Mutations at the putative active cavity of styrene monooxygenase: Enhanced activity and reversed enantioselectivity

Journal of Biotechnology 161 (2012) 235–241

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Mutations at the putative active cavity of styrene monooxygenase: Enhanced activity and reversed enantioselectivity Hui Lin a,b , De-Fang Tang a , Abeer Ahmed Qaed Ahmed a,b , Yan Liu a , Zhong-Liu Wu a,∗ a Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Environmental Microbiology Key Laboratory of Sichuan Province, P.O. Box 416, Chengdu 610041, PR China b Graduate University of the Chinese Academy of Sciences, Beijing 100049, PR China

a r t i c l e

i n f o

Article history: Received 6 January 2012 Received in revised form 5 June 2012 Accepted 8 June 2012 Available online 11 July 2012 Keywords: Biocatalysis Styrene monooxygenase Protein engineering Rational design Chiral epoxide

a b s t r a c t Styrene monooxygenase (SMO) catalyzes the first step of styrene degradation, and also serves as an important enzyme for the synthesis of enantiopure epoxides. To enhance its activity, molecular docking of styrene was performed based on the X-ray crystal structure of the oxygenase subunit of SMO to identify three amino acid residues (Tyr73, His76 and Ser96) being adjacent to the phenyl ring of styrene. Variants at those positions were constructed and their enzymatic activities were analyzed. Three mutants (Y73V, Y73F, and S96A) were found to exhibit higher enzymatic activities than the wild-type in the epoxidation of styrene, while retaining excellent stereoselectivity. The specific epoxidation activity of the most active mutant S96A toward styrene and trans-␤-methyl styrene were 2.6 and 2.3-fold of the wild-type, respectively. In addition, the Y73V mutant showed an unexpected reversal of enantiomeric preference toward 1-phenylcyclohexene. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Styrene monooxygenase (SMO) is an enzyme involved in the upper catabolic pathway of styrene degradation (Bestetti et al., 2004; Mooney et al., 2006). It is a member of the class E flavoprotein monooxygenases, which is composed of the oxygenase (StyA) and the reductase (StyB) domains. StyA catalyzes the epoxidation of alkenes, and StyB catalyzes the two-electron reduction of FAD (Kantz et al., 2005; Kantz and Gassner, 2011; Otto et al., 2004). The native substrate of SMO is styrene, which can undergo asymmetric epoxidation to form the single enantiomer of (S)-styrene oxide (Fig. 1) (Bernasconi et al., 2000; Di Gennaro et al., 1999; Lin et al., 2010; Panke et al., 2002; Park et al., 2006; Tischler et al., 2010; Toda et al., 2012). Since it is well recognized that enantiopure epoxides are extremely important building blocks in fine chemical industry, the development of efficient synthesis methods for enantiopure epoxides has been a fundamental research area in asymmetric synthesis. Considerable effort has been made by synthetic chemists including the Noble Price-winning work of Sharpless epoxidation which allows the asymmetric epoxidation of prochiral allylic

∗ Corresponding author at: Chengdu Institute of Biology, Chinese Academy of Sciences, 9 South Renmin Road, 4th Section, Chengdu, Sichuan 610041, PR China. Tel.: +86 28 85238385; fax: +86 28 85238385. E-mail address: [email protected] (Z.-L. Wu). 0168-1656/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2012.06.028

alcohols. However, for nonactivated terminal alkenes, such as styrene, chemo-catalyzed asymmetric epoxidation suffers from insufficient selectivity and/or impractical reaction temperature (Lin et al., 2011a). Therefore, the excellent selectivity of SMO has attracted much interest in the synthesis of chiral epoxides, and SMO-containing recombinant Escherichia coli has been extensively investigated. The process for the production of (S)-styrene oxide has been scaled up by using recombinant E. coli cells expressing the SMO from Pseudomonas fluorescens VLB120 and economic assessment shows that bioprocess performs best in terms of production costs compared with other three chemical alternatives (Kuhn et al., 2010; Panke et al., 2002; Schmid et al., 2001). In addition, with continuous effort in the functional studies and the identification of novel SMOs, the substrate spectrum of SMO has been expanded. The majority of SMOs characterized so far have shown epoxidation activity toward a variety of substituted styrene derivatives and heterocyclic styrene analogues, and the SMO from Pseudomonas sp. LQ26 also takes non-conjugated secondary allylic alcohols as substrates (Bernasconi et al., 2000, 2004; Lin et al., 2011a,b,c; Park et al., 2005; van Hellemond et al., 2005). Although SMOs display excellent enantioselectivity in most cases and could serve as a good complementary or better approach to existing chemo-catalytic methods, it remains highly desired to improve their catalytic activity in order to enhance its potential for scaled-up production. The X-ray crystal structure of the dimeric FAD-specific oxygenase subunit of the SMO from Pseudomonas putida S12 (PDB ID:

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H. Lin et al. / Journal of Biotechnology 161 (2012) 235–241 Table 1 Primers used in site-directed mutagenesis. Mutation Oligonucleotide sequencesa

Fig. 1. Epoxidation of styrene catalyzed with styrene monooxygenase (SMO).

3IHM) has recently been released without a substrate (Ukaegbu et al., 2010). However, its overall structure homology to that of p-hydroxybenzoate hydroxylase (PHBH) from P. fluorescens (PDB ID: 1CC4) provides insight into the putative substrate and flavinbinding pockets, which has assisted the search for mutants with altered substrate specificity through molecular docking of ˛ethylstyrene into the putative active cavity for the SMO from Pseudomonas sp. LQ26. The investigation of several rationally designed mutants at positions 43–46 leaded to one mutant with substrate preference toward the bulkier substrate (Qaed et al., 2011). However, all of those designed mutants display decreased enzymatic activity, probably as a consequence of reduced FAD binding affinity, because residues 43–46 are located in the center of the putative substrate access channel connecting the postulated styrene and FAD binding cavities and might be involved in FAD binding or could interact with the flavin ring (Feenstra et al., 2006; Ukaegbu et al., 2010). Another study on the engineering of the SMO from P. putida CA-3 has been performed by screening an error-prone PCR library using indigo assay, resulting in mutants exhibiting higher rates of epoxide formation (Gursky et al., 2010). However, this method carries the risk of generating mutants with increased activity only toward the analog substrate indigo, but not the target substrate styrene (Gursky et al., 2010; Zhang et al., 2009). In the current work, to investigate amino acid substitutions that might enhance the catalytic efficiency of SMO, a rational design approach was undertaken, which relied on molecular docking assisted by the AutoDock program and focused on those amino acid residues that might interact with the phenyl ring of styrene. The SMO from Pseudomonas sp. LQ26 (designated as StyAB2) was used as the parental enzyme since it has been well studied in our laboratory as a highly selective biocatalyst (Lin et al., 2010, 2011b,c; Qaed et al., 2011), and its high homology with other SMOs from the genus of Pseudomonas would facilitate the docking study. This strategy led to several SMO mutants with increased enzymatic activities toward styrene, and one mutant displayed reversed enantioselectivity toward the substrate 1-phenylcyclohexene. 2. Materials and methods 2.1. Chemicals The substrates styrene, trans-␤-methyl styrene, 2-vinylpyridine and 1-phenylcyclohexene were purchased from Alfa Aesar (Tianjin, China). Racemic styrene oxide (1S, 2S)-1-phenylpropylene oxide and (1R, 2R)-1-phenylpropylene oxide were purchased from Sigma–Aldrich (St. Louis, MO, USA), and used as standard products. Other standard products including racemic 1-phenylcyclohexene oxide and 2-(oxiran-2-yl)pyridine were synthesized from the corresponding alkenes according to the literatures (Fieser and Fieser, 1967; Hanzlik et al., 1976). Other reagents were purchased from general suppliers and were used without further purification. 2.2. Docking studies The X-ray crystal structure of the oxygenase subunit of SMO (SMOA) from P. putida S12 was available from the PDB database (PDB ID: 3IHM). The amino acid sequence of this subunit shares

Y73F

5 -CCA TCT GAT GAA TTC GGT TTC TTT GGC CAC TAC TAC TAT G-3 5 -C ATA GTA GTA GTG GCC AAA GAA ACC GAA TTC ATC AGA TGG-3

Y73V

5 -CCA TCT GAT GAA TTC GGT GTC TTT GGC CAC TAC TAC TAT G-3 5 -C ATA GTA GTA GTG GCC AAA GAC ACC GAA TTC ATC AGA TGG-3

Y73S

5 -CCA TCT GAT GAA TTC GGT TCC TTT GGC CAC TAC TAC TAT G-3 5 -C ATA GTA GTA GTG GCC AAA GGA ACC GAA TTC ATC AGA TGG-3

H76N

5 -GAA TTC GGT TAC TTT GGC AAC TAC TAC TAT GTC GGC G-3 5 -C GCC GAC ATA GTA GTA GTT GCC AAA GTA ACC GAA TTC-3

H76V

5 -GAA TTC GGT TAC TTT GGC TAC TAC TAC TAT GTC GGC G-3 5 -C GCC GAC ATA GTA GTA GTA GCC AAA GTA ACC GAA TTC-3

H76A

5 -GAA TTC GGT TAC TTT GGC GCC TAC TAC TAT GTC GGC GG-3 5 -CC GCC GAC ATA GTA GTA GGC GCC AAA GTA ACC GAA TTC-3

S96A

5 -CTC AAG GCC CCG GCC CGC GCT GTC G-3 5 -C GAC AGC GCG GGC CGG GGC CTT GAG-3

S96L

5 -CTC AAG GCC CCG CTC CGC GCT GTC GAC-3 5 -GTC GAC AGC GCG GAG CGG GGC CTT GAG-3

S96T

5 -C AAG GCC CCG ACC CGC GCT GTC G-3 5 -C GAC AGC GCG GGT CGG GGC CTT G-3

a

The nucleotide changes are underlined.

an 89% identity with the same subunit of the SMO from Pseudomonas sp. LQ26. To prepare the structure for docking, chain B of the StyA homodimer and all water molecules of SMOA were removed, and charges and non-polar hydrogen atoms were added using MGLTools 1.5.4. AutoDock 4.0 was used for docking, and the docking parameters were kept to their default values in general (Morris et al., 1998) except that the grid spacing was changed to 0.275, the number of AutoDock 4 GA runs was increased from 10 to 50, and the docking grids were set as 22 × 32 × 22 A˚ for styrene and 28 × 32 × 28 A˚ for 1-phenylcyclohexene. The 50 independent runs from AD4 were analyzed in MGLTools 1.5.4 and the results were visualized using the program Pymol. 2.3. Construction of point mutations Site-directed mutagenesis was performed according to the QuikChange® site-directed mutagenesis protocol (Stratagene, La Jolla, CA) using the plasmid pETAB (Lin et al., 2010) encoding the wild-type StyAB2 (GenBank ID: GU593979) as the template. The sequences of mutagenic oligonucleotide primers (Table 1) were synthesized by Shanghai Invitrogen Life Technologies. The PCR product was treated with 20 U of Dpn I at 37 ◦ C for 2 h and transformed into DH5˛ competent cells. The successful introduction of the desired mutations was confirmed by sequencing at Shanghai Invitrogen Life Technologies. 2.4. Expression of the wild-type and mutant StyAB2 in E. coli BL21 E. coli strain BL21(DE3) containing the constructed plasmids was used to produce the wild-type and mutant StyAB2. Single colonies were picked up and grown overnight at 37 ◦ C in LuriaBertani broth containing 50 ␮g kanamycin/ml. For each mutant and the wild-type, two single colonies were picked and cultivated to make two independent heterologous expressions. The overnight culture (2 ml) was inoculated into Terrific Broth (200 ml) containing 50 ␮g kanamycin/ml in a 500 ml flask and incubated at 37 ◦ C for 3 h followed by 18 h incubation at 20 ◦ C with gyratory shaking at 220 rpm. The cells were harvested by centrifugation, washed twice with potassium phosphate buffer (0.1 M, pH 7) and stored at 4 ◦ C. To determine the expression levels of the wild-type and mutant enzymes, crude cell extracts were analyzed by SDS-PAGE, then the

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oxygenase subunit was purified using Profinity IMAC Ni-Charged resin (Bio-Rad, Hercules, CA, USA) as described previously (Lin et al., 2010). Then, the purified proteins were analyzed by SDS-PAGE. The protein content was determined with a commercial BCA Protein Assay kit using bovine serum albumin as a standard (Beyotime, Beijing, China). 2.5. Biotransformation with whole cells and product analysis The harvested recombinant E. coli BL21 cells expressing the wild-type and mutants with a cell dry weight (CDW) of 0.1 g were resuspended in a biphasic system (Lin et al., 2010; Panke et al., 1999) of 10 ml potassium phosphate buffer (100 mM, pH 6.5) containing 10% (v/v) bis-(2-ethylhexyl) phthalate (BEHP) with the addition of 10 mg styrene or 1-phenylcyclohexene. For 2-vinyl pyridine, a monophasic system without BEHP resulted in better conversion and thus was applied instead of the biphasic system. The reaction was carried out at 30 ◦ C for 4 h with shaking at 230 rpm and terminated by extraction with ether. The organic phases were combined, dried with anhydrous sodium sulfate, concentrated under vacuum, and subjected to GC and chiral HPLC analysis. Specific epoxidation activities were measured using whole cells following the literatures (Bae et al., 2008; Park et al., 2006). Briefly, recombinant E. coli BL21 cells with 0.5 g CDW/L were resuspended in 4 ml potassium phosphate buffer (100 mM, pH 7.0) containing glucose (5 g/L), and incubated at 30 ◦ C for 10 min before the addition of 1.5 mM substrate (30 mM stock solution of styrene or trans-␤methyl styrene in ethanol). The reaction was continued for 5 min. The mixture was extracted with ether containing 0.1 mM dodecane as an internal standard and analyzed with gas chromatography (GC). One unit (U) is defined as the activity that produces 1 ␮mol of oxide per min. GC analysis was performed on a Fuli 9790 II system connected to a flame ionization detector using column BP5 (30 m × 0.22 mm ID × 0.25 ␮m film thickness, SGE Analytical Science, Australia) to determine the conversion of each substrate (styrene, trans-␤-methyl styrene, 2-vinylpyridine or 1phenylcyclohexene) to the corresponding epoxide. Enantiomeric excesses were determined using chiral HPLC on a Shimadzu LC 20-AD (Shimadzu, Japan) with a PDA detector using Daicel Chiralpak AS-H column for styrene oxide (hexane:2-propanol = 90:10, 0.5 ml/min, tR (R) 10.27 min, tR (S) 10.67 min), Chiralcel AD-H column for 2-methyl-3-phenyloxirane (hexane:2-propanol = 90:10, 1 ml/min, tR (R) 4.14 min, tR (S) 4.78 min), or Chiralcel OD-H column for 1-phenylcyclohexene oxide (hexane:2-propane = 99:1, 0.5 ml/min, tR (R,R) 12.53 min, tR (S,S) 13.67 min) and 2-(oxiran-2yl)pyridine (hexane:2-propane = 95:5, 0.5 ml/min, tR (R) 15.05 min, tR (S) 15.83 min).

Fig. 2. Orientation of styrene docked into the putative active site of SMO (PDB ID: 3IHM). Substrate is shown in green in the stick mode. Residues His76, Ser96 and Tyr73 are shown in sky blue, and residues Arg43, Leu44, Leu45 and Asn46 are shown in purple in the stick mode. The figure was generated using the program Autodock and displayed using the program Pymol.

the target sites for rational design. In addition, unlike the residues 43–46, residues Tyr73, His76 and Ser96 are away from the putative FAD binding channel, which would avoid negative impacts on catalytic activity caused by reduced FAD binding (Feenstra et al., 2006; Ukaegbu et al., 2010). Therefore, residues Tyr73, His76 and Ser96 were modified using site-directed mutagenesis to investigate their effects on the enzymatic activity and enantioselectivity. Three amino acid substitutions were designed for each site in a way to reflect typical changes in size and hydrophobicity of the side chain of the residue. Tyr73 was replaced with Phe, Val and Ser. Compared with Tyr, Phe lacks the hydroxyl group while retaining the aromatic structure; Ser lacks the aromatic structure while retaining the hydroxyl group; and Val lacks both the hydroxyl group and the aromatic structure, but retains part of the steric hindrance. His76 was replaced with Asn, Val and Ala, all of which lack the electronically charged side chain, but contain a polar but uncharged residue (for Asn), or representative hydrophobic residues (for Val and Ala). Ser96 was replaced with Ala, Leu and Thr. Compared with Ser, Thr retains the hydroxyl group with an additional methyl group; and Leu and Ala lack the hydroxyl group and are representative hydrophobic residues with varied side chain sizes.

3. Results The SMOA structure from P. putida S12 has been released without substrate and FMN. Based on the putative substrate-binding center of SMOA as well as our previous work, which has shown the residues 43–46 being close to the ␣-substitute of ˛-ethylstyrene, the native substrate styrene was docked into SMOA using AutoDock 4.0. The returned 50 results were analyzed in the MGL tools 1.5.4, and the highest scoring conformer with the vinyl group of styrene adjacent to the residues 43–46 was shown in Fig. 2. The amino acid residues Tyr73, His76 and Ser96, which form the bottom of the substrate-binding pocket, were found to be adjacent to the benzene ring of styrene with their side chains facing the ˚ respectively substrate with distances of 7.15, 11.93 and 9.48 A, (Fig. 2). It is well recognized that residues adjacent to the substrate play a critical role in catalytic activity, and thus commonly act as

Fig. 3. Biotransformation of styrene by the wild-type and mutant StyAB2. The reaction was carried out at 30 ◦ C for 4 h with 0.1 g cell dry weight (CDW) resuspended in a biphasic system of 10 ml potassium phosphate buffer (100 mM, pH 6.5) containing 10% (v/v) bis-(2-ethylhexyl) phthalate (BEHP) with the addition of 10 mg styrene. Activities were normalized as percentages of the activity of the wild-type. Each column represents the mean (SD) of triplicate assays.

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Fig. 4. Specific epoxidation activities of the wild-type and mutant StyAB2 toward styrene and trans-␤-methyl styrene. The reaction was carried out at 30 ◦ C for 5 min with 0.5 g CDW/L resuspended in 4 ml potassium phosphate buffer (100 mM, pH 7.0) containing glucose (5 g/L) and 1.5 mM substrate. Each column represents the mean (SD) of triplicate assays.

All the constructed mutants listed in Fig. 3 were functionally expressed in E. coli at a level similar to the wild-type. Their activities were examined in the epoxidation of styrene in the biphasic reaction for 4 h (Lin et al., 2010; Panke et al., 1999). All active mutants retained excellent enantioselectivity, yielding the product (S)-styrene oxide with >99% ee. Mutants Y73F, Y73V and S96A exhibited higher activities than the wild-type, while other mutants showed lower activities or even major deleterious effect (Fig. 3). The best mutant S96A displayed a relative activity of 180% (Fig. 3). Specific epoxidation activities were then measured for the most active mutants when the assays were carried out in an aqueous system for 5 min (Panke et al., 1998; Park et al., 2006). The results confirmed the increased enzymatic activity of mutants Y73F, Y73V and S96A for the epoxidation of styrene, as well as for trans␤-methyl styrene (Fig. 4) without any negative impact on their enantioselectivities. The whole cell specific epoxidation activity of the wild-type StyAB2 was 66.5 U/g CDW, which was comparable to that of the other SMOs measured under similar conditions, such as that from Pseudomonas sp. VLB120 (79 ± 5 U/g CDW) and P. putida SN1 (55 ± 5 U/g CDW) (Panke et al., 1998; Park et al., 2006). The specific epoxidation activities of the most active mutant S96A toward styrene and trans-␤-methyl styrene were 2.6 and 2.3-fold of the wild-type, respectively (Fig. 4). For substrates 2-vinyl pyridine and 1-phenylcyclohexene, the changes in activities were varied for the mutants Y73F, Y73V and S96A. Only S96A and Y73V displayed slight increases in the 2-vinyl pyridine and 1-phenylcyclohexene conversions, respectively (Table 2). Interestingly, the asymmetric epoxidation of 1-phenylcyclohexene catalyzed with the Y73V mutant displayed reversed enantioselectivity compared to the wild-type, resulting in

Table 2 Substrate conversion and enantiomeric excess for the bioepoxidation of 2-vinyl pyridine and 1-phenylcyclohexene using SMO mutants after 4 h reaction.

N

O O

Mutant

Conversion (%)

ee (%)

Conversion (%)

ee (%)

WT Y73F Y73V S96A

57 58 38 64

>99 (S) >99 (S) >99 (S) >99 (S)

7 3 10 4

71 (S,S) 33 (S,S) 60 (R,R) 50 (S,S)

the (R,R)-enantiomer with 60% enantiomeric excess (Table 2), while the epoxidation of styrene, 2-vinylprydine and trans-␤-methyl styrene catalyzed with the same mutant yielded (S)-enantiomers with >99% ee, the same as the wild-type. 4. Discussion Crystal structure-based rational design of proteins focuses on a small number of variants and directly tests the substrates of interest to avoid the screening of a huge number of mutants using substrate analogues. This method has proven to be efficient for the improvement of a variety of enzymatic properties (Bornscheuer and Pohl, 2001; Schmidt et al., 2009; Voigt et al., 2001). In this work, three potentially critical residues in the SMO from Pseudomonas sp. LQ26 were proposed according to structure-based molecular modeling, and three mutants, Y73F, Y73V and S96A, were

Fig. 5. A section of a multiple-sequence alignment of StyA with oxygenases from various resources. The alignment was performed with the program DNAMAN. P. putida, Pseudomonas putida; R. opacus, Rhodococcus opacus; N. farcinica, Nocardia farcinica; A. aurescens, Arthrobacter aurescens; D. acidovorans and Delftia acidovorans are shown. Amino acid residues at positions 73 and 96 are shadowed to show the natural existence of Val73 and Ala96 in several putative SMOs and in one self-sufficient one-component SMO from R. opacus 1CP.

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Fig. 6. Two orientations of 1-phenylcyclohexene docked into the putative active site of SMO (PDB ID: 3IHM). Those orientations are expected to generate the corresponding (S,S)-enantiomer (A) or (R,R)-enantiomer (B). The substrate is shown in the ball and stick mode, and the adjacent amino acid residues within 4 A˚ are shown in the stick mode. The docked conformer was generated using the program Autodock and displayed using the program Pymol. The access of reduced FAD and oxygen is marked with arrows in orange.

identified to exhibit higher activity in styrene epoxidation compared to the wild-type enzyme. Amino acid residues at positions 73 and 96 are the same for all SMOs originating from the genus Pseudomonas, as well as the one from the metagenome (van Hellemond et al., 2007), i.e. Tyr and Ser, respectively. However, it is noteworthy that self-sufficient one-component SMOs have Ile and Ala at the corresponding positions, respectively, which includes the StyA2B from Rhodococcus opacus 1CP (Tischler et al., 2009) and two putative SMOs from Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 (Fig. 5). Furthermore, for several putative SMOs, both the amino acid substitutions Y73V and S96A exist naturally (Fig. 5), indicating that these substitutions have already been explored by natural evolution, although whether they would indicate higher activity in native proteins is correctly unknown. Experimental studies on those putative enzymes might provide more information on the structure–functional relationship of SMOs in the future. Hydrophobic interaction appeared to be critical at position 73. Replacement of the Tyr residue with Phe or Val increased the activity, while loss of the hydrophobic side chain led to significantly impaired activity for the Y73S mutant. In fact, the putative substrate binding site of SMO is completely buried within the protein core, surrounded by more than ten hydrophobic residues and only four hydrophilic residues. The high hydrophobicity is regarded as being consistent with the hydrophobic nature of the substrate styrene (Ukaegbu et al., 2010). On the other hand, the size of the side chain of the residue at position 96 dramatically affects the

enzymatic activity of SMO. The mutant S96T only added an additional methyl group on the side chain compared with the wild-type, but lost most of the enzymatic activity (Fig. 3). The substitution of Ser with Ala was well accepted and resulted in increased activity, while larger residue such as Leu led to a complete loss of activity (Fig. 3). Surprisingly, when the mutants S96T, S96A and S96L were modeled using the SWISS-MODEL version 8.05 (Kiefer et al., 2009) and applied in the automatic docking of styrene, no difference was found in terms of the binding energy, intermolecular energy, internal energy or torsional energy for all three resulting docking complexes compared with the wild-type. Based on the mechanism of this biocatalytic epoxidation reaction, the reactive cavity should accommodate not only styrene, but also the reduced FAD and oxygen, and the binding of substrate is indeed affected by the presence of FAD (Ukaegbu et al., 2010). The docking model where only styrene occupies the cavity could only provide limited information and may not reflect subtle changes in protein structure. It could be hypothesized that the replacement of Ser with larger residues such as Leu might affect FAD binding indirectly or weaken the interaction of reduced FAD with the substrate through the subtle movement of the substrate in the active pocket pushed by the steric hindrance of the side chain. In addition, residues Tyr73 and Ser96 are located at the same domain of the oxygenase subunit of the SMO on the ␤3- and ␤4-sheet, respectively, and are very ˚ Therefore, the amino close to each other with a distance of 3.41 A. acid substitution with large side chain at position 73 might cause

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distortion of the local structure within the limited space, and thus result in major deleterious effects on the catalytic reaction. On the contrary, smaller side chains at either position 73 or 96 may benefit the intermolecular interaction causing higher activity in the mutants Y73V and S96A. However, the double mutant combining the beneficial substitutions of Y73V and S96A did not show cumulative effect, and its enzymatic activity toward styrene remained similar to that of the mutant S96A (data not shown). The result indicates that the S96A mutation might have created enough space for the proper orientation of the ␤-sheets. Therefore, further reduction of the size of the side chain at position 73 would have little beneficial effect. In the majority of cases in this study, the mutations did not affect the enantioselectivity of the enzyme. Therefore, we assumed that the putative active cavity of SMO should be strictly shaped by surrounding residues and the mutations could hardly change the orientation of the substrate. The reversal of enantioselectivity for the mutant Y73V during the epoxidation of 1-phenylcyclohexene was unexpected because all other experimentally assigned SMOs display the same enantioselectivity (Bernasconi et al., 2000; Di Gennaro et al., 1999; Lin et al., 2010; Panke et al., 1998; Park et al., 2005; Tischler et al., 2009; van Hellemond et al., 2005), and the overall structure of the active cavity of SMOs should not be flexible enough to generate complementary enantiomers. Moreover, the mutants from directed evolution were also reported to produce (S)-enantiomers (Gursky et al., 2010). Based on the fact that no reversal of stereoselectivity was observed for the Y73V mutant with the substrates styrene, 2vinylprydine or trans-␤-methyl styrene, we hypothesized that this reversal of stereoselectivity might be due to the structural flexibility of this particular substrate, 1-phenylcyclohexene, which contains a cyclohexenyl group that adopts a half-chair conformation with C2 symmetry. The representative binding modes for 1-phenylcyclohexene into the putative active site of SMO resulting from automated docking are shown (Fig. 6A and B). Unlike styrene or styrene derivatives which displayed distinct preference to one single orientation in automated docking, and often produced enantiopure oxide with >99% ee (Lin et al., 2011b), the symmetric cyclohexenyl group in 1-phenylcyclohexene apparently resulted in much reduced stereoselectivity as the two highest scoring conformers in Fig. 6 were estimated by the program with very similar energy and docking scores. Since the putative substrate binding cavity is located at the bottom of the FAD binding site (Ukaegbu et al., 2010), oxygen and reduced FAD would come from the same direction for both conformers (Fig. 6A and B). Therefore, the (S,S) and (R,R)-enantiomers of the oxide product would be achieved from the two conformers shown in Fig. 6A and B, respectively. The biotransformation results indicated that the conformer in Fig. 6A should be slightly preferred by the wild-type enzyme, leading to the formation of (1S, 2S)-1-phenylcyclohexene oxide with medium enantioselectivity (71%ee). The replacement of tyrosine with valine may have weakened the strong ␲–␲ interactions between the substrate and enzyme by the loss of the phenyl group from the side chain at position 73 and increased the flexibility of the active pocket. In addition, the size of the pocket may have expanded due to the reduced volume of the side chain, which may also facilitate the reversal of enantiomeric preference. However, the active site of the Y73V mutant should not have changed significantly as its selectivity toward styrene, 2-vinylprydine and trans-␤-methyl styrene remained the same as the wild-type. Therefore, the stereoswitch of the Y73V mutant toward 1-phenylcyclohexene was apparently triggered by subtle changes in the protein structure, which only took effect for this particular substrate with a symmetric cyclohexenyl group and having two conformers with similar energy in the automatic docking study.

In conclusion, three amino acid substitutions, Y73F, Y73V, and S96A were identified via a rational design approach to enhance the catalytic efficiency of SMO. These residues are located in the putative active pocket of the enzyme and could possibly interact with the phenyl ring of the native substrate, styrene. One of the mutants, Y73V, displayed reversed enantiomeric preference toward the substrate 1-phenylcyclohexene while retaining the same enantioselectivity toward other substrates. The results extended the knowledge of the structural–sequence relationship of SMOs and demonstrated that structure-based rational design was an efficient approach to altering the characteristics of this enzyme.

Acknowledgements This work was supported by the National Natural Science Foundation of China (20802073 and 21072183), the 100 Talents Program and the West Light Foundation of the Chinese Academy of Sciences, and the Organization for Women in Science for the Developing World (to A.A.Q.A.), formerly known as the Third World Organization for Women in Science.

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