzymeafterincubatingitat4°Cfordifferenttimeperiods.

AcknowledgementsA.R.‐S.wassupportedbyafellowshipfromCoordenaçãodeAperfeiçoamentode

Pessoal de Nível Superior (CAPES/PDSE).We thankMalgorzataMurawska for

helpwith obtaining chiral HPLC separations. Thisworkwas supported by the

European Union 7th framework projects Metaexplore (KBBE‐2007‐3‐3‐05,

222625) and Kyrobio (KBBE‐2011‐5, 289646) and by NWO (Netherlands

OrganizationforScientificResearch)throughanECHOgrant.

Author contributions: RJF and DIC constructed and characterized the

enzymevariants. RJF,DBJ andHJWdesigned the laboratory experiments.HJW

and DIC adapted the design algorithms and performed the computational

screening. HJW, SJW and DBJ designed the in silico approach. PAJ and ARS

performed additional enzyme characterization. WS performed kinetic

resolutionsandtogetherwithBLFcorrectedthemanuscript.RJF,HJW,DICand

DBJwrotethepublication.

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