© 2009 Nature America, Inc. All rights reserved. University, Sendai, Japan. and Cellular Modeling Group, EML Research gGmbH, Heidelberg, Germany. Republic. 1 ( 1,3-dichloroprop-1-ene and polymers liquid polysulfone of duction and extractive agent. TCP is used as a chemical intermediate in a as use theand pro formulation manufacture, its of a result as ment non-natural compound is TCP ( conversion their catalyzing of capable to time evolve have sufficient had not microorganisms because ment Industrial Revolution the are often since recalcitrant and persist in the humans environ by gener biosphere the into introduced anthropogenically substrates ated contrast, In toxins. substances, potential occurring including naturally metabolizing of capable enzymes targets. engineering protein and loci evolutionary both as tunnels access of importance the highlight and evolution directed The the results demonstrate power with design of combining rational nase from dehaloge connecting haloalkane (DhaA protein the engineered site of the active tunnels access the in located residues targeted we here in the or its close vicinity are most frequently predicted as hot spots for mutagenesis are located puter-guided site-directed mutagenesis by com prepared constructs fine-tuning for and spot” “hot residues these of mutagenesis systematic for used is evolution directed then stabilization, state transition and binding in participating activities very efficient two-step approach for proteins engineering with desired Combining rational protein design with directed evolution provides a design with directed evolution may be a valuable strategy for refining catalytic properties of enzymes with buried active sites. and shifted the rate-limiting step to the release of products. Engineering access tunnels by combining computer-assisted protein promoting activated complex formation. Kinetic analyses confirmed that the mutations improved bond carbon-halogen cleavage These changes apparently enhance activity with TCP by decreasing of accessibility the active site for water molecules, thereby large aromatic residues at two out of three randomized positions and two positions modified by site-directed mutagenesis. buried active site with bulk solvent by rational design and randomized them by directed evolution. The most active mutant has compound haloalkane mutants dehalogenase with up to 32-fold higher activity than wild type toward the toxic, recalcitrant anthropogenic Engineering enzymes to degrade anthropogenic compounds efficiently is challenging. We obtained Rebecca C Wade PavlovaMartina substrate anthropogenic an degrading for strategy a as tunnels access dehalogenase Redesigning nature CH nature Received 20 January; accepted 28 May; published online 23 August 2009; Loschmidt Laboratories, Loschmidt Institute Laboratories, of Experimental Biology and National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno, Czech 2 ). In addition, it is produced as in quantities a significant byproduct Living organisms have typically had millions of years to evolve evolve to years of millions had typically have organisms Living 2 1–7 Department Department of Physical Chemistry and Center for Biomolecules and Complex Molecular Systems, Palacký University, Olomouc, Czech Republic. Rhodococcus Rhodococcus rhodochrous E . Computer modeling is initially used to identify residues residues to used identify is initially . modeling Computer 1 MIC ,2,3-trichloropropane (TCP) using a new strategy. We identified key residues in access tunnels connecting the A L BIOLOGY L 3 1 , Masataka Tsuda , 5 5 , Klvana Martin These These authors contributed equally to this work. should Correspondence be addressed to J.D. ([email protected]).

advance online publication online advance 1 ), which is released into the environ 9 NCIMB13064) with bulk NCIMB13064) solvent. . However, in the study presented 8 . The amino acid residues that 4 1 , Yuji Nagata , 5 , Zbynek Prokop 10,11 . One such toxic, such .One 4 & Jiri Damborsky

doi:10.1038/nchembio.20 4 Department Department of Life Environmental Sciences, Graduate School of Life Sciences, Tohoku 1 , Radka Chaloupkova ------

C176Y Y273F (M2)) were obtained that were 4 times and 3.5 times times 3.5 and times 4 were that obtained were (M2)) Y273F C176Y version dhaA base catalytic a by activated molecule water a by hydrolyzed alkyl- covalent a intermediate. This of alkyl-enzyme intermediate is subsequently formation and halide a of displacement bond, of atom carbon a carbon-halogen the of to on cleavage leading substrate, halogenated the nucleophile a of attack nucleophilic via site in proceeds the active reaction ing The environment. dehalogenation site and its surround the active between can exchange ligands which tunnel slot the and tunnel main the as to (referred nels domains protein two between located worldwide spp. dehaloge haloalkane nase DhaA the is TCP against activity detectable have to reported dehalogenase first The mechanism. hydrolytic a by enzymes dehalogenase alkane to 2,3-dichloropropane-1-ol (DCL, ( slowly be can TCP conditions, laboratory environment groundwater the in time of periods long for persist to likely is TCP years, 100 to up extend to estimated is conditions groundwater under half-life its that gen. Further, given carcino human a be to anticipated is and groundwater, and water drinking water, surface in concentrations low in detected been has 2-(chloromethyl)oxirane (also called epichlorohydrin, epichlorohydrin, called (also 2-(chloromethyl)oxirane example, compounds—for chlorinated other of manufacture the in DNA shuffling and error-prone PCR have been applied to the the to applied been have PCR error-prone and shuffling DNA 1 5 gene to improve the kinetic properties of DhaA for TCP con TCP for DhaA of properties kinetic the improve to gene , which has been isolated from various strains of of strains various from isolated been has ,which 14,24 1 . Two evolved double point mutants (G3D C176F and and C176F (G3D mutants point double evolved Two . 5 16–20 . The active site of DhaA is an occluded cavity cavity occluded an is DhaA of site active The . 1 , Pavel Banas 1 4 , which cleave carbon-halogen bonds bonds , cleave carbon-halogen which 4 Rhodococcus Rhodococcus rhodochrous ) ) by the action of microbial halo 2 2 , Michal Otyepka 1 , with two major access tun access major two ,with k cat = 0.08 s 0.08 = 1 t r a 3 . However, under −1 Rhodococcus 2 ) converted converted ) 2 L C I 3 ) through through ) 3 ) 2 Molecular Molecular 2 1 , 3 2 . . TCP TCP .

s e  ------

© 2009 Nature America, Inc. All rights reserved. in ~8% and ~13% of the simulations, respectively). We in respectively). ~8% and the ~13% of simulations, hypothesized (occurring slot mutant M2 the and enzyme the wild-type the both through of tunnel released be could the that indicated also and enzyme 4 in wild-type the with (~70%) simulations RAMD 24 out of 17 in occurred tunnel main the through product the of release ( M2 in tunnel than enzyme main the wild-type the in via frequently more released was DCL that cated indi calculations The bottlenecks. possible identify to and solvent bulk with site active buried the connecting tunnels access the probe to simulations RAMD using studied was mutant M2 the and DhaA wild-type the from DCL product dehalogenation the of release The tunnels access in spots hot of mutagenesis Saturation RESULTS enzyme. wild-type natural TCP,the toward to the compared activity higher 32-fold showed which of active most activities higher with variants protein unique 25 Weobtained screening. by identified were activities improved with mutants and constructed were genes mutant of libraries exhaustive mutagenesis, saturation by targeted were tunnels access the in spots hot These mutagenesis. for residues key the identify and site active (RAMD) TCP conversion. We applied random acceleration molecular dynamics 1 Fig. design and directed evolution in an iterative manner ( DhaA main of the tunnel narrowed phenylalanine or tyrosine bulkier type a by Cys176 wild the in CAVER using tunnels mutants access the and the of comparison Quantitative solvent. surrounding the with site active buried the connecting nel the tun access Instead, the of mouth state. the modify to found was transition mutation C176Y the of stabilization the the or in site substrate active the of positioning the alter not did mutations the experiments in evolution directed obtained these mutants the in improved for basis structural the elucidate to calculations mechanical quantum and simulations dynamics 176. We molecular used at position have recently stitution a sub carried mutants evolved independently both that noteworthy is It enzyme. wild-type the than TCP with respectively, active, more ( mutagenesis. for selected residues green, tunnel; slot the to corresponding pathway release product blue, tunnel; main the to corresponding pathway release product yellow, domain; cap white, domain; Gray, main DhaA. ( simulations. computer by mutagenesis 1 Figure t r a  to entry ligand’s the for importance greater has tunnel slot the that b a ) Ball and stick model of the residues selected for mutagenesis. for selected residues the of model stick and ) Ball In the present study, we combined several methods of rational rational of methods several combined we study, present the In

out of 8 RAMD simulations (~50%) with M2. The simulations simulations The M2. with (~50%) simulations RAMD 8 of out ) to obtain DhaA mutant enzymes with significantly improved improved significantly with enzymes mutant DhaA obtain to )

L C I Hot spot residues lining the access tunnels selected for for selected tunnels access the lining residues spot Hot 2 7 to simulate the release of the product from the enzyme’s enzyme’s the from product the of release the simulate to s e 2 5 . 2 6 revealed that the substitution of of substitution the that revealed b a ) Cartoon model of wild-type wild-type of model ) Cartoon l135 Supplementary Table 1 Supplementary 2 5 . The results indicated that that indicated results The . W141 Supplementary Supplementary L246 V245 C176 ). The The ). - - - were used as starting proteins for directed evolution experiments experiments evolution directed for proteins starting as used were M3 and M2 mutants The Y273F). C176Y (W141F M3 mutant triple Y273F) (C176Y previously described M2 mutant More was constructed. specifically, into a was W141F inserted double ing to the phenylalanine) most ( conservative substitution contain library a therefore, separate to unfolding; lead often protein ing the diameter of the slot tunnel. However, mutations of tryptophan by Trp141isoleucine. substituted is one the of key determin residues is 246 position in leucine and valine; and phenylalanine alanine, by valine, tuted by phenylalanine and serine; methionine, substi valine in is position 245 141 is substituted by position in tryptophan substituted threonine; and is phenylalanine 135 position in isoleucine (ref. HLD-II subfamilies three dehalogenases: the haloalkane of of one of alignment sequence multiple a in were analyzed acid amino their propensities computer simulations, ( mutagenesis for spots hot as (Val245) tunnel main the and site active the tunnel, the slot of interface the at and Leu246) and (Trp141 site active the and tunnel slot the of interface the at (Ile135), tunnel slot the in located residues selected we TCP. Thus, toward the activity with increased mutant’s associated is importance increased this that and enzyme, wild-type the in than mutant M2 the in site active the from exit or dichroism spectra of the wild-type DhaA and its variants. its and DhaA wild-type the of spectra dichroism ( 2 Figure no and comparable, was level expression The analysis. further extracts for cell-free obtain to and wild-type than higher variants activity mutant with unique 25 the carrying cultures the of expression R254A. mutation, spontaneous a carried 27) GTA A in (variant to triplet TTT. codon the library from clone One change two-base a requires phenylalanine to valine of mutation method mutagenesis saturation the of to bias the technique, a such evolution as directed routine error-prone PCR, due by obtained have been never probably would mutations the of Some than the template ( activity ants higher with detailed characterization. Sequencing revealed 25 unique mutant vari to more subjected and were isolated cultures to wild-type relative ity The wild-type. 51 clones exhibiting the highest improvement in activ than to activity have appeared higher activity, but a proportion small activity such no or dehalogenating little had (96%) TCP. for toward library the in mutants Most screened and expressed were variants were picked, A from and clones 5,273 the DhaA libraries total of final against TCP activity enhanced with Mutants respectively. clones, 2,705 and 2,568 contained B and A Libraries 246. and 245 135, positions desired the at mutations point possible all introduce to used was sets primer degenerate with A libraries to leading a Va a 33 31 27 21 19 17 M3 M2 WT ) Summary of mutations in selected DhaA variants. ( variants. DhaA selected in mutations of ) Summary To assess the mutability of the sites identified as hot spots by the the by spots hot as identified sites the of mutability the Toassess Small-scale cultivation was used to compare the level of DhaA DhaA of level the compare to used was cultivation Small-scale r iant advance online publication online advance

Characteristics of mutants with enhanced activity against TCP. against activity enhanced with mutants of Characteristics 135 W C V Y F F L I I I 141 W W W W F F F F Fig. Fig. 176 Residue C Y Y Y Y Y Y Y Y Y 1

and B, respectively. Saturation mutagenesis mutagenesis Saturation respectively. B, and ). 245 M M V V V F F F 246 L L L L I I I I I 273 Y F F F F F F F F

b nature nature Mean residue ellipticity (×103 deg cm2 dmol–1) –8 –4 12 Supplementary Table Supplementary 2 0 4 8 185 29). In this alignment, alignment, this In 29). ch 200 3 Wa b 1 0 e 4 . For instance, . the For instance, ) Far-UV circular circular ) Far-UV mic , resulting in the the in resulting , v 215 elength (nm) a 230 l biology 19 17 M3 M2 WT 245 260 33 31 27 21 2 ). - - - - - 8

© 2009 Nature America, Inc. All rights reserved. frequency from mutant 21 to mutant 31—that is, in the direction of of direction the in is, 31—that mutant to 21 mutant from frequency ( variants the all of tions simula in observed was tunnel main the through TCP. release DCL better understanding of the molecular basis for enhanced activity with gain to 31) and 27 (21, experiments mutagenesis from obtained ants vari active most the and M2 the variant template the enzyme, with wild-type conducted were DCL of egress the of simulations RAMD against TCP activity enhanced of basis Molecular haloalkanes. (>C3) activity longer toward reduced and haloalkanes, C2-C3 1,2-disubstituted toward activity specific in increase systematic a showed 31 variant the and ( measured also were substrates halogenated catalyzed by DhaA and the the wild-type 31 variant with several other tively, enzyme. than The that rates of the of dehalogenation wild-type by quantified 31, and 21, 27 variants the of efficiency catalytic the but clones, tested all for wild-type. the of that than higher times 32 is a has 31 variant The mutants. M3 higher have ( substrate a as TCP deter with mined were parameters kinetic state steady The detail. in acterized showed than higher activity the templates M2 and M3, and were char were ( analyzed activities specific of lytic properties of the constructed and evolved protein variants in terms acid residues contributing to the amino of number overall the on particularly enzymes, mutant the of effect on the packing of amino acid residues in the secondary structure to the wild type. This indicates that the inserted mutations have a small the variants exhibit an increase in can be seen at type the second negative peak, where of the CD spectra wild- the and mutants the between compactly.differences fold Small they that suggesting enzyme, wild-type the of that to identical nearly ( nm 195 at peak positive one tested enzyme variants show two negative peaksthe all at of 208 spectra and The 222structure. nm,secondary and and folding their affected variants purified the by carried mutations the whether assess to used l mg 20 of yield purification average an with homogeneity to purified and overexpressed were 33) and 31 27, 21, TCP 19,(17, toward activity highest the with clones the and M2, M3 ( enzyme wild-type the of The relative of the activity mutants was up to 40-fold higher than that difference significant among mutants was observed (data not shown). glycine bufferatpH8.6and37°C. for K a k 31 27 21 M3 M2 WT M2 WT Variant Table 1 nature CH nature increasing activity. The release of DCL through the slot tunnel appears Data publishedpreviously cat The three variants with the highest activity (21, 27 and 31) clearly clearly 31) and 27 (21, activity highest the with variants three The Circular dichroism (CD) in spectra the far-UV region werespectral b b b a , catalyticconstant; b b b a m andfork

Steady Steady state kinetic parameters of wild-type DhaA and its variants 1.26 1.26 ± 0.07 1.02 ± 0.06 0.55 ± 0.04 0.07 ± 0.01 0.14 ± 0.04 0.04 ± 0.01 k cat E k cat MIC 0.28 0.08 were cat (s values than the wild-type enzyme and both M2 and and M2 both and enzyme wild-type the than values −1 A ≤ ) L BIOLOGY L 25% and K 14 m . Kineticconstantsweremeasuredin50mMNaHCO , Michaelisconstant;WT, wildtype;Mut,mutant. k cat Supplementary Fig. 4 Fig. Supplementary 1.2 1.2 ± 0.1 1.1 ± 0.1 1.2 ± 0.2 1.0 ± 0.1 0.9 ± 0.1 1.0 ± 0.2 ≤ K / 15%, respectively. K m Supplementary Table 2 Table Supplementary 1.0 2.2 (mM) m Fig.2

, is 12, 23 and 26 times higher, respec higher, times 26 and 23 12, is , advance online publication online advance α Table  -helical content. The improved cata k b 222 222 nm Supplementary Fig. 2 Supplementary cat ). The spectra of the variants are variants the of ).spectra The value equal to 1.26 s 1.26 to equal value k 1 cat 1,050 1,050 ± 105 ). The variants 21, 27 and 31 31 and 27 21, variants ).The b values (8 to 35%) compared Data fromthisstudy. Kineticconstantsweremeasuredin100mM / 458 458 ± 83 156 ± 48 927 927 ± 100 K 70 70 ± 12 40 ± 13 m 280 (s 36 −1 Supplementary Fig. 3 Fig. Supplementary K −1 ( ), but with decreasing decreasing with ),but m M Fig. Fig. 2 is slightly increased increased slightly is −1 ). The wild-type, wild-type, The ). ) a ). ). (Mut/WT) rel. 25.5 13.8 31.5 3 −1 -NaOH bufferatpH9.4and30°C.Thes.d. 1.8 3.5 1.0 3.5 1.0 k , which ,which cat

), ------(Mut/WT) rel. the the buried active site. The dynamics of water molecules were therefore restricts the exchange of water molecules between the bulk solvent and the of introduction large hydrophobic residues into the access tunnels DhaA. Attime, same the TCP by of wild-type dehalogenation for ing lations, suggesting that this product release is unlikely to be rate limit of the access tunnels lowered the frequency of product egress in simu in the two positions modified by site-directed mutagenesis. Narrowing matic in residues two out as the of positions well three as randomized on previous pre-steady state kinetic studies—that is, on DhlA with with DhlA on is, studies—that kinetic state pre-steady previous on cycle, reaction the of before or in beginning the step forming the chloride the ion product ( at lies DhaA wild-type the by TCP of step conversion slowest the that suggests formation DCL and 0.04 ± of number 0.01 s of constant rate a with 0.07 ± 0.01 s observed was products both of formation ( TCP excess with DhaA wild-type the of ing mix rapid after formation DCL or ion chloride was either for burst observed of sign No mutagenesis. the by affected steps identify to nistic insight into the performance of these catalytic two enzymes and conducted with the DhaA wild-type and the 31 mutant to gain mecha change conformational slow the TCP rate of conversion by the 31 hydrolysis nor is mutant neither rate in the presence of DhaA the wild-type with experiments of effect no since mutant, 31 the in TCP of conversion the in step rate-limiting the of shift a indicates result of 2 fraction the Increasing state. transition a in site single a to due is effect isotope was linear,inventory that the solvent kinetic suggesting ( mutant 31 the by TCP of conversion the enzyme. wild-type the in than stable more is complex activated the and solvent, bulk the from site active the shield mutant 31 the in tunnels narrowed The mutant. 31 the of NACpopulation in was the lower 6.7-fold type wild than in the ( complex activated the of formation hinder thus and the hinder access water sterically of TCPmolecules to the nucleophile Asp106. The competition leads to reduced formation of the NAC since the active site compete with TCP for with the interaction nucleophile, ( solvent bulk from molecules water to accessible easily is DhaA wild-type of site the active that revealed analysis the 31 This mutant. and of DhaA wild-type the of simulation (MD) dynamics molecular production by 2 ns of followed (EMD) dynamics molecular equilibration 2 ns of during (NAC), configuration near-attack so-called the orientation, analyzed together with the stability of the substrate TCP in the reactive 1.2 1.1 1.2 1.0 0.9 1.0 0.5 1.0 H Proton inventory was performed to investigate rate limitation in in limitation rate investigate to performed was inventory Proton K 2 O results in increased enzymatic activity of the 31 mutant. This This mutant. 31 the of activity enzymatic increased in results O m

Fig. 3 Fig. rel. (Mut/WT) −1 26.3 23.2 11.5 k 1.8 3.9 1.0 7.8 1.0 cat a , well the with which corresponds steady state turnover ) and that increasing numbers of water molecules in in molecules water of numbers increasing that and ) / K m

2 H −1 ( simulations (~13%) eight of out one only in occurred mutant this of site active DCL the from of Release V245F. and C176Y I135F, substitutions: bulky three of because release DCL to amenable least the is mutant 31 The substitution. I135V the to due 27 mutant in 2b pathway and substitution, W141F the of result a as 21 mutant in pathway 2c mutants: these in pathways other via exits erentially pref DCL although 27, and 21 mutants in occur can it However, mutation. I135F the be to activity toward TCP, contains the large aro large the TCP, contains toward activity 2 Supplementary TableSupplementary 1 O further suggests that the step determining . The absence of burst for both chloride . ion for burst of chloride The both absence The 31 mutant, which shows the highest highest the shows which mutant, 31 The

3 completely prevented in mutant 31 by 31 mutant in prevented completely 1 . Transient kinetic experiments were were experiments kinetic Transient . 3 1 . The increase of the catalytic .the catalytic of The increase 2 Fig. 3 Fig. H 2 Fig. 3 Fig. O has been detected in in detected been has O d ). , inset). The proton proton The , inset). t r a d ). Instead, linear linear ).Instead, Fig. 3b Fig. Fig. 3 L C I d ). Based , c ). The ).The s e  ------

© 2009 Nature America, Inc. All rights reserved. oratory: rational protein design and directed evolution. The for The evolution. directed and design protein rational oratory: lab the in substrates catalytic non-natural toward the enzymes of enhance and performance be evolution can rapid such approaches mimic major to Two applied mutations. traits of phenotypic number new small of a by introduction the on depends ability the ­generally but rapidly, so functions new years evolve can few involved a teins within them with contact the chemicals following less) (or acquiring anthropogenic organisms degrade of to cases ability documented many are There DISCUSSION ( product a of release the is mutant by this TCP catalyzed with reaction the for step steps chemical is for limiting the 31 mutant; instead, the rate-limiting s 0.07 ± (1.26 kinetics the with well correspond which of constants rate 1.42 with ± 0.31 and 1.36 phase, ± 0.18 s state steady linear a by followed was s 1.5 ± 5.2 and 2.3 ± 5.8 of rates observed with DCL, and ions chloride both for identified was burst rapid mixing of the 31 mutant with upon excess TCP,contrast, In bond. a clear pre-steady state carbon-chlorine the of cleavage by limited be to DhaA wild-type the by catalyzed conversion TCP the expected bond—we faster than cleavage the of be carbon-halogen significantly ( 1,3-dibromopropane ( 1-chlorohexane ( 1,2-dichloroethane study a previous from are DhaA wild-type H in activity the between ratio The activity. dehalogenase the on E-R.Cl complex; enzyme-substrate E.R-Cl, substrate; R-Cl, enzyme; free E, (blue). mutant 31 the and (red) DhaA wild-type the by catalyzed reactions the ( ion chloride the to fits best the represent lines dashed and Solid kinetics. ( clarity. for shown are hydrogens polar Only mutant. 31 the in TCP for NAC the and DhaA wild-type in ( atoms. oxygen the on Å centered 0.5 of a radius have waters representing Spheres (blue). waters structural by occupied are positions internal discrete only and closed, is mutant 31 the of cavity the (red); solvent bulk to accessible is DhaA wild-type the of cavity site active The simulations. MD during solvent bulk for sites 3 Figure t r a  generating for approach versatile and promising most the is mer

c are indicated by (•). ( (•). by indicated are b a

− NAC distance (Å) 12 10 , alkyl-enzyme intermediate; E.Cl intermediate; , alkyl-enzyme

2 4 6 8 L C I Structural basis of enhanced activity against TCP. Comparison of the wild-type DhaA (left) and the 31 mutant (right). ( (right). mutant 31 the and (left) DhaA wild-type the of TCP. against Comparison activity enhanced of basis Structural 0 Fig. 3 Fig. 0.5 s e WT Time (ns) 7 b ) and bromocyclohexane ( ) bromocyclohexane and ) Effect of water molecules on the NAC distance of two reacting atoms in the active site during MD simulations. Snapshots shown shown Snapshots simulations. MD during site active the in atoms reacting two of distance NAC the on molecules water of ) Effect 1.0 d ). 5 ) and 1,2-dibromoethane ( 1,2-dibromoethane and ) 9 −1 c ) ) Snapshots from MD simulations of the nonreactive configuration of TCP competing with waters for the nucleophile in the the in nucleophile the for waters with competing TCP of configuration nonreactive the of simulations MD from ) Snapshots 1.5 3 ). These findings suggest that none of the the of none that suggest findings These ). 1 , in which substrate binding was found to found was binding substrate , which in −1 for chloride ions and DCL, respectively, 2.0 −1 k cat , respectively ( respectively , value observed in steady state state steady in observed value − 3 .ROH, enzyme-product complex; Cl complex; enzyme-product .ROH,

1 NAC distance (Å) 3 . 10 12 4 2 4 6 8 . It is unclear how the pro the how unclear is It . 0 8 ) 0.5 3 3 , and on DhaA with , with on and DhaA Fig. 3 Fig. 6 Time (ns) 31 ) 3 1.0 2 , on LinB with with LinB on , d ). The burst burst The ). 1.5 2 O O ( V 2.0 H ) and in D in ) and - - -

 ) and DCL ( DCL ) and − and ROH, products. The inset illustrates the solvent kinetic isotope effect effect isotope kinetic solvent the illustrates inset The products. ROH, and f u mtn wt hget ciiy oad C ( TCP toward k activity highest with mutant our of cost for essential be would which system, continuous-flow a in radation 0.14 sufficient s ( TCP have with efficiency M2 catalytic nor and activity enzyme catalytic wild-type the neither However, performance catalytic improved further with Y273F) C176Y (M2, one and variants positive potentially 14 provided PCR error-prone by created library second-generation A conversion. showing TCP improved C176Y) (M1, one only and clones, 10,000 of out variants shuffling DNA by created DhaA mutants of library random a which in study previous a in than more and clones conversion—significantly TCP improved positive with 51 sequences unique 25 yielded clones ~5,000 of library mutant A mutants. viable isolate to required space sequence the reduced tially substan mutagenesis combinatorial for protein the in sites of specific Identification evolution. directed and design molecular assisted DhaA by wild-type ‘focused directed evolution’ combining computer- TCP for than efficiency catalytic higher 26-fold and activity catalytic catalytic new DhlA-producing microorganisms have survived, grown and shown shown and grown survived, have microorganisms DhlA-producing plant purification groundwater scale 1,2 in ( 1,2-dichloroethane for DhlA dehalogenase haloalkane the of those k cat cat 2 In this study, we constructed a DhaA variant with 32-fold higher higher 32-fold with variant DhaA a constructed we study, this In d c O O ( / - K

E +R-CI = 3.3 s 3.3 = advance online publication online advance activities, while the latter seems to be the best way to optimize optimize to way best the be to seems latter the while activities, Concentration (mM) effective, full-scale bioremedial processes. The catalytic 0.2 0.4 0.6 0.8 1.0 1.2 −1 - m V 0 dichloroethane–mineralizing microorganisms used in a full- a in used microorganisms dichloroethane–mineralizing W107 obs , 100 s 1,050 = 0 N41 k 

properties of existing enzymes existing of properties cat ) was plotted versus various various versus plotted ) was ) kinetic data. Reaction scheme shows rate-limiting steps for for steps rate-limiting shows scheme Reaction data. ) kinetic 1 0 / V k −1 K 2.0 1 H k m / –1 , V = 40 s ob k Time (s) E.R-C % cat s −1 4.0 2 / H d K M TCP 2 ) Rate-limiting step from the pre-steady state state pre-steady the from step ) Rate-limiting I O 1 −1 m 00 = 6,200 s 6,200 = k M −1 D106 2 6.0 k –2 ) are of the same order of magnitude as as magnitude of order same the of are ) −1 E-R.CI and 156 s 8.0 – −1 k 1 1 3 M k 4 H 3 –3

yielded 30 potentially positive positive potentially 30 yielded 5 −1 Concentration (mM) 2 0.2 0.4 0.6 0.8 1.0 1.2 . The haloalkane dehalogenase .dehalogenase The haloalkane −1 O/ nature nature E.CI M 0 2 0 ), which has proven utility utility proven has which ), 8 H . −1 – W107 .R 2 1 0 N41 , respectively) for biodeg O ratios. The data for for data The O ratios. OH V a H ) Accessibility of active active of ) Accessibility / ch 0.5 V k ob 4 Time (s) % k s e –4 mic 2 H E +CI k k 2 cat 1 O cat 1.0 00 a 12 s 1.26 = = 0.04 and and 0.04 = TCP l –

D106

+

biology constants RO 1.5 H

−1 1 4 - ­ , .

© 2009 Nature America, Inc. All rights reserved. The structures of the mutant proteins were modeled using the mutagenesis mutagenesis the using modeled were proteins mutant the of structures The sequence. DhaA wild-type the recover to structure crystal the in putationally com were made substitutions point 1CQW). code Three Bank Data (Protein the free enzyme of a of variant structure on were the crystal based its mutants simulation. and modeling Molecular METHODS architecture. site active the of by disruption not to does lead functionality of loss replacement their since mutagenesis for targets good represent site active the outside located residues tunnel sites. The active buried with biocatalysts new for engineering methodology applicable may be generally a valuable, evolution directed by mutagenesis exhaustive simula with and combined tions modeling computer by tunnels access lining spot residues hot identifying that indicate results The solvent. surrounding the with cavity site active buried the connecting routes access fying can be improved substrates by toward modi anthropogenic enzymes anatomy, physicochemical properties and dynamics. ligand and water passage through access tunnels for optimizing tunnel the utility of simultaneously randomizing several residues that influence access tunnels to alter enzymes’ activities. Here we have demonstrated ties or activities mutations in access single-point tunnels carrying with modifiedenzymes specificities, several of enantioselectivi variants describing Papers been shown to affect the substrate specificity of cytochromeLeu177 P450sin the haloalkane dehalogenase LinB mentally by exhaustive site-directed mutagenesis the of tunnel residue activity and specificity logenases’ substrate influencedehaaccesssubstantially­proposedhaloalkanetunnelsthat attack. hinder nucleophilic the sterically strongly 1,2-position the in substitutents since halogen dehalogenases, haloalkane the by substrates 1,2-halogenated of conversion substitution–mediated nucleophilic for bimolecular the important are state transition the of stabilization and complex the activated of formation Precise substrates. for these cleavage bond pro and ethanes panes, in carbon-halogen of accordance the with proposed limitation 1,2-halogenated other toward activity increased has also it that showed mutant 31 the for mapping Substrate release. product disfavors and site active occluded the in catalysis favors that trade-off the documents in 31 the mutant release to tion product the step from reac the chemical the of switch rate-limiting The observed ously previ reported DhaA—as wild-type the by TCP conversion for step rate-limiting the is cleavage bond carbon-halogen that simulations age cleav bond carbon-halogen inhibit molecules water additional but step, hydrolytic the for co-substrate a as site active the must in present be molecule water One cavity. site active occluded the to ecules mol water of access restricts pathways entrance its of the that modification showed complex and 31, enzyme-substrate its variant of of simulations MD simulations RAMD in event rare a Product site. was active release occluded targeted most the positions to five leading of mutagenesis, out by four in residues aromatic large the tunnel access main the rowing progress. in currently is work this in identified dehalogenase mutant the carrying bacterium five a of TCP-degrading least the plant. Construction of the startup after years at for efficiently 1,2-dichloroethane degrade to potential nature CH nature In summary, we provide experimental evidence that the activity of of the activity that evidence In summary, experimental we provide we structures, crystal of analysis comparative previous Following nar of importance apparent the on study, focusing present the In 36–38 3 1 —and —and that this step is improved significantly in the 31 mutant. . Rapid quench-flow analysis confirmed from indications MD E MIC 40,43–49 A L BIOLOGY L support the general applicability of redesigning

2 5 advance online publication online advance , the most active mutant 31 contains 31 contains mutant , active most the The RAMD simulations of DhaA and and DhaA of simulations RAMD The 3 4 9 0 and verified this experi this verified and . Access tunnels have also 27,41,42

------. induced with IPTG ( IPTG with induced were microcultures The plates. agar LB on made was wells 96 the of replica a and h, 16 least at for °C 37 at r.p.m. 120 at shaken were plates The controls. wild-type four and vector pAQN the with controls negative M2, four of trols 100 and medium LB containing plate 96-well a of wells separate in resuspended Methods in are the described mutagenesis saturation of 1,056). Details (diversity DhaA245-6 and 32) (diversity DhaA135 sublibraries: separate two create to designed were synthetic pairs primer degenerate of Twooligonucleotide set a oligonucleotides. using out carried was 246 and 245 135, tions are in mutagenesis listed for used oligonucleotides The (Toyobo). polymerase Plus KOD with PCR inverted of principle the using mutagenesis site-directed by ( M3 and M2 denoted mutants two and type wild Mutagenesis, seeding and propagation of the libraries. (ref. 157° > chlorine leaving the and carbon attacked the oxygen, atom carbon attacked the and oxygen nucleophilic the between distance the (i) class: reaction this for previously proposed conditions geometric 0.5 ps the using two-parameter every evaluated was trajectories MD calculated the along NACs of presence consisted of 2 simulations ns of EMD followed by 2 ns MD.of production The MD. by were classical studied The on reactivity site the molecules water active of product release are provided in the simulations the RAMD of Details Scientific). (DeLano PyMol 0.99 of module genase activity was assayed by a previously described method was assayed by described a activity previously genase assay,Activity state kinetics. state steady and pre-steady the in described as recorded were samples at process 4 and was storage performed °C. protein of purification CD spectra (pH 7.5). The enzymes were sterilized and stored in the same buffer. The entire were fractions pooled and dialyzed overnight against 50 mM phosphate buffer proteins were washed out with buffer containing 500 mM . The active 500 mM sodium chloride and 10 mM imidazole). Unbound and weakly bound (50 buffer,buffer mM phosphate resin in pH the 7.5, equilibrating containing to the were bound enzymes His-tagged The Biotech). Pharmacia (Amersham sepharose a chelating using performance purified high with further packed was column extract affinity HiTrap cell-free The h. 1 for g 21,000 centrifuga at after tion used was supernatant The PLC). Gallenkamp (Sanyo 150 of concentration final a to IPTG 0.5 of mM. The cells were addition harvested and disrupted by the sonication using a Soniprep by initiated was °C 30 at expression enzyme of induction The °C. 37 at LB of purification) (for l 2 in coli the by vector pAQN a in purification. and Overexpression analysis. and for wereused isolated further The plasmids subsequently media. LB of ml 3 inoculate to used were colonies replica corresponding and identified, were activity in improvement significant most the exhibiting tures color. purple deep a formed controls negative and Wells microcul containing for 16 h. Active colonies produced a yellow bright color, while inactive colonies change in pH. The microplates were sealed with parafilm and incubated at 25 °C ity was therefore detected by adding a phenol red dye that changes color upon a medium is reduced rapidly by the production of protons. buffered Dehalogenating weakly the activ of pH The proton. a and ion chloride a alcohol, primary are a products reaction the molecule, and substrate per is consumed molecule water One substrate. the from ion chloride a of displacement hydrolytic the catalyze dehalogenases Haloalkane mM. 10 of concentration apparent an to 25 concentration final at red phenol buffer, NaHEPES mM mM 20 (1 buffer assay (pH 8.2). 10 mutant library were defrosted, and cell pellets were lysed in 1 mM HEPES buffer High-throughput microplate screening. for r.p.m. 1,800 °C. at −80 at stored and min 35 centrifugation by °C 4 at pelleted °C, 30 at h 5 further a BL21 cells with plasmids were cultured in 10 ml (for cell-free extract) and extract) in 10 ml were cell-free (for cultured plasmids with cells BL21 µ g g ml Supplementary Table 3 Supplementary . The individual colonies were picked with sterile toothpicks and and toothpicks sterile with picked were colonies individual The . −1 ampicillin ( ampicillin µ l l of cell free extract was transferred into 140 1 1 ≤ ) at a final concentration 0.5 mM and cultivated for for cultivated and mM 0.5 concentration final a at ) 3.41 Å and (ii) the angle formed by the nucleophilic nucleophilic the by formed angle the (ii) and Å 3.41 1 tac 0 ). Each 96-well plate contained eight positive con positive eight contained plate ). 96-well Each promotor under the control of of control the under promotor . Saturation mutagenesis at amino acid posi acid at amino mutagenesis . Saturation The The µ Supplementary Methods Supplementary g ml g dhaA The microplates with the propagated Supplementary Methods Supplementary −1 ). The TCP substrate was added added was substrate TCP The ). gene variants were transcribed transcribed were variants gene 2 SO 4 , 1 mM EDTA, ,mM 1 8.2,and pH Fig. 2 Fig. The His-tagged DhaA t r a µ a Haloalkane dehalo Haloalkane ) were constructed constructed were ) l l of phenol red ( lacI 5 Supplementary Supplementary 0 , by measuring . The effects of q . L C I 36). Escherichia Escherichia . s e 1 2  ) ------

© 2009 Nature America, Inc. All rights reserved. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprintsandpermissions/. http://npg.nature.com/ at online available is information permissions and Reprints Published online at http://www.nature.com/naturechemicalbiology/. contributed tothe of paper. the writing tools; biology molecular J.D. interpreted data and mutants.designed All authors R.C.W. contributed the RAMD modeling tool; M.T. and Y.N. contributed solvent isotopic kinetic effect measurements; P.B. and M.O. mutants; designed measurements;kinetics R.C. CD performed spectroscopy measurements and modeling molecular and mutants; designed Z.P. pre-steadyperformed state M.P. measurements;mutagenesis performed and M.K.activity performed AUTHOR CONTRIBUTION Centre Brno for computational resources. Agriculture, Forestry, and Fisheries, Japan. We acknowledge the Supercomputing Education, Culture, Sports, Science, and Technology, Japan and of the Ministry (EST.CLG.980504),Organization and Grants-in-Aid from of the Ministry the of Czech Academy Sciences of (IAA401630901), the North Atlantic Treaty Agency the of Czech Republic and (201/07/0927 203/08/0114), the Grant Agency the Education of Czech Republic (LC06010 and MSM0021622412), the Grant Promotion Science.of We acknowledge from financial support of the Ministry Foundation and M.P. acknowledges a scholarship from the Japan for Society manuscript. R.C.W. acknowledges the the gratefully of Klaus support Tschira U. Bornscheuer (University Greifswald, Germany) for reading this critical of We thank V. de Lorenzo (Centro Nacional de Biotecnologia, Spain) and A on the Note: Data a of Protein part as study. deposited was previous (1CQW) study. a previous DhaA of of structure part crystal the as Bank: deposited was (AF060871) codes. Accession are analysis data and the in described design experimental the of Details (BioLogic). ment instru 37 °C a (pH using in quench-flow 8.6) QFM buffer a 400 rapid glycine at performed were experiments quench-flow Rapid measurements. velocity constants of DhaA wild and type mutants with TCP were determined by initial taining TCP and increasing concentrations of assays in (pH buffer 8.6) glycine con activity by were performing determined as the release of 1 is defined activity enzymatic of One unit sulfate. ammonium and ferric anate thiocy mercuric with nm 460 at spectrophotometrically ions halide released t r a  cknow

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