Design and realization of a tailor-made enzyme to modify the molecular recognition of...

Design and realization of a tailor-made enzyme to modify the molecularrecognition of 2-arylpropionic esters by Candida rugosa lipase

Fabrizio Manetti a, Daniela Mileto b, Federico Corelli a, Simonetta Soro c,Cleofe Palocci c, Enrico Cernia c, Ilaria D'Acquarica d, Marina Lotti b,

Lilia Alberghina b, Maurizio Botta a;*a Dipartimento Farmaco Chimico Tecnologico, Universita© degli Studi di Siena, Via Aldo Moro snc, I-53100 Siena, Italy

b Dipartimento di Biotecnologie e Bioscienze, Universita© degli Studi di Milano-Bicocca, Piazza della Scienza 2, I-20126 Milan, Italyc Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universita© di Roma `La Sapienza',

Piazzale Aldo Moro 5, I-00185 Rome, Italyd Dipartimento di Chimica, Universita© di Roma `La Sapienza', Piazzale Aldo Moro 5, I-00185 Rome, Italy

Received 10 February 2000; received in revised form 18 July 2000; accepted 25 July 2000

Abstract

Within a research project aimed at probing the substrate specificity and the enantioselectivity of Candida rugosa lipase(CRL), computer modeling studies of the interactions between CRL and methyl ( þ )-2-(3-benzoylphenyl)propionate(Ketoprofen methyl ester) have been carried out in order to identify which amino acids are essential to the enzyme/substrateinteraction. Different binding models of the substrate enantiomers to the active site of CRL were investigated by applying acomputational protocol based on molecular docking, conformational analysis, and energy minimization procedures. Thestructural models of the computer generated complexes between CRL and the substrates enabled us to propose that Phe344and Phe345, in addition to the residues constituting the catalytic triad and the oxyanion hole, are the amino acids mainlyinvolved in the enzyme^ligand interactions. To test the importance of these residues for the enzymatic activity, site-directedmutagenesis of the selected amino acids has been performed, and the mutated enzymes have been evaluated for theirconversion and selectivity capabilities toward different substrates. The experimental results obtained in thesebiotransformation reactions indicate that Phe344 and especially Phe345 influence CRL activity, supporting the findings ofour theoretical simulations. ß 2000 Elsevier Science B.V. All rights reserved.

Keywords: Tailor-made enzyme; Lipase; Molecular modeling; Mutagenesis ; 2-Arylpropionic ester; Molecular recognition

1. Introduction

In recent years the crystallographic data for sev-eral enzymes have become available [1^9], providingthree-dimensional structures of their active sites. Fur-

ther, the development of sophisticated molecularmodeling software has allowed computational studiesof the interactions between the amino acids of thecatalytic site and di¡erent substrates [10]. In conjunc-tion with the experimental results obtained from ki-netic and structure^activity relationship (SAR) stud-ies, these theoretical approaches can lead to thedevelopment of more general models, which areable to rationalize the substrate^enzyme interactions

0167-4838 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 7 - 4 8 3 8 ( 0 0 ) 0 0 1 8 5 - 0

* Corresponding author. Fax: +39-0577-234333;E-mail : [email protected]

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Biochimica et Biophysica Acta 1543 (2000) 146^158www.elsevier.com/locate/bba

and thus allow a more detailed description of enzy-matic reactions and provide support to protein engi-neering studies [11].

The binding site of CRL consists of three majorregions that are responsible for the enantiomeric dis-crimination by the enzyme: (i) the catalytic triad(common to all the serine proteases and responsiblefor the hydrolytic reaction of the substrate) formedby the residues Ser209, His449 and Glu341; (ii) theoxyanion hole (which stabilizes by hydrogen bondingthe negative charge present on the oxyanion oxygenof the tetrahedral intermediate) formed by the back-bone NH atoms of Gly124, Ala210, and Gly123 (it islikely that the NH group of Gly123 participates inhydrogen bond formation to the oxyanion [12,13]);and (iii) the long, narrow, hydrophobic tunnel, char-acterized by an L shape, which projects ¢rst towardthe core of the protein, then bends toward the pro-tein surface. This tunnel has been characterized asthe binding site of the acyl moiety of the ligandand can accommodate a fatty acid as long as 18carbon atoms or even more. In addition, a hydro-phobic pocket de¢ned by Phe296, Leu297, Phe344and, more deeply, Phe345 represents an additionalregion contributing to the interactions with sub-strates [14].

Based on the knowledge of both the topography ofthe active site of CRL and the three-dimensionalstructures of the complexes between the enzymeand the two enantiomers of one of its inhibitors((1R)- and (1S)-menthyl hexylphosphonate) deter-mined by X-ray crystallography [13,14], the inhibi-tion process was modeled by a computational proto-col starting from the tetrahedral oxyanionintermediate (referred to as `¢rst tetrahedral oxyan-ion intermediate') which is ¢rst formed along theaccepted pathway for ester hydrolysis catalyzed byserine proteases [14^16].

Molecular modeling studies led to the rationaliza-tion of the enzyme stereoselectivity on the basis ofthe spatial arrangement of both the catalytic residuesand substrates, and to the identi¢cation of two ami-no acids mainly involved in the enzyme^substrateinteractions. Mutation of these residues resulted indecreased enantioselectivity, which was explained interms of an increased enzymatic conversion of the R-enantiomer representing the poorer substrate for thewild-type lipase. These data suggest that proposed

amino acids (particularly Phe345) play an importantrole in determining enzymatic activity and enantiose-lectivity. Moreover, on the basis of these ¢ndings,our model could represent a promising tool for theguidance of further studies on the structure/functionrelationship of the CRL catalytic environment in-volved in the hydrolysis of 2-substituted propionicacid esters.

2. Materials and methods

2.1. Molecular modeling

All calculations and graphical manipulations wereperformed on a Silicon Graphics Indigo workstationusing the software package MacroModel/BatchMin[17] equipped with the AMBER* all-atom force ¢eld.

The enzyme models used in this study were derivedfrom the coordinates of the structures labelled 1lpmand 1lps in the Brookhaven Protein Data Bank,which represent the complexes between CRL and(1R)-menthyl hexylphosphonate and (1S)-menthylhexylphosphonate, respectively, re¢ned to 2.2 Aî res-olution [13,14]. Both substrates link covalently to theOQ of catalytic serine (Ser209) and have S stereo-chemistry at the phosphorus atom as a consequenceof the nucleophilic attack by Ser209 from the re faceof the ester [13].

To create initial coordinates for the docking stud-ies, the two calcium ions, the three N-acetylglucos-amine residues and all water molecules of the crystalstructure were removed and excluded from the cal-culations. Hydrogen atoms were added in their ideal-ized positions at the pH 7.9 used for the kinetic mea-surements by means of InsightII Biopolymersoftware [18], in such a way as to protonate allNH2 groups (Lys and Arg side chains, and the N-terminal Ala1), and deprotonate all COOH groups(Glu and Asp side chains, and the C-terminalVal534).

In the present study, the computational step cor-responding to the molecular docking approach issimpli¢ed, since the functional groups of the sub-strates interacting with the enzyme and the aminoacid residues of the catalytic machinery are wellknown in advance. Thus, the docking problem isessentially reduced to locate the possible conforma-

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tions of the ligand within the active site of the en-zyme. In particular, the phosphonyl group in the X-ray crystal structures was used to guide the buildingof the tetrahedral oxyanion intermediates providinga good template to adjust torsional angles of the R-and S-enantiomers of the substrate. Based on the¢nding that the catalytic serine attacks from the reface of the ester, the carbonyl carbon of the ligandwas covalently bound to the oxygen of the hydroxylgroup of the Ser209 to form the tetrahedral oxyanionintermediate with the proper stereochemistry. Thehydrogen atom of the Ser209 hydroxyl group wasthen transferred to the sp2 NO2 nitrogen atom ofthe imidazole ring of the side chain of His449, ren-dering it positively charged. The oxyanion generatedby attack of Ser209 was directed toward the oxyan-ion hole formed by the backbone NH atoms ofGly124 and Ala210, and the oxygens of the leavingalcohol and Ser209 side chain were positioned sothat all the catalytically essential hydrogen bondswere formed. Finally, the aromatic portion of theinhibitor was positioned in the mouth of the hydro-phobic tunnel (according to the rule for predictingthe enantioselectivity of CRL toward 2-substitutedcarboxylic acids [19]) in such a manner to avoidany unfavorable van der Waals (vdW) interaction.

The complexes generated were then submitted to astatistical conformational search with Monte CarloMultiple Minimum (MCMM) method involving therotatable bonds of the ligand and the catalytic serineside chain (Fig. 1). MCMM causes an input structureto be modi¢ed by random changes in its torsion an-gles as given by the TORS command. Values of 0³and 180³ are respectively the minimum and maxi-mum angular increments to be added or subtractedfrom the current dihedral angles. It is important tonote that conformational mobility of the active siteresidues was not a complicating factor since it hasbeen shown that these residues undergo only minor

structural changes upon the binding of di¡erent in-hibitors. For instance, X-ray di¡raction data high-lighted that the structures of the CRL^menthyl hex-ylphosphonate complexes show no signi¢cantdi¡erences in the polypeptide backbone with respectto the unliganded open conformation of the enzyme,and the positions of the catalytic residues are essen-tially identical to those in the ligand-free lipase[13,14].

The least square superimposition routine was se-lected (COMP command) to compare each new com-plex with all conformers previously found in order toeliminate duplicate minima. The chirality checkingcommand (CHIG) was used to reject any structurewhose chirality has been changed by the minimiza-tion.

Because of the large number of atoms in the mod-el, to correctly optimize the complex of the ligandsand CRL, the following constraints had to be im-posed: (a) a subset, centered on the ligand and com-prising only the substrate and a shell of residuessurrounding the binding site of the enzyme within aradius of 8 Aî from the ligand, was created and sub-jected to energy minimization. The substrate and allamino acid side chains of the shell were uncon-strained during energy minimization to allow for re-orientation and thus proper hydrogen-bonding geo-metries and vdW contacts; (b) all the atoms externalto the subset remained ¢xed, even if their non-bondinteractions with all the relaxing atoms have beencalculated.

Two intramolecular distances were monitored dur-ing the docking experiments in order to ensure thatthe relative orientations of the residues of the cata-lytic triad remained compatible with the postulatedmechanism of the hydrolytic reaction. These were thedistances between the hydrogen atom bound to theNO2 of the imidazole ring of His449 and both the OQoxygen atom of Ser209 and the oxygen atom of theleaving alcoholic group.

Energy minimization of the complexes was per-formed using the Polak^Ribie©re conjugate gradientmethod until the derivative convergence was 0.05kJ/Aî mol. The default non-bonded cuto¡ protocolemployed by the program was modi¢ed. Thus, avdW cuto¡ of 12.0 Aî , an electrostatic cuto¡ of 20Aî , and a hydrogen bonding cuto¡ of 2.5 Aî wereutilized for all calculations.

Fig. 1. Dihedral angles submitted to a statistical conformationalsearch.

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Conformational search and energy minimizationprocedures generated some CRL^ligand complexeswhere the interactions (i.e., hydrogen bonds) of thesubstrate with the oxyanion hole and with His449were broken. Structures lacking all the catalyticallyessential hydrogen bonds involving the imidazolering of His449 were considered as non-productivetetrahedral oxyanion intermediates and, accordingly,were no further investigated.

2.2. DNA mutagenesis and production of recombinantproteins (rCRLs)

Mutagenesis was performed on a LIP1-encodingsynthetic gene cloned in pPICKB (Invitrogen, SanDiego, CA) obtained as previously described(pPIC-slip1) [20].

Site-directed mutagenesis was carried out by over-lap extension using the polymerase chain reaction(PCR) technique [21]. The ¢rst PCR step was per-formed using 50^100 ng pPIC-slip1 as the templateand 50 pmol mutagenic/wild-type 24^30-mer oligo-nucleotides as primers. Ampli¢cation was carriedout using 2.5 U Pfu TURBO Polymerase (Strata-gene, Heidelberg, Germany) in a ¢nal volume of100 Wl and the following PCR program: initial dena-turation (94³C, 3 min), 25 cycles of denaturation(94³C, 1 min), annealing (55³C, 1 min), extension(72³C, 1 min), and a ¢nal extension (72³C, 5 min).PCR products were separated on 1.2% agarose geland eluted using QUIAquik Gel Extraction kit(Quiagen, Germany). Fifty ng of DNA from the ¢rstampli¢cation step were directly used in the secondPCR step performed as above.

Mutagenized gene fragments were checked by se-quencing, digested with HpaI and EcoRI, and clonedin the pPIC-slip1 plasmid replacing the correspond-ing wild-type sequence. Recombinant plasmids wereampli¢ed in Escherichia coli JM101 (Promega, Mad-ison, WI). Transformation was as described by Sam-brook et al. [22]; transformants selection was on LBplates (2% peptone, 1% NaCl, 1% yeast extract, 2%agar) containing 25 Wg/ml Zeocin.

Plasmid DNA was extracted from E. coli cells us-ing QUIAGEN-tip 100 kit (Quiagen).

Five to ten ng DNA were linearized with SacI andused to transform Pichia pastoris X-33 (wild-type;Invitrogen) by the Bicine Yeast Transformation pro-

tocol [23]. Transformants were selected on YEPDplates (2% glucose, 1% yeast extract, 2% peptone,2% agar) containing 100 Wg/ml Zeocin and testedon small scale for lipase production.

Fermentation of the best performing clones wascarried out in 500 ml BMGY (1% glycerol, 2% pep-tone, 1% yeast extract) inoculated with 25 ml of anovernight culture. Induction of lipase expression wasobtained by adding twice a day 1% methanol for4 days.

Supernatants were collected by centrifugation andconcentrated by tangential £ow ¢ltration using Mini-tan Ultra¢ltration system (Millipore, Bedford, MA).The membrane sheet used for the concentration was:(1) Durapore 0.45 mm in order to eliminate remain-ing cells after supernatant recovery; (2) Polysulfone50 000 NMWL for the concentration. Supernatantswere analyzed by sodium dodecyl sulfate^polyacryl-amide gel electrophoresis using Mini-PROTEAN IIElectrophoresis Cell Biorad (Bio-Rad, Hercules, CA)[24].

2.3. Biocatalyzed reactions of wild-type and mutatedrecombinant enzymes (rCRLs) on arylpropionicacid esters

The rCRLs solutions (wild-type, Phe344Val,Phe345Val, and Phe344,345Val) were lyophilizedand the powders so obtained were characterized asfar as their lipolytic activity, protein content [25] andspeci¢c activity before using them in biocatalyzedhydrolysis reactions of arylpropionic esters. The lip-olytic activity was assayed by alkalimetric ¢nal titra-tion. The assay mixture, containing 2.5 ml of bu¡er(20 mM Hepes, 2 mM EDTA, pH 7.6), 0.5 ml oftributyrin and 0.1 ml of enzyme solution (150 mg/ml), was incubated at 37³C under magnetic stirring(300 rpm) for 30 min. The reaction mixture wasstopped with 2.5 ml of acetone/ethanol mixture 1:1(v/v) and titrated with 0.05 M NaOH in the presenceof phenolphthalein as the indicator using an auto-matic burette (Methrom).

rCRLs were employed as catalysts in hydrolysisreactions of the methyl esters of two arylpropionicacids, namely ( þ )-2-(3-benzoylphenyl)propionic acid(Ketoprofen) and ( þ )-2-(6-methoxy-2-naphthyl)pro-pionic acid (Naproxen), using essentially the sameprocedure reported by Sih et al. [26]. The methyl

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esters were synthesized using Brook's method [27]starting from racemic acids (Naproxen was kindlyprovided by Alfa Wasserman, and Ketoprofen waspurchased from Sigma).

To 150 mg of rCRLs in 1 ml of 0.2 M phosphatebu¡er (pH 7.9), the substrate (244 mg Naproxenmethyl ester or 268 mg Ketoprofen methyl ester)was added. The resulting suspension was energeti-cally stirred at 40³C for 168 h and then centrifugedfor 20 min at 600 rpm. The precipitate was washedwith 0.2 M phosphate bu¡er and centrifuged againto collect the water insoluble ester. Supernatant andwashing were combined and acidi¢ed to pH 2.0 withHCl and the precipitate was collected by ¢ltration toa¡ord the acid. No hydrolysis was observed at pH7.9 and 40³C in the absence of enzyme. The samehydrolysis reactions were performed in a biphasicsystem (bu¡er/CHCl3) in an attempt to increase thereaction rate by emulsioning the substrate organicsolution in aqueous medium, but no increase in thereaction rate was indeed observed.

Enzymatic conversion of Ketoprofen and Naprox-en methyl esters to the corresponding acids, enantio-meric excess (e.e.p %) and enantiomeric ratio (E, cal-culated according to Chen et al. [28]) weredetermined by enantioselective normal-phase high-performance liquid chromatography (NP-HPLC).Analytical liquid chromatography was performedon a Waters chromatograph equipped with a RheodyModel 7725i 20 Wl injector and two Model M510solvent-delivery systems. The detector used was aModel M490 programmable multi-wavelength detec-tor (Waters Chromatography, Division of Millipore,Milford, MA). Chromatographic data were collectedand processed using the Millennium 2010 Chroma-tography Manager software (Waters Chromatogra-phy). All the analyses for Naproxen samples wereperformed on the broadly applicable (R,R)-ULMOchiral stationary phase (CSP) column (250U4.6mm i.d., from Regis Technologies, Morton Grove,IL), at a £ow rate of 2.0 ml/min. The mobile phasewas 0.1% tri£uoroacetic acid in 2% 2-propanol/n-hexane (v/v). Retention times obtained for the con-sidered analytes were the following: Naproxen meth-yl ester (unresolved) = 3.95 min; (S)-Naprox-en = 14.07 min; (R)-Naproxen = 18.27 min.

For Ketoprofen samples, we performed a tandemarrangement between the above-mentioned CSP and

the commercially available Chiralcel OJ chiral sta-tionary phase column (250U4.6 mm i.d., from DaicelChemical Industries, Tokyo, Japan), at a £ow rate of1.0 ml/min. The mobile phase was 0.5% acetic acid in7% 2-propanol/n-hexane (v/v). Retention times ob-tained for the considered analytes were the follow-ing: Ketoprofen methyl ester (unresolved) = 22.28min; (R)-Ketoprofen = 28.02 min; (S)-Ketopro-fen = 32.58 min. Chromatographic runs were allmonitored by UV detection at V 254 nm and25 þ 1³C.

3. Results and discussion

3.1. Molecular modeling

Based on the X-ray structure of the CRL^menthylhexylphosphonate complexes, a model of the CRL^methyl ( þ )-2-(3-benzoylphenyl)propionate (the sub-strate) complexes was built and used for the rationaldesign of site-directed mutagenesis experiments onCRL.

In agreement with kinetic and computational stud-ies previously reported [29^31], showing the S-enan-tiomer as the fast-reacting substrate, the complexbetween the S-enantiomer of the substrate and theenzyme proved to be 24 kJ/mol more stable than thecorresponding complex involving the R-enantiomer.Although the calculated value of the energy di¡er-ence of the ¢rst tetrahedral oxyanion intermediates isnot in agreement from a quantitative point of viewwith that experimentally determined1 and corre-sponding to about 8 kJ/mol, considering that thiskind of calculation is usually complicated by thehigh negative energy values of the enzyme^ligandcomplexes and their £uctuations during the simula-tions [32], the qualitative agreement reached bymeans of molecular mechanics simulations can beconsidered as a good result. In fact, current modelingmethods are extremely useful as a qualitative tool,

1 We have compared the di¡erence in energy of the minimizedstructures of the ¢rst tetrahedral oxyanion intermediate modelswith vvG# values calculated from kinetic data on the basis of thefollowing equation: vvG# =3RTlnE, where E is the enantiomer-ic ratio.

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but quantitative predictions (for example, the degreeof enantioselectivity) are still not reliable [33].

The analysis of the above-mentioned complexeshighlighted a di¡erent orientation of the ligandwith respect to the starting docking geometry as aconsequence of tetrahedral oxyanion intermediatebuilding and subsequent energy minimization. In

the case of the S substrate, both the acyl chain andthe alcoholic moiety are directed out of the activesite, while the methyl group at the stereogenic centeris directed toward the entrance of the hydrophobictunnel (Fig. 2, top). On the contrary, the methylgroup in the R-enantiomer is out of the tunnel anddirected toward a region de¢ned by Gly124 and the

Fig. 2. Stereo representation of the CRL^substrate ¢rst tetrahedral oxyanion intermediate complexes of the S-enantiomer (top) andthe R-enantiomer (bottom) of methyl 2-(3-benzoylphenyl)propionate. The tetrahedral intermediate, including Ser209, is shown inblack, and the hydrogen-bonding network involving the catalytic serine and the oxyanion hole (Gly123, Gly124, and Ala210) is drawnby thin gray lines. Both the fast-reacting S-enantiomer and the slow-reacting R-enantiomer lost a hydrogen bond between the His449residue of the catalytic triad and the OQ atom of Ser209. The leaving alcoholic chain of both enantiomers points out of the activesite. In this view, the hydrophobic tunnel points into the plane of the paper, and the amino acid residues constituting the mouth ofthis tunnel (Met213, Leu304, Phe345, and Phe415) are depicted. (Top) Both the acyl and the alcoholic moieties of the S-enantiomerare directed out of the active site, while the methyl group at the chiral carbon atom points toward the entrance of the hydrophobictunnel. Three hydrogen bonds between the oxyanion hole and the oxyanion were found. (Bottom) The methyl group at the stereogeniccenter of the R-enantiomer is directed toward Gly124 and Phe296. Gly123 lost a hydrogen bond with the oxyanion due to a confor-mational rearrangement focused on the Gly122^Gly124 sequence.

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aromatic side chain of Phe296. As a consequence ofthe orientation of the methyl group at the R stereo-genic center, a conformational rearrangement of ami-no acid main chains ranging from Gly122 to Gly124occurs, which contributes (together with a slightlydi¡erent spatial position of the oxyanion oxygen)to a loss of a hydrogen bond between oxyanionand Gly123 NH. Superposition of all heavy atomsand all hydrogens attached to heteroatoms in thesequence Gly122^Gly123^Gly124 led to a rootmean square (rms) value of 0.35 Aî , with a distancebetween the Gly123 amide hydrogens of the twocomplexes calculated to be 0.7 Aî . Moreover, the dif-ference between the oxyanion oxygen^Gly123 amidehydrogen distances in the two complexes was foundto be 1.3 Aî . On these bases, one may conclude thatonly minor conformational changes within theGly122^Gly124 sequence occur upon binding of theR-enantiomer, which led to an incomplete hydrogenbonding network for the complex with CRL.

In addition, the aromatic portion of the substrate,as a result of a rotation around the Cchiral^Caromatic

bond, is forced to point toward Phe344, whose aro-matic ring undergoes a rotation of about 120³. In asimilar way, at the bottom of the hydrophobic pock-et ¢lled by the substrate, Phe345 aromatic ringmoves toward Phe296 and Leu297 assuming a per-pendicular orientation with respect to the substratebenzoyl moiety (Fig. 2, bottom) without any pro¢t-able interaction. Mutual position of these two phenylmoieties are consistent with a possible tilted T inter-action, but the ring center-ring center distance (5.7Aî ) is higher than the optimum value of about 5 Aî

[34].On the other hand, although also the aromatic

portion of the S substrate is located inside thesame hydrophobic pocket de¢ned by residuesHis449, Phe415, Phe344, Phe345, Phe296, andLeu297, the overall dimension of this cavity remainssmaller than that where the aromatic portion of theR substrate lies, due to the fact that no reorientationof the amino acid aromatic side chains occurred. Inparticular, the benzoyl portion of the S substrate isable to arrange itself in a tilted T orientation [34]with respect to the Phe345 aromatic side chain con-stituting the bottom of the pocket, which providespro¢table Z-interactions between the enzyme andthe ligand. In this case, the planes of the two aro-

matic rings de¢ne a dihedral angle of 87³, the cen-troids of the rings being at a distance of around 5.1Aî .

The methyl group of the alcoholic portion of theS- and R-enantiomers is oriented toward Glu208main chain, and Gly124, respectively.

The most signi¢cant di¡erences between the gen-erally accepted mechanism of hydrolysis and ourmodel are related to the imidazole ring of His449.In fact, His449 NO2^H group in each of the twocomplexes is not able to form a hydrogen bondwith OQ atom of catalytic serine due to a 75³H9OQ^Ser209 bond angle. We feel that the lack ofthe NO2^H9OQ hydrogen bond between His449 andSer209 is not to be considered as a key element in theanalysis of the results. In fact, although the hydrogenbond between NO2 and OQ is able to favor the nucle-ophilic attack of serine to the carbonyl carbon atomof the substrate as well as the transfer of the hydro-gen atom of the hydroxyl group of the catalytic ser-ine to the imidazole ring of the His449, leading to theformation of the ¢rst tetrahedral oxyanion inter-mediate, it should be considered that the computa-tional simulation has been performed at that step ofthe enzymatic pathway when the ¢rst tetrahedraloxyanion intermediate has been already formed.

A second binding mode, with the benzoyl moietyof the substrate interacting with the enzymatic hy-drophobic tunnel, was also found. The (S)-enantiom-er^enzyme complex showing the aromatic portion ofthe substrate located into a region surrounded byMet213, Phe125, Gly414, Phe415, and, more deeply,Pro246 and Leu 413, is 9 kJ/mol more stable then thecorresponding (R)-enantiomer^enzyme complex. Thealcoholic moiety of the substrate is directed towardthe Gly124 main chain without any interaction, andis linked by hydrogen bond to His449 NO2. The samegroup of the corresponding R-enantiomer occupies apocket surrounded by Glu208, Ser209, Ala210, andGly122 main chains, and the methyl group at thestereogenic carbon is located in a wide region with-out any speci¢c interaction. In contrast, the methylgroup at the S stereogenic center gives rise to thesame interactions highlighted for the R-enantiomerin the other model.

In addition, although we cannot claim that an ex-haustive search was performed, the low-energy con-former was found repeatedly over the course of the

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calculations with an average replication of ¢ve times.Further attempts were made to locate additionalpoints on the potential energy hypersurface. In par-ticular, repetition of the calculation for several addi-tional times from di¡erent starting geometries of theinhibitor did not alter the result.

We could not determine which mode of binding isthe most favored for each compound on the basis ofthe sole energy calculation, because of the intrinsicinaccuracy of molecular mechanics calculations (seeabove) and because the entropic contribution to thepotential energy of complexes in which the substrateis di¡erently oriented in the active site cannot beassumed to be the same. Thus, although in the caseof the second binding model, a better correlation ofthe calculated energy di¡erence between complexesinvolving di¡erent enantiomers (about 9 kJ/mol)and experimentally determined enantioselectivity(E = 27.2, corresponding to a vvG# of about 8 kJ/mol) was found, it was not possible to highlightstructural properties in the complexes belonging tothis binding model able to explain the enantioprefer-ence of CRL. On the basis of these considerations,we feel that the enantiopreference for the S-enan-tiomer that has emerged in this theoretical studycould be attributed to the lack of a hydrogen bondbetween the R substrate and the residues of the oxy-anion hole, as shown in the ¢rst binding model de-scribed. Accordingly, we suggest that the lack of thecomplete hydrogen bonding network, crucial for the¢ne tuning of the catalytic activity, might account forthe slower reaction rate of R-enantiomer.

In conclusion, the above reported computationalstudy led us to propose a binding model not in ac-cordance with the rule for predicting the enantiopref-erence of the CRL toward 2-substituted carboxylicacid [19], based on the proposal that the largest sub-stituent at the stereocenter of the substrate is coor-dinated to the active site tunnel. In the binding modewe suggest, both the acyl chain and the alcohol moi-ety of the R- and S-enantiomers of methyl 2-(3-ben-zoylphenyl)propionate are directed out of the activesite, nevertheless the catalytically crucial hydrogenbonds are all retained. A similar CRL^ligand bindingmode has been published also by Holmquist et al.[30] and reported to be the consequence of the stericfeatures of the ligand. In particular, if the largestsubstituent at the chiral center of the substrate is

too bulky to ¢t into the active site tunnel, the sub-strate may be bound in this alternate binding mode.

Our computational experiments predicted a rea-sonable ¢rst tetrahedral oxyanion intermediate ge-ometry for the enantiomer processed by CRL andled to identify a second, di¡erent geometry for thenot hydrolyzed enantiomer. Both the complexes be-tween CRL and either S- or R-antipode of methyl 2-(3-benzoylphenyl)propionate, as obtained by model-ing experiments, are in agreement with the acceptedmechanism of ester hydrolysis catalyzed by serineprotease as well as with the classical model explain-ing the CRL enantioselectivity in terms of the size,shape, and topology of the binding site. Althoughthe enantiopreference of lipases and esterases hasbeen attributed to the spatial arrangement of thecatalytic residues on the basis of structural data (X-ray structure of CRLînhibitor complexes) [13,14],some other aspects could play an important role inthe mechanism of the hydrolysis. The ¢nding thatboth R- and S-enantiomers seem to be recognizedby CRL has been discussed recently when the con-cept of transition-state model has been introduced toexplain the enantiopreference of lipases. It has beensuggested that the enantioselectivity displayed in theresolution of racemic esters be mainly governed bythe energy di¡erence in the diastereomeric forms ofthe postulated transition states, re£ected by the tet-rahedral intermediates.

The stereoelectronic theory, developed by Des-longchamps [35], has transformed the aforemen-tioned thermodynamic concepts into geometric con-straints on the three-dimensional structure of thetetrahedral intermediates. On the basis of the stereo-electronic theory, the ester CÔ bond can be cleavede¤ciently if both the two remaining oxygens of thetetrahedral intermediate have their lone-pair orbitalantiperiplanar to the breaking CÔ bond. This the-oretical observation has been supported by semiem-pirical molecular orbital calculations performed onlipase-catalyzed transesteri¢cation reactions by Emaet al. [36], which have con¢rmed that the gaucheconformation of the transition states (and apparentlyof the tetrahedral intermediates) has a lower energy(few kJ/mol) with respect to a theoretically possibleanti conformation. Accordingly, when the R-sub-strate^CRL and S-substrate^CRL complexes are an-alyzed on the basis of the stereoelectronic theory, it

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can be noted that only the last complex gives rise tooutput structures where the ligand shows the gaucheconformation required for the hydrolysis reaction tooccur. However, there are no evident structural rea-sons to support these ¢ndings, since no structuralelement in the R-enantiomer^CRL complex preventsthe conversion of the substrate from the anti to thegauche conformation. It is likely that an energeticbarrier to this conversion does exist, but it cannotbe highlighted by docking experiments.

3.2. Site-directed mutagenesis on CRL substratebinding cavity

With the aim of gaining further insight on thebinding of the substrate to CRL, computer generatedstructures of the complexes between the enzyme andthe ligand enantiomers have been used for the ration-al design of site-directed mutagenesis experiments onCRL structure. We have focused our attention onresidues predicted by the model to be close to theactive site and in contact with the substrate. In par-ticular, the amino acids involved in this study arePhe344 and Phe345. Phe344, exposed to the solventin the CRL open form [6], is located at the entranceof the hydrophobic pocket where it may be involvedin the initial recognition and transport of hydropho-bic substrates into the binding site. This residue has adegree of conformational freedom, allowing its phe-nyl group to move from the protein surface towardthe mouth of the hydrophobic pocket, partially oc-cluding it. On the other hand, Phe345 represents partof the mouth of the hydrophobic tunnel, in associa-tion with Met213, Leu304 and Phe415. The introduc-tion of the Val residue by site-directed mutagenesiswas intended to provide some insight about the pos-sible role of the side chains in the wild-type complex.

The mutations were chosen according to the fol-lowing considerations. (i) A previously published pa-per by our group [10] highlighted that the CH groupat 4-position of the Phe345 aromatic ring stericallyinteracts with the methyl group at the stereogeniccenter of the methyl (R)-2-(6-methoxy-2-naphthyl)-propionate, accounting for both the stereochemicalpreference and the high enantioselectivity of CRL.Substitution of Phe345 by Val removes these stericalclashes because of the shortening of the side chain,providing favorable hydrophobic interaction between

the methyl group on the chiral carbon of the sub-strate and the isopropyl moiety of the mutated ami-no acid. (ii) In the model of binding with both theacyl chain and the alcohol moiety of the substratedirected out of the active site, when the con¢gurationof the ligand was inverted from S to R, some con-formational rearrangement occurred due to unfavor-able steric contacts involving both the Phe344 andPhe345 aromatic rings. From this point of view,the suggested mutations could provide some addi-tional space useful for the R substrate orientationinside the catalytic site.

In the light of these considerations, both of theproposed mutations were expected to enhance thea¤nity of CRL toward the R-enantiomer, with aconsequent decrease of the e.e. values.

Three di¡erent plasmids were built in order to in-troduce in the LIP1 sequence the mutation Phe344V-al, Phe345Val, and the double substitution

Fig. 3. Site-directed mutagenesis by overlap extension [21].Thick arrows indicate double-stranded DNA, thin arrows indi-cate oligos. The mutagenesis site is represented by small circles.

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Phe344,345Val (Figs. 3 and 4). The introduction ofeach mutation by overlap extension polymerasechain reaction required four di¡erent primers andtwo subsequent ampli¢cation steps. The two externalprimers (A and B in Fig. 3) were common in eachexperiment and complementary to the wild-type se-quence, whereas the internal primers (C and D inFig. 3) were designed as to insert the desired muta-

tion and silent mutations in order to generate diag-nostic restriction sites. The recombinant expressionvectors carrying the two single and the double muta-tions were introduced in P. pastoris as described inSection 2. Lipase-secreting transformants were se-lected by gel electrophoresis analysis of culturesupernatants. Cultures expressing mutant lipase pro-teins were grown as described. After 4 days of fer-

Fig. 4. Partial nucleotide sequence of the LIP1 synthetic gene. (A) The region replaced in pPICslip1 to introduce mutations is includedbetween the HpaI and EcoRI sites (arrows). The external oligonucleotides (non-mutagenic) are indicated in dashed boxes. The graybox spans the region containing the two phenylalanine codons (in bold) subjected to mutagenesis. (B) Oligonucleotide sequences de-signed for PheCVal mutations. First and third lines, complementary mutagenic oligonucleotides; second line, wild-type sequence. Mu-tagenized phenylalanine codons are boxed. Bold letters indicate nucleotide substitutions, where lowercase letters indicate a silent muta-tion.

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mentation, cells were removed by centrifugation andconcentrated culture supernatants analyzed in gelelectrophoresis (not shown) before use in the reactiondescribed in the following where the wild-type re-combinant enzyme was used as the control.

3.3. rCRLs catalyzed hydrolysis reactions ofarylpropionic acid esters

Wild-type and mutated recombinant enzymes werecharacterized for their lipolytic activity, protein con-tent, and speci¢c activity (Table 1). Kinetic experi-ments performed using the recombinant (wild-typeand mutated) CRLs on 2-arylpropionic estersshowed a decreased enantioselectivity of the hydro-lysis reaction catalyzed by CRL mutants. In partic-ular, while the result obtained from Naproxen meth-yl ester by using either CRL wild-type or thePhe344Val mutant were not signi¢cantly di¡erent interms of e.e.p values of Naproxen (90.91% and90.15%, respectively, Table 2), a marked decreaseof the e.e.p value (73.47%) of Naproxen was ob-served in the experiment involving the Phe344,345Val

mutant. e.e.-Values for enzymatic hydrolysis of Ke-toprofen methyl ester are in agreement with thistrend, as similar e.e.p values (89.28% and 86.28%)were obtained for Ketoprofen in the hydrolysis reac-tion catalyzed by CRL wild-type and Phe344Val mu-tant, respectively, while the experiment involving thePhe344,345Val mutant gave a 61.84% e.e.p value.

In a similar way, while the result obtained fromboth Naproxen and Ketoprofen esters by using ei-ther CRL wild-type or the Phe344Val mutant werenot appreciably di¡erent in terms of percent conver-sion of substrates (Naproxen methyl ester: 32 vs. 33for wild-type and Phe344Val, respectively; Ketopro-fen methyl ester: 33 vs. 38 for wild-type andPhe344Val, respectively; Table 2), a marked decreaseof the percent values of conversion of Naproxen andKetoprofen esters (18% and 9%, respectively) wasobserved in the experiment involving thePhe344,345Val mutant. These results have been con-¢rmed when Phe345Val was used as catalyst in Ke-toprofen and Naproxen methyl esters hydrolysis:Naproxen showed 9% of conversion and 42% e.e.p ;no reaction was observed in the case of Ketoprofen.

Table 2Hydrolysis of Naproxen and Ketoprofen methyl esters catalyzed by CRLs

Substrates (methyl esters) Candida rugosa lipases Conversion (acid mmol/mgprotein)U103

Conversion (%) e.e.p (%) þ S.D.a Eb

Naproxen Wild-type 7.59 32 90.91 þ 0.03 32.0Naproxen Phe344Val 13.70 33 90.15 þ 0.02 29.9Naproxen Phe344,345Val 3.79 18 73.47 þ 0.05 7.6Naproxen Phe345Val 0.30 9 42.19 þ 0.05 2.6Ketoprofen Wild-type 4.85 33 89.28 þ 0.06 27.2Ketoprofen Phe344Val 9.36 38 86.28 þ 0.05 23.0Ketoprofen Phe344,345Val 1.08 9 61.84 þ 0.01 4.5Ketoprofen Phe345Val ^ ^ ^ ^aStandard deviation of six replicate injections.bCalculated according to Chen et al. [28].

Table 1Lipolytic activity, protein content and speci¢c activity of rCRLs used in this study

Candida rugosa lipases Lipolytic activitya (WEq acid/min) Protein concentration (Wg/ml) Speci¢c activity (WEq acid/min per mg)

Wild-type 10.2 121 84Phe344Val 15.4 54 284Phe344,345Val 0.1 108 1Phe345Val 1.5 320 5aHydrolysis reaction of tributyrin as substrate in phosphate bu¡er at 37³C and pH 7.9.

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This trend in the substrate conversion could be ra-tionalized on the basis of the pro¢table interactionsmainly involving Phe345 of the hydrophobic pocketof the enzyme, as reported above in the descriptionof the (S)-substrate^CRL complex. In fact, computa-tional experiments performed on the Phe344,345Valmutated enzyme show that the substitution of thePhe345 aromatic side chain with the isopropyl one,causes the loss of the Z-interactions between the ar-omatic moieties, thus reducing the overall stability ofthe complex.

In summary, experimental data indicated that thepresence of Phe344Val mutation did not substan-tially in£uence the enzyme activity, while the singlePhe345Val and the double mutation Phe344,345Valdetermined a signi¢cant decrease in enzyme activityand selectivity. In this regard, biological assays sup-port the molecular modeling suggestions highlightingthe role of Phe345 in the stereochemical preference ofCRL for the S-enantiomer in both the CRL^methyl2-(6-methoxy-2-naphthyl)propionate and the CRL^methyl 2-(3-benzoylphenyl)propionate complexes.

Acknowledgements

This research was partially supported by the ECProgram BIOTECHNOLOGY (BIO4-CT96-0005).The authors are indebted to Professor F. Gasparrini,University of Rome `La Sapienza', for his help insetting up the analytical methodology for substrateseparation. The authors also thank all their partnersin the EC Project for helpful discussions. Support bythe Progetto Finalizzato Biotecnologie of the ItalianNational Research Council to L.A. is also acknowl-edged.

References

[1] Z.S. Derewenda, U. Derewenda, G.G. Dodson, J. Mol. Biol.227 (1992) 818^839.

[2] U. Derewenda, A.M. Brzozowski, D.M. Lawson, Z.S. Dere-wenda, Biochemistry 31 (1992) 1532^1541.

[3] D. Blow, Nature 351 (1991) 444^445.[4] A.M. Brzozowski, U. Derewenda, Z.S. Derewenda, G.G.

Dodson, D.M. Lawson, J.P. Turkenburg, F. Bjorkling, B.Huge-Jensen, S.A. Patkar, L. Thim, Nature 351 (1991) 491^494.

[5] L. Brady, A.M. Brzozowski, Z.S. Derewenda, E. Dodson,G.G. Dodson, S. Tolley, J.P. Turkenburg, L. Christiansen,B. Huge-Jensen, L. Norskov, L. Thim, U. Menge, Nature343 (1990) 767^770.

[6] P. Grochulski, Y. Li, J.D. Schrag, F. Bouthillier, P. Smith,D. Harrison, B. Rubin, M. Cygler, J. Biol. Chem. 268 (1993)12843^12847.

[7] H. van Tilbeurgh, L. Sarda, R. Verger, C. Cambillau, Na-ture 359 (1992) 159^162.

[8] F.K. Winkler, A. D'Arcy, W. Hunziker, Nature 343 (1990)771^774.

[9] M.M.G.M. Thunnissen, E. Ab, K.H. Kalk, J. Drenth, B.W.Dijkstra, O.P. Kuipers, R. Dijkman, G.H. de Haas, H.M.Verheij, Nature 347 (1990) 689^691.

[10] M. Botta, E. Cernia, F. Corelli, F. Manetti, S. Soro, Bio-chim. Biophys. Acta 1337 (1997) 302^310.

[11] M. Botta, E. Cernia, F. Corelli, F. Manetti, S. Soro, Bio-chim. Biophys. Acta 1296 (1996) 121^126.

[12] M. Cygler, D.J. Schrag, Biochim. Biophys. Acta 1441 (1999)205^214.

[13] P. Grochulski, F. Bouthillier, R.J. Kazlauskas, A.N. Serreqi,J.D. Schrag, E. Ziomek, M. Cygler, Biochemistry 33 (1994)3494^3500.

[14] M. Cygler, P. Grochulski, R.J. Kazlauskas, J.D. Schrag, F.Bouthillier, B. Rubin, A.N. Serreqi, A.K. Gupta, J. Am.Chem. Soc. 116 (1994) 3180^3186.

[15] M. Norin, F. H×¡ner, A. Achour, T. Norin, K. Hult, Pro-tein Sci. 3 (1994) 1493^1503.

[16] R.J. Kazlauskas, Trends Biotechnol. 12 (1994) 464^472.[17] Programs MacroModel and BatchMin, version 4.5, Colum-

bia University, New York, NY.[18] InsightII Software, version 97.0, Molecular Simulation, Inc.,

San Diego, CA.[19] S.N. Ahmed, R.J. Kazlauskas, A.H. Morinville, P. Grochul-

ski, J.D. Schrag, M. Cygler, Biocatalysis 9 (1994) 209^225.[20] S. Brocca, C. Schmidt-Dannert, M. Lotti, L. Alberghina,

R.D. Schmid, Protein Sci. 7 (1998) 1415^1422.[21] S.N. Ho, H.D. Hunt, R.M. Horton, J.K. Pullen, L.R. Pease,

Gene 77 (1989) 51^59.[22] H. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning.

A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY, 1989.

[23] R.J. Klebe, J.V. Harriss, Z.D. Sharp, M.G. Douglas, Gene25 (1983) 333^341.

[24] U.K. Laemmli, Nature 227 (1970) 680^685.[25] O.H. Lowry, N.T. Rosenbrough, A.L. Farr, R.J. Randall,

J. Biol. Chem. 193 (1951) 265^269.[26] Q.M. Gu, C.S. Chen, C.J. Sih, Tetrahedron Lett. 27 (1986)

1763^1766.[27] M.A. Brook, T.H. Chan, Synthesis 3 (1983) 201^203.[28] C.S. Chen, Y. Fujimoto, G. Girdankas, C.J. Sih, J. Am.

Chem. Soc. 104 (1982) 7294^7299.[29] I.J. Colton, S.N. Ahmed, R.J. Kazlauskas, J. Org. Chem. 60

(1995) 212^217.[30] M. Holmquist, F. H×¡ner, T. Norin, K. Hult, Protein Sci. 5

(1996) 83^88.

BBAPRO 36260 8-11-00


[31] M. Norin, K. Hult, A. Mattson, T. Norin, Biocatalysis 7(1993) 131^147.

[32] F. H×¡ner, T. Norin, K. Hult, Biophys. J. 74 (1998) 1251^1262.

[33] R.J. Kazlauskas, Curr. Opin. Chem. Biol. 4 (2000) 81^88.[34] W.L. Jorgensen, D.L. Severance, J. Am. Chem. Soc. 112

(1990) 4768^4774.

[35] P. Deslongchamps, P. Atlani, D. Frehel, A. Malaval, Can. J.Chem. 50 (1972) 3405^3412.

[36] T. Ema, J. Kobayashi, S. Maeno, T. Sakai, M. Utaka, Bull.Chem. Soc. Jpn. 71 (1998) 443^453.

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