University of Groningen

Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifidafusca by Mutational AnalysisDudek, Hanna M.; Gonzalo Calvo , de, Gonzalo; Torres Pazmino, Daniel; Stepniak, Piotr;Wyrwicz, Lucjan S.; Rychlewski, Leszek; Fraaije, Marco W.; Stępniak, PiotrPublished in:Applied and environmental microbiology

DOI:10.1128/AEM.00687-11

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2011, p. 5730–5738 Vol. 77, No. 160099-2240/11/$12.00 doi:10.1128/AEM.00687-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Mapping the Substrate Binding Site of Phenylacetone Monooxygenasefrom Thermobifida fusca by Mutational Analysis£†

Hanna M. Dudek,1 Gonzalo de Gonzalo,1 Daniel E. Torres Pazmiño,1# Piotr Stępniak,2Lucjan S. Wyrwicz,2,3 Leszek Rychlewski,2 and Marco W. Fraaije1*

Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4,9747 AG Groningen, The Netherlands1; Bioinfobank Institute, Limanowskiego 24A, 60-744 Poznań, Poland2; and

Laboratory of Bioinformatics and Systems Biology, M. Skłodowska-Curie Cancer Centre andInstitute of Oncology, WK Roentgena 5, 02-781 Warsaw, Poland3

Received 26 March 2011/Accepted 18 June 2011

Baeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Amongthese enzymes, phenylacetone monooxygenase (PAMO) from Thermobifida fusca is the only protein showingremarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only alimited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO,the substrate binding site of this protein has not yet been defined. In this study, a structural model ofcyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and comparedwith the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may accountfor its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants inorder to verify the role of these positions. Extensive substrate screening revealed several mutants thatdisplayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxi-dations. Further substrate profiling resulted in the identification of mutants with improved catalytic propertiestoward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromisedin comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within theactive site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme.These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases towardan expanded substrate scope.

Enzymes have been gaining increasing attention as efficientand selective catalysts to be used in synthetic chemistry.Baeyer-Villiger monooxygenases (BVMOs) comprise a groupof enzymes that are particularly interesting for synthetic appli-cations. These biocatalysts employ molecular oxygen as a mildoxidant to oxidize carbonylic compounds. Apart from catalyz-ing Baeyer-Villiger reactions, BVMOs are capable of oxidizinga range of heteroatoms (e.g., sulfur, nitrogen, and boron).Furthermore, they often perform these reactions with highchemo-, regio-, and enantioselectivity (5, 15, 30). The use ofoxygen, which is a cheap and clean oxidant, and their diversityof catalyzed reactions make BVMOs attractive candidates forbiocatalytic processes.

The identification of phenylacetone monooxygenase (PAMO) inthe moderately thermophilic bacterium Thermobifida fusca hasbrought about a breakthrough in the research on BVMOs (11).PAMO is a thermostable enzyme that can easily be expressedin Escherichia coli and purified. Besides, its crystal structure

has been solved as the first structure of a BVMO (17). WhilePAMO shows excellent stability, even in the presence of or-ganic solvents (6, 28), its substrate specificity is rather re-stricted. The enzyme accepts mainly small aromatic ketonesand sulfides (7, 26), whereas the oxidations of bulkier ketonesoccur with lower activity and selectivity (27). However, theunique robustness of PAMO and the availability of its three-dimensional structure could facilitate the redesign of this en-zyme in order to alter the substrate scope. It would be valuableto obtain PAMO variants displaying the same stability butpresenting a relaxed substrate specificity and, thus, acceptingalicyclic or bulky substrates.

Members of different enzyme classes, including BVMOs,have been successfully modified by directed evolution. Cyclo-hexanone monooxygenase (CHMO) from Acinetobacter sp.NCIB 9871 (3) was subjected to random mutagenesis, andmutants with improved enantioselectivity were identified (20,21). In a similar fashion, a BVMO from Pseudomonas fluo-rescens DSM 50106 was evolved toward increased enantiose-lectivity (16). The elucidation of the crystal structure of PAMOhas laid the foundation for more rational engineeringstudies. A homology model of cyclopentanone monooxygenase(CPMO) from Comamonas sp. strain NCIMB 9872 (13) basedon the structure of PAMO supported a redesign study ofCPMO by which the enantioselectivity of the enzyme could beimproved (4). Recently, the Reetz group described a few casesin which residues in the active site of PAMO were targeted bysemirandom mutagenesis (22, 23, 33). They obtained a numberof mutants that act on substrates not accepted by the wild-type

* Corresponding author. Mailing address: Laboratory of Biochem-istry, Groningen Biomolecular Sciences and Biotechnology Institute,University of Groningen, Nijenborgh 4, 9747 AG Groningen, TheNetherlands. Phone: 31-50-3634345. Fax: 31-50-3634165. E-mail: m.w.fraaije@rug.nl.

# Present address: R&D Department, Genencor InternationalB.V., a Danisco Division, Archimedesweg 30, 2333 CN Leiden, TheNetherlands.

† Supplemental material for this article may be found at http://aem.asm.org/.

£ Published ahead of print on 1 July 2011.

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PAMO. As an example of rational engineering of PAMO,shortening an active-site loop by the deletion of residues S441and A442 resulted in a variant accepting bulkier substrates (2).Moreover, a model of CPMO built on the basis of the PAMOstructure inspired the preparation of the mutant M446G,which turned out to have a different substrate scope than thewild-type enzyme (31).

It should be noted that the molecular basis of the narrowsubstrate specificity of PAMO is not clear. The crystal struc-ture of PAMO lacks any substrate, product, or inhibitor fromwhich the binding site for substrates can be inferred. Takinginto account the complex catalytic mechanism of BVMOs,probably involving domain movements (19, 29), it is hard tospeculate on how substrates enter and bind in the active site.Some clues about the substrate binding site originated fromprevious mutagenesis studies. Residues belonging to the ac-tive-site loop spanning the region 440 to 446 have been repeat-edly found to play a role in the substrate specificity and theenantioselectivity of PAMO (2, 22, 23, 31). On the other hand,examination of the crystal structure reveals that this regionprobably forms only part of the binding site. Nevertheless,detailed knowledge of the residues interacting with the sub-strate in the active site is necessary to efficiently alter proper-ties such as the substrate range or the enantioselectivity ofPAMO by enzyme engineering.

To address the above-described problem, we strove for adetailed comparison of PAMO and CPMO structures. Thelatter enzyme can catalyze Baeyer-Villiger oxidations of nu-merous aliphatic and aromatic substrates, including small ali-cyclic ketones, which are not accepted by PAMO (13). Weprepared a homology model of CPMO based on the crystalstructure of PAMO. A comprehensive inspection of the activesites of these two enzymes let us identify 15 residues thatdiffered between PAMO and CPMO (Fig. 1). We hypothesizedthat a subset of these residues is involved in the substraterecognition. In a systematic site-directed mutagenesis study,we have mapped the active site of PAMO with respect tosubstrate specificity. By exchanging amino acids at the selectedpositions for their counterparts from CPMO, several mutantswith significantly altered substrate scope and enantioselectivity

have been identified. These findings indicate that the respec-tive residues interact with substrates during catalysis. Betterunderstanding of the substrate specificity determinants inPAMO opens up new routes for more effective redesign of thisenzyme.

MATERIALS AND METHODS

Enzymes and reagents. Oligonucleotide primers for mutagenesis were pur-chased from Sigma. PfuTurbo DNA polymerase was from Stratagene. DpnI wasfrom New England BioLabs. E. coli TOP10 cells were obtained from Invitrogen.Yeast extract and Bacto tryptone for preparation of culture media were pur-chased from Becton, Dickinson & Company. Substrates 1 to 8, 10, and 12 to 14(see Fig. 3), as well as ethyl benzoate (product 1a), d-valerolactone (product 3a),and other chemicals, were obtained from Acros Organics, Sigma-Aldrich, Merck,Julich Chiral Solutions GmbH, Clontech, and Roche Diagnostics GmbH. Sulfide9 was prepared as described previously (25), while ketone 11 was synthesized bytreating 1-(4-chlorophenyl)propan-2-one with methyl iodide and NaOH in abiphasic water/CH2Cl2 system. Racemic sulfoxides 5a to 8a were prepared bytreatment of the starting sulfides with H2O2 in methanol at room temperature(yields higher than 80%). Compound 11a was obtained by acetylation of 1-(4-chlorophenyl)ethanol with acetic anhydride and pyridine. Ketoester 12a wasachieved according to the literature (7). His-tagged phosphite dehydrogenaseused for regeneration of NADPH was overexpressed in E. coli TOP10 from apBAD plasmid containing a codon-optimized ptxD gene and purified on a Ni21-Sepharose column (GE Healthcare).

Homology modeling and structural analysis. The sequence of CPMO re-trieved from GenBank (gi?62286566) was submitted to the Protein StructurePrediction MetaServer (http://meta.bioinfo.pl) (12), which joins various homol-ogy modeling and threading algorithms. The results obtained were ranked ac-cording to the 3D-Jury method as described previously (12). The mappingsobtained were submitted to Modeller (9).

Construction of PAMO mutants. Single mutants were prepared byQuikChange site-directed mutagenesis using as a template a modified pBADplasmid containing the pamO gene fused to a C-terminal His tag (11). Theconstruction of the mutants with mutations M446G, Q152F/L153G, and Q152F/L153G/M446G has been reported before (31). The mutagenic primers used inthis study are listed in Table S1 in the supplemental material. Prior to transfor-mation, a DpnI-treated reaction mixture was purified using a NucleoSpin extractII kit (Macherey-Nagel). The introduced mutations were verified by sequencing(GATC Biotech, Konstanz, Germany). Mutations of two or three neighboringresidues were typically introduced in one QuikChange reaction. The Q152Fmutant was obtained by curing the mutation of L153G in the Q152F/L153Gmutant. Multiple mutants were constructed by introducing mutations in consec-utive rounds of mutagenesis. In each round, a plasmid with the verified sequencewas used as a template. The triple mutant with mutations Q152F/L153G/M446Gwas a starting point for the construction of the 8-fold mutant (V54I/C65V/I67T/Q93W/Q152F/L153G/I339S/M446G) and the 7-fold mutant (Q152F/L153G/S441A/A442G/S444C/M446G/L447P). The 12-fold mutant was obtained by re-combination of the 8-fold mutant with the 7-fold mutant using PvuI sites. The 10-and 11-fold mutants were achieved by reversing the Q152F and/or L153G mu-tations in the 12-fold mutant.

Screening of mutants. The activities of the mutants on ketones 1 to 4 weretested in reactions with whole cells. For these reactions, E. coli strains trans-formed with plasmids containing the wild-type or mutant pamO genes weregrown in CellStar 24-well plates (Greiner Bio-One). First, 1 ml of Luria-Bertanimedium supplemented with 50 mg ml21 ampicillin was dispensed into each wellof a plate and inoculated with a glycerol stock of the respective PAMO mutant.The wild-type PAMO, the inactive mutant R337A, and a well with no cells wereincluded on every plate. Next, the plate was covered with an AeraSeal film (ExcelScientific, Inc.) to reduce evaporation of the medium and incubated overnight at37°C with shaking. On the next day, 20 ml of a preculture from each well was usedto inoculate 2 ml of Luria-Bertani medium supplemented with 50 mg ml21

ampicillin and 0.02% (wt/vol) L-arabinose for the induction of protein expressionin a 24-well plate. The plate was covered with an AeraSeal film and incubatedovernight at 37°C. On the third day, the 2-ml cultures were transferred into 10-mlPyrex glass tubes, and a substrate (as 1 M stock in dimethyl sulfoxide [DMSO])was added to a final concentration of 10 mM. After incubation at 37°C for 24 h,the reaction mixtures were extracted with 1 ml of tert-butyl methyl ether sup-plemented with 0.1% (vol/vol) mesitylene as an internal standard, dried overanhydrous magnesium sulfate, and analyzed by gas chromatography (GC).

Oxidations of thioanisole (substrate 5) were tested using cell extracts. Precul-

FIG. 1. Active-site residues of PAMO targeted in the mutagenesisstudy. FAD is shown in black. For clarity, only the side chains of aminoacid residues are presented. Proposed mutations are indicated. Theschematic was prepared using PyMol software and the structure ofPAMO (PDB ID 1W4X_A).

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tures were prepared in 24-well plates as described above. On the next day, 40 mlof a preculture from each well was used to inoculate 4 ml of LB mediumsupplemented with 50 mg ml21 ampicillin and 0.02% (wt/vol) L-arabinose in atest tube. The tubes were incubated overnight at 37°C, after which the cells wereharvested by centrifugation, resuspended in 1 ml of 50 mM Tris-HCl, pH 7.5, anddisrupted by sonication. Each reaction mixture was comprised of 450 ml of a cellextract, 2.5 mM thioanisole, 1% (vol/vol) DMSO, 100 mM NADPH, the coen-zyme regeneration system (20 mM phosphite and 2 mM phosphite dehydroge-nase), and 50 mM Tris-HCl, pH 7.5, up to 2 ml. In a control reaction mixture, thecell extract was replaced with 450 ml Tris buffer. The reaction mixtures wereincubated and extracted as described above and analyzed by GC.

Since PAMO shows higher activity at slightly basic pH, wild-type PAMO andthe selected mutants (A442G, S441A, I67T, L443F, M446G, and I67T/L338P/A435Y/A442G/L443F/S444C) were tested for conversion of substrate 4 at pH9.0. For this, cell extracts were prepared and reaction mixtures were set up asdescribed above, except that 50 mM Tris-HCl, pH 9.0, was used.

Protein expression and purification. Wild-type and mutant PAMO proteinswere overexpressed and purified by affinity chromatography as described previ-ously (8). The extinction coefficients of flavin adenine dinucleotide (FAD) boundin PAMO mutants were determined as described previously (11). UV and visible-light absorption spectra were collected on a Perkin-Elmer Lambda Bio40 spec-trophotometer.

Conversions with isolated enzymes. Isolated enzymes were used to analyzeconversions of compounds 5 to 14. The reaction mixtures were typically com-prised of 2.5 to 10 mM substrate, 4 mM enzyme, 200 mM NADPH, 20 mMsodium phosphite, 3 mM phosphite dehydrogenase, and 50 mM Tris-HCl. Thereactions were carried out at pH 9.0, except for substrates 11 and 12, for whichpH 7.5 was used. The oxidations were usually performed at 30°C and 200 rpm.For substrates 9 and 13, reactions were also tested at 37°C. The conversion levelsand enantiomeric purity of the products were assessed by GC or high-perfor-mance liquid chromatography (HPLC).

GC and HPLC analyses. A Shimadzu GC 2014 chromatograph equipped with anAT5 column (30 m by 0.25 mm by 0.25 mm; Grace) and a Hewlett Packard HP 6890GC system with an HP1 column (30 m by 0.32 mm by 0.25 mm; Hewlett Packard)were employed to identify product formation catalyzed by the mutants. Enantiose-lective oxidations of compounds 2 and 11 were analyzed on a Hewlett Packard HP6890 chromatograph equipped with a chiral column, the Chiralsil Dex CB (25 m by0.32 mm by 0.25 mm; Varian), while a Chiraldex G-TA column (30 m by 0.25 mm by0.25 mm; Alltech) was used to separate enantiomeric products of oxidations ofcompounds 5 and 12. A Chiralcel OD column (25 cm by 0.46 cm; Daicel) wasemployed for the separation of compounds 6a to 8a. The products of conversion ofketone 2 were assigned by comparing them with the products of reactions catalyzedby CHMO (1), CPMO (18), and 4-hydroxyacetophenone monooxygenase (14). Anoxidation product of ketone 4 was identified by comparing it with the product of a

reaction catalyzed by the M446G mutant of PAMO (24). The absolute configura-tions of sulfoxides 5a to 8a and esters 11a to 12a were assigned by comparison of theGC or HPLC retention times with the published data (7, 27). The temperatureprograms used for separation and retention times of substrates and products in GCand HPLC are in Tables S2 and S3, respectively, in the supplemental material.

Determination of melting temperatures. The ThermoFAD method (10) wasemployed to measure the melting temperatures of the wild-type PAMO and selectedmutants. Fifty-microliter samples containing pure proteins diluted with 50 mMTris-HCl, pH 7.5, to a final concentration of 16 mM were placed in a 96-wellreal-time (RT)-PCR plate (Bio-Rad Laboratories). Measurements were conductedusing a MyiQ real-time PCR detection system (Bio-Rad Laboratories), which allowsexcitation in a range of 475 to 495 nm. The RT-PCR apparatus was equipped witha fluorescence emission filter of 515 to 545 nm. Unfolding was conducted in a rangefrom 20°C to 90°C. Fluorescence was recorded after every 0.5°C temperature in-crease, followed by a 10-s stabilization step. Each measurement was performed induplicate. As this method does not determine the equilibrium constant for thefolded and unfolded enzyme, the derived melting temperatures are indicated asapparent melting temperature, T9m. The T9m values were derived from the peaks ofthe first derivatives of fluorescence intensity.

RESULTS

Comparative analysis of the CPMO model and the PAMO

crystal structure. On the basis of the PAMO structure (PDBID 1W4X_A), a homology model of CPMO (41% of sequenceidentity) was calculated with Modeller. According to the 3D-Jury results, the protein was modeled on the basis of an align-ment prepared by the FUGUE server (32). The model ofCPMO was superimposed onto the PAMO structure. Theisoalloxazine moiety of the FAD cofactor was used as a refer-ence for the reaction center. Residues in the closest proximity(8 Å) to the C4a of the FAD cofactor were annotated andscreened for differences. The assumed 8-Å radius covered theregion surrounding the active site and identified 49 aminoacids, of which 30 were facing the surface of the cavity, andonly 12 of those were variable when comparing PAMO andCPMO. Thus, they could represent determinants of substratespecificity (marked on the alignment in Fig. 2; also see Fig. S1in the supplemental material). By comparison with CPMO, the

FIG. 2. Sequence alignment of PAMO and CPMO. Mutations proposed after the initial analysis are indicated with an asterisk (*), whilemutations proposed after the analysis of the new structure of PAMO are indicated with a pound sign (#).

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suggested mutations were V54I, C65V, I67T, Q93W, Q152F,L153G, I339S, S441A, A442G, S444C, M446G, and L447P.During the course of this study, the structure of PAMO (19a)in complex with both FAD and NADPH has been elucidated,which enabled further refinement of the residues flanking theactive site. As a result, the following additional mutants wereproposed: L338P, A435Y, and L443F (Fig. 2; also see Fig. S1in the supplemental material).

Substrate profiling using whole cells or cell extracts. Basedon the structural analysis described above, we prepared 15single and 15 multiple mutants (containing 2 to 12 substitu-tions) of PAMO in which selected residues were replaced byamino acids occurring at the respective positions in CPMO(Fig. 2). These mutants were initially screened in conversionscatalyzed by whole cells or cell extracts for activity against fourketones and one sulfide (Fig. 3A). The results are summarizedin Table 1. Only one of the single mutants, the L153G mutant,was found to be inactive with all of the tested substrates.Inactivation was also observed for the 12-fold mutant whichcontains this substitution. One single mutant, the A435Y mu-tant, was active only with bicyclo[3.2.0]hept-2-en-6-one (sub-strate 2). All of the other single mutants oxidized substrates 1,2, and 5. None of the mutants tested showed activity in theoxidation of cyclopentanone (substrate 3). The only mutantable to convert 1-indanone (substrate 4) was the M446G mu-tant, yielding the unexpected lactone 1-isochromanone (com-pound 4b) as the only product (Fig. 4). This unique reactivityof the M446G mutant has already been reported (24).

Interestingly, the L338P mutant, in which a drastic substitu-tion was introduced next to the catalytically essential R337,presented activity comparable to that of the wild-type enzyme.However, when this mutation was combined with I67T or moreamino acid substitutions, we observed a significant drop in theactivity in the Baeyer-Villiger reactions. For the I67T/L338P,I67T/L338P/A435Y/A442G, and I67T/L338P/A435Y/A442G/L443F/S444C mutants, no oxidation or only traces of esterscould be observed, while the formation of sulfoxide 5a, albeitof poor enantiomeric purity, was detected.

Remarkably, for a number of mutants, the enzymatic regio-and enantioselectivities were altered. The Baeyer-Villiger ox-idation of the racemic compound 2 can result in the formation

of four products: two enantiomers of the “normal” (product2a) and two enantiomers of the “abnormal” (product 2b) lac-tones (Fig. 4). The wild-type PAMO produces both the “nor-mal” and “abnormal” lactones in a ratio of about 3:1, with highenantiomeric excesses (ee) in favor of the (1S,5R)-enantio-mers. The I67T and S441A mutants yielded the two regioiso-mers in almost equal amounts. This trend, although less pro-nounced, was also observed for the V54I and L443Fmutants. Furthermore, the Q152F, A435Y, and A442G mu-tants generated compound 2b as the main product. Whenthe enantioselectivity in the oxidation of compound 2 wasanalyzed, it turned out that all of the mutants producedmainly the (1S,5R)-enantiomer of compound 2a. Interest-ingly, some mutations led to a decrease in the enantioselec-tivity of product 2b (I67T, S441A, and L443F), while threemutants (Q152F, A435Y, and A442G) showed preferencetoward the opposite (1R,5S)-enantiomer.

In some situations, combining mutations resulted in syner-gistic effects. When the S441A mutant, which displayed the lowenantioselectivity for product 2b, was merged with the A442Gvariant, showing little preference for the (1R,5S)-enantiomer,the resulting S441A/A442G mutant catalyzed the productionof the (1R,5S)-enantiomer of product 2b with high opticalpurity. This effect was even more striking for the double mu-tant I67T/L338P: combining two mutants selective for the(1S,5R)-enantiomer of product 2b created an enzyme produc-ing the opposite enantiomer. Furthermore, the fact that sev-eral multiple mutants (Q93W/A442G/S444C/M446G/L447P, Q93W/S441A/A442G/S444C/M446G/L447P, andS441A/A442G/S444C/M446G/L447P) feature very similarenantioselectivity for ketone 2 illustrates that the effects ofmutations depend very much on the context. The A442G mu-tation, which was the only substitution increasing the selectivityfor the (1R,5S)-enantiomer of product 2b that was present inall three mutants, did not appear to be able to account by itselffor such a high ee value for the (1R,5S)-enantiomer of product2b of the multiple mutants. The S441A mutation, which byitself is less selective for the (1R,5S)-enantiomer of product 2b,was missing in the Q93W/A442G/S444C/M446G/L447P mu-tant. This indicates that one or more of the other mutations(S444C, M446G, or L447P), which did not have much effect in

FIG. 3. Substrates used in the whole-cell screening (A) and in conversions with the isolated enzymes (B).

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the single mutants, caused this significant change in the enan-tioselectivity in the multiple mutants. This observation is in linewith the predicted role of the mutated residues: together, theyare involved in shaping the substrate binding cavity.

Sulfoxidation of thioanisole (substrate 5) by the wild-typePAMO leads to the formation of the (R)-methyl phenylsulfoxide with a moderate ee and conversion level. Severalmutants afforded significantly increased activity for this sul-fide (I67T, A442G, L443F, M446G, I67T/L338P, and S441/A442G). For two of the mutants tested, the opposite enan-tiomeric preference was observed: I67T and A442G formedthe (S)-sulfoxide with high optical purities. Interesting ef-fects occurred when the multiple mutants were tested withthis sulfide. When the substitution Q152F was introducedinto the A442G or S441A/A442G mutant, the resulting en-zymes showed a change in the enantiomeric preference,forming the (R)-sulfoxide. Moreover, the addition of themutation I67T (along with C65V, which, in contrast, did notseem to have an effect) to the triple mutant with mutationsQ152F/S441A/A442G resulted in a significant change in the

FIG. 4. (A) The Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one (racemic compound 2) leads to the formation of the “nor-mal” (2a) and “abnormal” (2b) lactones. (B) Similarly, 1-indanone (4)can be oxidized to the “normal” (4a) and “abnormal” (4b) products.

TABLE 1. Oxidations of substrates 1 and 2 catalyzed by the PAMO mutants in whole cells and oxidations ofsubstrate 5 catalyzed by cell extractsa

Description of enzyme RA (%)b of

substrate 1

Substrate 2c Substrate 5c

RA (%)b 2a:2bd ee (%) (2a/2b)e RA (%)b ee (%)

Wild type 100 (12%) 100 (9%) 73:27 94/82 100 (28%) 26 (R)V54I 65 54 58:42 90/76 38 34 (R)C65V 94 54 74:26 92/84 74 22 (R)I67T 16 33 54:46 84/28 140 74 (S)Q93W 65 97 70:30 91/78 89 27 (R)Q152F 35 17 30:70 66/(-)84 12 NDL153G #3 #3 ND ND #3 NDL338P 59 34 74:26 88/80 82 13 (R)I339S 81 74 71:29 93/86 62 42 (R)A435Y #3 25 29:71 46/(-) $90 #3 NDS441A 73 62 50:50 88/2 110 22 (R)A442G 75 96 30:70 89/(-)14 270 68 (S)L443F 65 75 59:41 92/8 150 28 (R)S444C 66 110 80:20 97/78 66 38 (R)M446G 49 27 65:35 84/21 250 93 (R)L447P 85 77 74:26 94/92 44 14 (R)I67T/L338P #3 8 60:40 59/(-)48 130 #3C65V/I67T 47 37 52:48 81/17 81 65 (S)C65V/I67T/Q93W 54 41 57:43 85/46 12 NDS441A/A442G 56 100 24:76 76/(-)61 170 30 (S)Q152F/A442G 37 54 23:77 75/(-)88 35 57 (R)Q152F/S441A/A442G 33 58 22:78 78/(-)87 40 50 (R)C65V/I67T/Q152F/S441A/A442G 31 48 23:77 61/(-)25 45 63 (S)Q93W/A442G/S444C/M446G/L447P 38 27 52:48 85/(-)86 13 NDS441A/A442G/S444C/M446G/L447P (5-fold mutant) 41 32 49:51 87/(-)89 62 $80 (R)Q93W/S441A/A442G/S444C/M446G/L447P 40 45 47:53 81/(-)91 14 NDI67T/L338P/A435Y/A442G #3 #3 ND ND 47 5 (R)I67T/L338P/A435Y/A442G/L443F/S444C #3 5 ND ND 23 #3V54I/C65V/I67T/Q93W/I339S/S441A/A442G/

S444C/M446G/L447P (10-fold mutant)5 26 40:60 85/(-)96 12 ND

V54I/C65V/I67T/Q93W/Q152F/I339S/S441A/A442G/S444C/M446G/L447P (11-fold mutant)

#3 #3 ND ND #3 ND

V54I/C65V/I67T/Q93W/Q152F/L153G/I339S/S441A/A442G/S444C/M446G/L447P (12-fold mutant)

#3 #3 ND ND #3 ND

a Substrate 1 is phenylacetone, substrate 2 is bicyclo@3.2.0#hept-2-en-6-one, and substrate 5 is thioanisole.b RA, relative activity. For a fair comparison, all incubations were for 24 h and relative activities are expressed as the rate of conversion normalized to that obtained

with wild-type PAMO. Absolute conversion values for the wild-type PAMO are given in parentheses.c ND, not determined.d Ratio of compound 2a to 2b.e In most cases, the (1S,5R)-enantiomer is the main product; (-) indicates that (1R,5S)-lactone is the main product.

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enantioselectivity, from 50% ee for the (R)-enantiomer to63% ee for the (S)-sulfoxide.

The 11-fold mutant, from which the deleterious substitutionL153G had been removed, still did not perform any oxidation.Upon curing the Q152F mutation, which in the single mutantcaused a significant decrease in the activity, small amounts ofBaeyer-Villiger oxidation and sulfoxidation products were ob-served. The regio- and enantioselectivities of this 10-fold mu-tant in the oxidation of ketone 2 were similar to those of the 5-and 6-fold mutants.

Substrate profiling using isolated mutant enzymes. Eight ofthe mutant enzymes featuring altered biocatalytic properties(V54I, I67T, Q152F, A435Y, A442G, M446G, Q152F/A442G,and S441A/A442G/S444C/M446G/L447P) were chosen for pu-rification and further characterization. As isolated enzymes,they were tested in conversions of a number of sulfides (sub-strates 6 to 10) and ketones (substrates 11 to 14), which are notrecognized or are poorly recognized by the wild-type PAMO.In addition, the mutants were applied in oxidations of prochi-ral (substrates 5, 6, 12) or racemic (substrate 11) compounds inorder to further assess their enantioselective behavior. Tables2 and 3 provide experimental data on these reactions.

In the case of thioanisole, the results from conversions bycell extracts and by isolated enzymes could be compared. In

general, the findings from the initial screening (Table 1) werein agreement with the results of the isolated enzyme conver-sions (Table 2). One outlier was found, the I67T mutant, whichproduced the (S)-sulfoxide in the initial cell extract-basedscreening, while it turned out to be selective for the (R)-enan-tiomer when the isolated enzyme was used. The reason for thischange in enantiomeric preference remains unclear. Previousstudies have shown that the medium conditions, e.g., pHor additives/solvents, can influence the enantioselectivity ofPAMO, which may suggest that intracellular components mayinfluence the enantioselectivity of this mutant (6, 34). In addi-tion, 68% ee was observed for the A442G mutant when usingthe cell extract, which was much higher than the 10% ee seenwhen using the isolated enzyme. Although overoxidation ofsulfides catalyzed by PAMO, namely, the formation of sul-fones, can take place, the detected levels of sulfones (5 to 10%)indicate that this second reaction could not affect the ee sig-nificantly. This was further supported by the results of theoxidation of racemic sulfoxide 5a, which was performed for thetested mutants: the enantiomeric ratio values (E) obtainedwere low, ranging from 2 to 16 (data not shown). Finally, asanalyzed in the time-resolved conversion of thioanisole by theA442G mutant, the enantiomeric excess of product 5a did notchange during the progress of the reaction (data not shown).

Conversions of the benzyl methyl sulfide (compound 6) bythe mutants were performed with usually good yields and highoptical purities, leading to (S)-benzyl methyl sulfoxide. Thetwo exceptions were the A442G and M446G mutants, forwhich only moderate ee values were obtained. The change inthe enantioselectivity of the A442G mutant appears to be con-sistent with the inverted enantioselectivity of this specific mu-tant toward thioanisole. The M446G mutant was already char-acterized to some extent, and the results of conversion ofsulfide 6, as well as substrate 5, obtained in the current inves-tigation are similar to the published data (31).

For testing whether mutants were more effective with rela-tively bulky substrates, compounds 7 and 8 were tested. Whilethese sulfides are poorly accepted by the wild-type PAMO,they were found to be converted more efficiently by severalmutants. The variants M446G and S441A/A442G/S444C/M446G/L447P were able to catalyze the sulfoxidation of benzylphenyl sulfide (substrate 7) with good yields and high ee values.

TABLE 2. Conversions of sulfides 5 to 8 by the isolated PAMO mutantsa

Description ofenzyme

Substrate 5 Substrate 6 Substrate 7 Substrate 8

cb (%) ee (%) Config cb (%) ee (%) Config cc (%) ee (%) Config cb (%) ee (%) Config

Wild type 70 34 R 97 97 S 5 ND ND 18 37 SQ152F 18 54 R 70 90 S #3 ND ND 36 40 SM446G 78 90 R 82 60 S 80 97 R 60 78 SV54I 70 50 R 95 96 S 13 46 R 51 45 SA442G 42 10 S 45 42 S 17 34 R 36 28 R5-Fold mutantd 71 50 R 96 82 S 75 92 R 25 56 SQ152F/A442G 25 75 R 92 93 S 7 ND ND 41 40 SI67T 40 25 R 97 95 S 28 93 R 25 19 SA435Y 20 72 R 97 97 S #3 ND ND 13 35 S

a All reactions were performed at 30°C. For all compounds, a small amount of sulfone was observed (#12%). c, conversion; Config, configuration; ND, notdetermined.

b Reactions were stopped after 24 h.c Reactions were stopped after 48 h.d The 5-fold mutant has mutations S441A/A442G/S444C/M446G/L447P.

TABLE 3. Conversion of ketones 11 and 12 by the isolatedPAMO mutantsa

Description ofenzyme

Substrate 11 Substrate 12

t (h) c (%) E cb (%) ee (%) Config

Wild type 0.5 36 20 61 82 RQ152F 1 43 44 #3 ND NDM446G 1 11 19 20 81 RV54I 1 10 11 8 ND NDA442G 0.5 34 17 8 ND ND5-fold mutantc 2 35 28 7 ND NDQ152F/A442G 8 56 12 #3 ND NDI67T 1 9 18 43 82 RA435Y 1 26 54 #3 ND ND

a All conversions were performed at 30°C. c, conversion; Config, configuration;ND, not determined.

b Reactions were stopped after 2 h.c The 5-fold mutant has mutations S441A/A442G/S444C/M446G/L447P.

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The I67T mutant also performed this reaction in a highlyselective manner, but the yield was only moderate. Further-more, a few mutants oxidized sulfide 8 with activities andenantioselectivity that were higher than those of the wild-typeenzyme. It is worth mentioning that the M446G mutant pro-duced 60% of the sulfoxide with an enantiomeric excess of78% in favor of the (S)-enantiomer, whereas the A442G mu-tant yielded the (R)-sulfoxide with 28% ee. While a number ofmutants showed increased activities with compounds 7 and 8,none of them were able to convert more challenging substrates,such as sulfides 9 and 10 or ketones 13 and 14, to a significantextent (,2%).

The wild-type PAMO was previously reported to oxidize3-phenylpentan-2,4-dione (substrate 12) with a good yield andoptical purity (7). Two of the mutants tested (M446G andI67T) catalyzed the oxidation with similar enantioselectivitybut lower conversion rates than the wild-type enzyme. In thekinetic resolution of the racemic benzylketone (compound 11),all of the biocatalysts preferentially oxidized the (S)-enan-tiomer. The mutants A435Y and Q152F displayed increased Evalues (54 and 44, respectively) compared to that of the wild-type PAMO (E 5 20), while for the rest of the mutants, theenantioselectivity achieved was similar to that of the wild-typeenzyme.

Expression level and thermostability of PAMO mutants.

The variants of PAMO with a high mutation load createdduring this research did not show compromised expressionlevels. In fact, the expression levels of all of the mutants con-structed have been assessed by SDS-PAGE, and no differencesbetween the wild-type PAMO and the mutants were observed.The multiple mutants, including the 12-fold mutant that wasfound to be inactive in Baeyer-Villiger reaction or sulfoxida-tion, were still able to incorporate FAD, as indicated by theA280/A441 ratio (Table 4). This demonstrates their ability tofold properly, although in the case of the M446G and S441A/A442G/S444C/M446G/L447P mutants and the 12-fold variant,the enzymes were not fully occupied with the cofactor. Thethermostability of the purified wild-type PAMO and the mu-tants was evaluated by the determination of apparent melting

temperatures by the ThermoFAD method (10). As can be seenfrom the data in Table 4, the single mutants did not suffer fromsignificant decreases in their apparent melting temperatureswhen compared to the relatively high T9m of the wild-typePAMO (60°C). The only exception to this trend was the A435Ymutant, for which a T9m of 55.5°C was observed. In this variant,an alanine residue was replaced by a bulky amino acid, whichcould affect the packing of residues inside the protein and,thus, its stability. The 5-fold S441A/A442G/S444C/M446G/L447P mutant displayed a T9m of 57°C. This loss of threedegrees in the T9m could be attributed to the additive effects ofthe substitutions A442G and M446G.

DISCUSSION

An extensive site-directed mutagenesis study was performedon PAMO. Fifteen residues in this enzyme were selected andmutated to amino acids present in CPMO at the correspondingpositions. The targeted positions are not conserved amongdifferent BVMOs (see Fig. S1 in the supplemental material),which supports the concept that they may account for theobserved differences in the substrate scope. Our data show thatmany of the introduced mutations influence the regio- andenantioselectivities of PAMO.

Oxidation of ketone 2 is often used as a probe for theselectivity of BVMOs. PAMO is able to produce both regioi-somers, similar to CHMO and unlike CPMO, which generatesmainly the “normal” lactone (product 2a) (1, 18). The “nor-mal” lactone is produced as the (1S,5R)-enantiomer byPAMO, similar to other BVMOs. PAMO also generates the(1S,5R)-enantiomer of compound 2b, in contrast to CPMOand CHMO, which provide the (1R,5S)-enantiomer of com-pound 2b (1, 18). The Q152F, A435Y, and A442G mutantshave manifested inverted enantioselectivity of product 2b,which makes them similar to CPMO and CHMO with respectto the enantioselective conversion of this substrate. However,their regioisomeric preference for the production of com-pound 2b rather than compound 2a is a unique propertyamong the BVMOs. Three other mutants (I67T, S441A, andL443F) generated similar amounts of both lactones, and theenantiomeric purity of compound 2b was lost. The drop in theenantioselectivity, even though it is an undesired property,could be interpreted as a step toward the inversion of theenantioselectivity. Therefore, it is reasonable to conclude thatmutations at these positions can be used to tune the enanti-oselectivity of PAMO. Lastly, a change of the regioselectivitywas observed for the V54I mutant, which produced similaramounts of compounds 2a and 2b while retaining the enantio-meric preference. In summary, our data confirm the hypothesisthat positions V54, I67, Q152, and A435, in addition to previ-ously analyzed S441, A442, L443, and M446 (22, 31), contrib-ute to the substrate specificity and the enantioselectivity ofPAMO.

It is worth noting that among the eight mutants tested asisolated proteins, all except I67T and A442G converted thio-anisole to the (R)-sulfoxide with an ee higher than that of thewild-type PAMO. Remarkably, the enantiomeric preference ofthe A442G mutant in oxidations of compounds 5, 6, and 8 wasdifferent than that of all the other mutants tested. The I67Tand M446G mutants oxidized sulfide 7 with excellent ee values,

TABLE 4. Absorbance ratios and apparent melting temperatures ofthe wild-type PAMO and the selected mutants

Description of enzyme A280/A441 T9m (°C)

Wild type 20 60Q152F 23 60A442G 22 59V54I 25 58I67T 27 60M446G ;50 58.5A435Y 21 55.5Q152F/A442G 26 595-Fold mutanta ;65 5710-Fold mutantb 22 NDc

12-Fold mutantd ;65 ND

a The 5-fold mutant has mutations S441A/A442G/S444C/M446G/L447P.b The 10-fold mutant has mutations V54I/C65V/I67T/Q93W/I339S/S441A/

A442G/S444C/M446G/L447P.c ND, not determined. The denaturation curve showed a gradual unfolding

from 30°C to 58°C with no clear transition.d The 12-fold mutant has mutations V54I/C65V/I67T/Q93W/Q152F/L153G/

I339S/S441A/A442G/S444C/M446G/L447P.

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while mutations Q152F and A435Y resulted in increased Evalues in the kinetic resolution of ketone 11. These data fur-ther support the idea that positions V54, I67, Q152, A435,A442, and M446 can be useful targets for tuning the enanti-oselectivity of PAMO in Baeyer-Villiger reactions or sulfoxi-dations.

As illustrated in Fig. 5, the above-mentioned amino acidscluster around the R337 residue, which has been found to beessential for catalysis (29). R337 in turn is predicted to interactwith NADP1, which is bound during the oxygenation step.S441, A442, L443, and M446 belong to a loop whose impor-tance for the substrate specificity of PAMO is well known (2,22, 31). A435 can also be considered a part of this extendedloop, and this residue points toward the putative binding site.While located close to the 440-to-446 loop as well, Q152 staysin the proximity of the ribose moiety of FAD, which indicatesthat it might interact with the cofactor and a mutation of Q152may affect the position of FAD. Similarly, a mutation at posi-tion V54 could influence the orientation of the flavin. I67 islocated on the opposite site of the isoalloxazine ring from theother residues discussed but also in the direct neighborhood ofthe flavin. Upon mutation to a polar residue, i.e., a threonine,this residue could form a hydrogen bond with the cofactor andthereby subtly change the architecture of the active site. Insummary, the residues found to influence the substrate speci-ficity and the selectivity of PAMO can directly or indirectly, viainteractions with the FAD cofactor, influence the positioningof a substrate in the active site. Nevertheless, it must be em-phasized that conclusions on structural effects of the mutationsshould be treated with caution, since it has been postulatedthat BVMOs adopt multiple conformations and undergo do-main rearrangement during the catalytic cycle (17, 19, 29).

The mutation of L153 to a glycine fully inactivates PAMO inBaeyer-Villiger and sulfoxidation reactions. Even though it wasalready known that the double mutant Q152F/L153G did notshow any Baeyer-Villiger activity (31), it was not clear at thattime which of the two mutations inactivates PAMO. Our re-sults demonstrate that the Q152F mutant could catalyze oxi-dations, while the substitution L153G was detrimental toPAMO either alone or in any combination. In this mutant, a

leucine, which is a relatively large amino acid, was exchangedfor a small one. Such a mutation could affect the orientation ofFAD, which must be tightly controlled in order to react withNADPH, oxygen, and a substrate. Strikingly, the correspond-ing glycine in CPMO could be mutated into much bulkierresidues, for example, a phenylalanine, a tyrosine, or a leucine,without impairing the catalytic properties and could even im-prove the enantioselectivity of the enzyme (4). Perhaps a moreconservative replacement of L153, such as L153I, could stillyield active enzyme, which may also display altered biocatalyticproperties.

Combining mutations into multiple mutants has in somecases resulted in unexpected properties. Most strikingly, thesubstitution L338P, which had virtually no effect in the singlemutant, led to almost a complete loss of activity when com-bined with other mutations. Furthermore, the highly mutatedvariants of PAMO (10 to 12 mutations) showed no or littleBaeyer-Villiger activity. This could be explained by the factthat some of the introduced mutations had negative effects onthe enzyme’s catalytic performance, and they resulted in severedecreases in its activity upon accumulation in one mutant.Meanwhile, it should be pointed out that the multiple mutantswere still viable with respect to expression and the binding ofthe flavin cofactor. This demonstrates again that PAMO isideally suited for enzyme redesign studies because it can tol-erate a high load of mutations.

Due to the chosen approach, which entails a limited capacityfor analysis, only certain specific substitutions were tested. Insome cases, quite dramatic changes were introduced (e.g.,A435Y and L447P), while the mutations in other variantscaused only slight changes (e.g., S444C and V54I). It could beargued that in order to get a complete view, one should per-form a site saturation mutagenesis on all of the active-siteresidues and assess the effects of all possible amino acid ex-changes. However, it would entail considerably more effort,particularly if more substrates were to be tested. Since wewanted to test as many as 15 positions with at least five sub-strates, the present approach appeared to be more appropri-ate. Certainly, our results have proven the concept of testingsingle substitutions to be valuable. We assume that if a muta-tion to a significantly different amino acid does not have anyeffect on the enzyme’s activity or selectivity, this position is notinvolved in interactions with a substrate. This appears to be thecase for the C65V, Q93W, I339S, and L447P mutants. On theother hand, if subtle mutations bring about a clear effect onthe enantio- or regioselectivity of the enzyme, as was the case for theV54I mutant, it emphasizes the importance of the position for sub-strate recognition and positioning in the active site.

A number of positions investigated in this paper, Q152,L153, L338, I339, and M446, had formerly been subjected topartial randomization in several PAMO mutant libraries, butno hits were found in the screening for activity with 2-phenyl-cyclohexanone (23). While one of these positions, namely,I339, seems not to be involved in substrate recognition in thepresent study, our data indicate that replacing the other resi-dues can affect the biocatalytic properties of PAMO. Appar-ently, the mutations of Q152, L338, and M446 tested in thecurrent project, which were also included in the libraries pre-pared in the research of Reetz and Wu, are not sufficient tointroduce activity against this specific compound. Moreover,

FIG. 5. A schematic representation of the active site of PAMO.Residues identified as hot spots for substrate specificity are displayedas sticks. Residue R337 that is essential for catalysis is also shown.FAD is presented as black sticks. The schematic was prepared usingPyMol software and the structure of PAMO (PDB ID 1W4X_A).

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the double Q93N/P94D mutant afforded a PAMO variant thatconverted 2-ethylcyclohexanone, as well as other 2- and 3-sub-stituted cyclohexanones, with high enantioselectivity (33). Inour investigation, the mutation of Q93W did not influence thesubstrate specificity or the enantioselectivity of PAMO. This isin line with the findings of Wu et al., who reported that noactivity on the above-mentioned substrates could be detectedwhen single mutants or single-position libraries were tested. Itsuggests that the effects of mutations at position 93 can only beobserved when an accompanying mutation, e.g., P94D, is in-troduced. In fact, the properties of the Q93N/P94D mutanthave been explained by a conformational change relatively farfrom the active site that translates into altered accessibility ofthe active site. This agrees with our conclusion that Q93 is notdirectly involved in substrate recognition.

This thorough site-directed mutagenesis study has allowedus to identify four positions in PAMO, V54, I67, Q152, andA435, which in addition to the previously known hot spots,such as S441, A442, L443, and M446, contribute to substratespecificity. With these data, the substrate binding pocket ofPAMO is becoming more defined, which will help to efficientlyengineer this enzyme to accept new substrates. As is clear fromour results, even single, arbitrarily designed substitutions ofindicated amino acids can result in mutants with increasedactivity and enantioselectivity. This supports the idea thatscreening libraries of PAMO randomized at identified posi-tions could bring greatly improved variants. Finally, the abilityof PAMO to accommodate numerous mutations highlights theexceptional robustness of this protein and underlines its suit-ability as a starting point for future directed-evolution experi-ments.

ACKNOWLEDGMENT

This work was supported by the EU-FP7 OXYGREEN project.

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