Journal of Biotechnology 161 (2012) 235–241

Contents lists available at SciVerse ScienceDirect

Journal of Biotechnology
journal homepage:

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,∗
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
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
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
© 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: (Z.-L. Wu).
0168-1656/$ – see front matter © 2012 Elsevier B.V. All rights reserved.

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:

and used as standard products. Materials and methods 2.5. Another study on the engineering of the SMO from P. / Journal of Biotechnology 161 (2012) 235–241 Table 1 Primers used in site-directed mutagenesis. and charges and non-polar hydrogen atoms were added using MGLTools 1. coli BL21 E. 2011). Zhang et al. 2. Mutation Oligonucleotide sequencesa Fig. 1976). 2010). pH 7) and stored at 4 ◦ C. 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. putida S12 was available from the PDB database (PDB ID: 3IHM). but not the target substrate styrene (Gursky et al. 2009). For each mutant and the wild-type. the number of AutoDock 4 GA runs was increased from 10 to 50. 2010).5.1 M. The cells were harvested by centrifugation. 2010. The sequences of mutagenic oligonucleotide primers (Table 1) were synthesized by Shanghai Invitrogen Life Technologies. Ukaegbu et al. fluorescens (PDB ID: 1CC4) provides insight into the putative substrate and flavinbinding pockets. and the docking grids were set as 22 × 32 × 22 A˚ for styrene and 28 × 32 × 28 A˚ for 1-phenylcyclohexene..c. 2006. Racemic styrene oxide (1S. 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. In the current work. The SMO from Pseudomonas sp.4 and the results were visualized using the program Pymol. AutoDock 4. crude cell extracts were analyzed by SDS-PAGE. 2-vinylpyridine and 1-phenylcyclohexene were purchased from Alfa Aesar (Tianjin. chain B of the StyA homodimer and all water molecules of SMOA were removed. and one mutant displayed reversed enantioselectivity toward the substrate 1-phenylcyclohexene. LQ26. this method carries the risk of generating mutants with increased activity only toward the analog substrate indigo.. However.. 2010. 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. 2010).4. then the . The 50 independent runs from AD4 were analyzed in MGLTools 1.275. 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. resulting in mutants exhibiting higher rates of epoxide formation (Gursky et al. coli strain BL21(DE3) containing the constructed plasmids was used to produce the wild-type and mutant StyAB2.. a rational design approach was undertaken. Qaed et al. 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.0 was used for docking. Chemicals The substrates styrene. to investigate amino acid substitutions that might enhance the catalytic efficiency of SMO. an 89% identity with the same subunit of the SMO from Pseudomonas sp. its overall structure homology to that of p-hydroxybenzoate hydroxylase (PHBH) from P. 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... Hanzlik et al. Docking studies The X-ray crystal structure of the oxygenase subunit of SMO (SMOA) from P. Other reagents were purchased from general suppliers and were used without further purification. washed twice with potassium phosphate buffer (0.. This strategy led to several SMO mutants with increased enzymatic activities toward styrene. two single colonies were picked and cultivated to make two independent heterologous expressions. La Jolla. To determine the expression levels of the wild-type and mutant enzymes. MO. However. 2. 2. all of those designed mutants display decreased enzymatic activity.1. Single colonies were picked up and grown overnight at 37 ◦ C in LuriaBertani broth containing 50 ␮g kanamycin/ml.. 3IHM) has recently been released without a substrate (Ukaegbu et al. 1998) except that the grid spacing was changed to 0. probably as a consequence of reduced FAD binding affinity.. Louis. Expression of the wild-type and mutant StyAB2 in E. 2. China). CA) using the plasmid pETAB (Lin et al. LQ26. putida CA-3 has been performed by screening an error-prone PCR library using indigo assay. 1967.3. 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. 2R)-1-phenylpropylene oxide were purchased from Sigma–Aldrich (St.2.. The PCR product was treated with 20 U of Dpn I at 37 ◦ C for 2 h and transformed into DH5˛ competent cells. 2011). and the docking parameters were kept to their default values in general (Morris et al. Epoxidation of styrene catalyzed with styrene monooxygenase (SMO).4. Lin et al. USA). 2010) encoding the wild-type StyAB2 (GenBank ID: GU593979) as the template.236 H.. To prepare the structure for docking. 1. 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. Construction of point mutations Site-directed mutagenesis was performed according to the QuikChange® site-directed mutagenesis protocol (Stratagene. trans-␤-methyl styrene. 2011b. and its high homology with other SMOs from the genus of Pseudomonas would facilitate the docking study.. 2S)-1-phenylpropylene oxide and (1R. The successful introduction of the desired mutations was confirmed by sequencing at Shanghai Invitrogen Life Technologies. However.

S) 13. pH 6. the target sites for rational design.67 min) and 2-(oxiran-2yl)pyridine (hexane:2-propane = 95:5. Val and Ala. dried with anhydrous sodium sulfate. or representative hydrophobic residues (for Val and Ala). His76 was replaced with Asn. His76 and Ser96.4. tR (S) 10. .05 min. but contain a polar but uncharged residue (for Asn). Specific epoxidation activities were measured using whole cells following the literatures (Bae et al. a monophasic system without BEHP resulted in better conversion and thus was applied instead of the biphasic system.22 mm ID × 0. 1 ml/min. 2006). and incubated at 30 ◦ C for 10 min before the addition of 1. and residues Arg43. 1999) of 10 ml potassium phosphate buffer (100 mM. Ser96 was replaced with Ala. Substrate is shown in green in the stick mode.5. 0. putida S12 has been released without substrate and FMN. 2.. Leu44. The organic phases were combined. 2006. 2-vinylpyridine or 1phenylcyclohexene) to the corresponding epoxide. Therefore. coli BL21 cells expressing the wild-type and mutants with a cell dry weight (CDW) of 0. Val and Ser. The figure was generated using the program Autodock and displayed using the program Pymol. Briefly. but retains part of the steric hindrance. His76 and Ser96 were modified using site-directed mutagenesis to investigate their effects on the enzymatic activity and enantioselectivity. and thus commonly act as Fig. 3. It is well recognized that residues adjacent to the substrate play a critical role in catalytic activity.1 g cell dry weight (CDW) resuspended in a biphasic system of 10 ml potassium phosphate buffer (100 mM.5 ml/min. Based on the putative substrate-binding center of SMOA as well as our previous work. or Chiralcel OD-H column for 1-phenylcyclohexene oxide (hexane:2-propane = 99:1. 3. The reaction was carried out at 30 ◦ C for 4 h with 0. One unit (U) is defined as the activity that produces 1 ␮mol of oxide per min. Ser96 and Tyr73 are shown in sky blue.. 11. tR (S) 4. For 2-vinyl pyridine. Results The SMOA structure from P.1 mM dodecane as an internal standard and analyzed with gas chromatography (GC).R) 12. the native substrate styrene was docked into SMOA using AutoDock 4. and Val lacks both the hydroxyl group and the aromatic structure. Hercules. residues Tyr73. Chiralcel AD-H column for 2-methyl-3-phenyloxirane (hexane:2-propanol = 90:10. 2010. Beijing.5 g CDW/L were resuspended in 4 ml potassium phosphate buffer (100 mM. His76 and Ser96 are away from the putative FAD binding channel.83 min). pH 6. Biotransformation with whole cells and product analysis The harvested recombinant E. The reaction was carried out at 30 ◦ C for 4 h with shaking at 230 rpm and terminated by extraction with ether. CA.67 min)..5 ml/min. Residues His76. Panke et al.0. and Leu and Ala lack the hydroxyl group and are representative hydrophobic residues with varied side chain sizes. Australia) to determine the conversion of each substrate (styrene.H. residues Tyr73. and subjected to GC and chiral HPLC analysis. concentrated under vacuum.5) containing 10% (v/v) bis-(2-ethylhexyl) phthalate (BEHP) with the addition of 10 mg styrene or 1-phenylcyclohexene. Thr retains the hydroxyl group with an additional methyl group. pH 7. Compared with Ser.. 2008. Ser lacks the aromatic structure while retaining the hydroxyl group. 2. Phe lacks the hydroxyl group while retaining the aromatic structure.48 A. Fig. 2010). tR (R) 15.15. The reaction was continued for 5 min. 0. Lin et al. Activities were normalized as percentages of the activity of the wild-type. tR (R. unlike the residues 43–46. recombinant E. coli BL21 cells with 0.14 min. Biotransformation of styrene by the wild-type and mutant StyAB2.5) containing 10% (v/v) bis-(2-ethylhexyl) phthalate (BEHP) with the addition of 10 mg styrene. 0. In addition. The amino acid residues Tyr73. Tyr73 was replaced with Phe. 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. GC analysis was performed on a Fuli 9790 II system connected to a flame ionization detector using column BP5 (30 m × 0. 2). which form the bottom of the substrate-binding pocket.5 mM substrate (30 mM stock solution of styrene or trans-␤methyl styrene in ethanol). The returned 50 results were analyzed in the MGL tools 1. tR (R) 4. all of which lack the electronically charged side chain. Each column represents the mean (SD) of triplicate assays. tR (S) 15. Park et al. Then. Compared with Tyr.78 min). Ukaegbu et al. were found to be adjacent to the benzene ring of styrene with their side chains facing the ˚ respectively substrate with distances of 7. 2010).93 and 9. (Fig. Enantiomeric excesses were determined using chiral HPLC on a Shimadzu LC 20-AD (Shimadzu.27 min.. Japan) with a PDA detector using Daicel Chiralpak AS-H column for styrene oxide (hexane:2-propanol = 90:10. The mixture was extracted with ether containing 0. which has shown the residues 43–46 being close to the ␣-substitute of ˛-ethylstyrene. the purified proteins were analyzed by SDS-PAGE. tR (S. SGE Analytical Science. 2. trans-␤-methyl styrene. and the highest scoring conformer with the vinyl group of styrene adjacent to the residues 43–46 was shown in Fig. Orientation of styrene docked into the putative active site of SMO (PDB ID: 3IHM). USA) as described previously (Lin et al. The protein content was determined with a commercial BCA Protein Assay kit using bovine serum albumin as a standard (Beyotime.25 ␮m film thickness. tR (R) 10.1 g were resuspended in a biphasic system (Lin et al. China). Leu and Thr.53 min.5. Leu45 and Asn46 are shown in purple in the stick mode.5 ml/min... / Journal of Biotechnology 161 (2012) 235–241 237 oxygenase subunit was purified using Profinity IMAC Ni-Charged resin (Bio-Rad. which would avoid negative impacts on catalytic activity caused by reduced FAD binding (Feenstra et al.0) containing glucose (5 g/L).

farcinica. yielding the product (S)-styrene oxide with >99% ee. 3). three potentially critical residues in the SMO from Pseudomonas sp. Y73V and S96A exhibited higher activities than the wild-type.S) 33 (S. The specific epoxidation activities of the most active mutant S96A toward styrene and trans-␤-methyl styrene were 2. respectively (Fig. In this work. For substrates 2-vinyl pyridine and 1-phenylcyclohexene..5 U/g CDW. Y73V and S96A.0) containing glucose (5 g/L) and 1. 2006). N.. 4) without any negative impact on their enantioselectivities. 2001. 4). 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. and three mutants. the changes in activities were varied for the mutants Y73F. The results confirmed the increased enzymatic activity of mutants Y73F. The reaction was carried out at 30 ◦ C for 5 min with 0.. such as that from Pseudomonas sp. respectively (Table 2). Each column represents the mean (SD) of triplicate assays. D. Y73V and S96A. Voigt et al.R) 50 (S.. Y73V and S96A for the epoxidation of styrene. 2009. 2010... Interestingly.R)-enantiomer with 60% enantiomeric excess (Table 2).5 g CDW/L resuspended in 4 ml potassium phosphate buffer (100 mM. The alignment was performed with the program DNAMAN. which was comparable to that of the other SMOs measured under similar conditions. Pseudomonas putida. P. as well as for trans␤-methyl styrene (Fig. 2001). The best mutant S96A displayed a relative activity of 180% (Fig. Specific epoxidation activities of the wild-type and mutant StyAB2 toward styrene and trans-␤-methyl styrene. putida. All the constructed mutants listed in Fig.. while other mutants showed lower activities or even major deleterious effect (Fig. opacus 1CP. Y73F. Their activities were examined in the epoxidation of styrene in the biphasic reaction for 4 h (Lin et al. were Fig. opacus.. acidovorans and Delftia acidovorans are shown. putida SN1 (55 ± 5 U/g CDW) (Panke et al. Lin et al. R. Only S96A and Y73V displayed slight increases in the 2-vinyl pyridine and 1-phenylcyclohexene conversions. Mutants Y73F. 2-vinylprydine and trans-␤-methyl styrene catalyzed with the same mutant yielded (S)-enantiomers with >99% ee. 3). while the epoxidation of styrene. 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. This method has proven to be efficient for the improvement of a variety of enzymatic properties (Bornscheuer and Pohl. 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. Rhodococcus opacus.5 mM substrate. the same as the wild-type. LQ26 were proposed according to structure-based molecular modeling.6 and 2. / Journal of Biotechnology 161 (2012) 235–241 Fig. A.S) the (R. Arthrobacter aurescens. 1999). Nocardia farcinica. 3 were functionally expressed in E. coli at a level similar to the wild-type. Park et al. 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. 1998.238 H. 2006). VLB120 (79 ± 5 U/g CDW) and P. 4. 5. the asymmetric epoxidation of 1-phenylcyclohexene catalyzed with the Y73V mutant displayed reversed enantioselectivity compared to the wild-type. The whole cell specific epoxidation activity of the wild-type StyAB2 was 66. Park et al. Panke et al. Schmidt et al. A section of a multiple-sequence alignment of StyA with oxygenases from various resources. 4.S) 60 (R. pH 7. . 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. aurescens.3-fold of the wild-type. All active mutants retained excellent enantioselectivity.

it is noteworthy that self-sufficient one-component SMOs have Ile and Ala at the corresponding positions. surrounded by more than ten hydrophobic residues and only four hydrophilic residues. while loss of the hydrophobic side chain led to significantly impaired activity for the Y73S mutant. respectively.. the putative substrate binding site of SMO is completely buried within the protein core. 5). as well as the one from the metagenome (van Hellemond et al. and the binding of substrate is indeed affected by the presence of FAD (Ukaegbu et al. In fact. 6. when the mutants S96T.H. indicating that these substitutions have already been explored by natural evolution. 2010). 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. the amino close to each other with a distance of 3. The docked conformer was generated using the program Autodock and displayed using the program Pymol. The mutant S96T only added an additional methyl group on the side chain compared with the wild-type. Two orientations of 1-phenylcyclohexene docked into the putative active site of SMO (PDB ID: 3IHM). 3). while larger residue such as Leu led to a complete loss of activity (Fig. and the adjacent amino acid residues within 4 A˚ are shown in the stick mode. but lost most of the enzymatic activity (Fig. The substitution of Ser with Ala was well accepted and resulted in increased activity.and ␤4-sheet. but also the reduced FAD and oxygen.. and are very ˚ Therefore. for several putative SMOs. 2009) and two putative SMOs from Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 (Fig.41 A. although whether they would indicate higher activity in native proteins is correctly unknown.. The docking model where only styrene occupies the cavity could only provide limited information and may not reflect subtle changes in protein structure. Based on the mechanism of this biocatalytic epoxidation reaction. 2007). acid substitution with large side chain at position 73 might cause . identified to exhibit higher activity in styrene epoxidation compared to the wild-type enzyme. which includes the StyA2B from Rhodococcus opacus 1CP (Tischler et al.S)-enantiomer (A) or (R. the reactive cavity should accommodate not only styrene. The access of reduced FAD and oxygen is marked with arrows in orange. Hydrophobic interaction appeared to be critical at position 73.. respectively. residues Tyr73 and Ser96 are located at the same domain of the oxygenase subunit of the SMO on the ␤3. / Journal of Biotechnology 161 (2012) 235–241 239 Fig. 2009) and applied in the automatic docking of styrene. internal energy or torsional energy for all three resulting docking complexes compared with the wild-type. Experimental studies on those putative enzymes might provide more information on the structure–functional relationship of SMOs in the future. Furthermore. Surprisingly. 5). i. Amino acid residues at positions 73 and 96 are the same for all SMOs originating from the genus Pseudomonas. 2010). Those orientations are expected to generate the corresponding (S. However. S96A and S96L were modeled using the SWISS-MODEL version 8. 3). no difference was found in terms of the binding energy. both the amino acid substitutions Y73V and S96A exist naturally (Fig. intermolecular energy. the size of the side chain of the residue at position 96 dramatically affects the enzymatic activity of SMO. respectively.R)-enantiomer (B).05 (Kiefer et al. On the other hand. Replacement of the Tyr residue with Phe or Val increased the activity.. Tyr and Ser.e. In addition. The substrate is shown in the ball and stick mode. The high hydrophobicity is regarded as being consistent with the hydrophobic nature of the substrate styrene (Ukaegbu et al. Lin et al.

3206–3216. Y. / Journal of Biotechnology 161 (2012) 235–241 distortion of the local structure within the limited space. A. the double mutant combining the beneficial substitutions of Y73V and S96A did not show cumulative effect.. J. the mutations did not affect the enantioselectivity of the enzyme. New York. 1999. the stereoswitch of the Y73V mutant toward 1-phenylcyclohexene was apparently triggered by subtle changes in the protein structure.. Bordoli.. Di Gennaro. Z. A. Shin. Nature of the reaction intermediates in the flavin adenine dinucleotide-dependent epoxidation mechanism of styrene monooxygenase. Feenstra. Sello.R)-enantiomers of the oxide product would be achieved from the two conformers shown in Fig.. oxygen and reduced FAD would come from the same direction for both conformers (Fig. and the overall structure of the active cavity of SMOs should not be flexible enough to generate complementary enantiomers. S. Kuhn.S) and (R.. A. Nallamothu... LQ26: effects of ␣. Applied and Environmental Microbiology 65. Liu. Improved biocatalysts by directed evolution and rational protein design. Journal of the American Chemical Society 98. H... Y. Vermeulen. The representative binding modes for 1-phenylcyclohexene into the putative active site of SMO resulting from automated docking are shown (Fig. 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.. Wu.. 1999.. 2010). three amino acid substitutions. P.. J. K. 2011a. Heinzle. E. Bioconversion of substituted styrenes to the corresponding enantiomerically pure epoxides by a recombinant Escherichia coli strain. 2001.. Bestetti. Commandeur.. Lin. 2011b). T.. 2008... Bosch... respectively. the 100 Talents Program and the West Light Foundation of the Chinese Academy of Sciences.L. G. Lin et al. S.. A new biocatalyst for production of optically pure aryl epoxides by styrene monooxygenase from Pseudomonas fluorescens ST.. Fieser. 2010.. Kiefer. S.Q.F. Fieser.. A. Biochemistry 50. F.T. 2011.. E. However. Michaely. Buhler. Lee. B. G. Schmid.. 2006.. 2794–2797.. M.Q. On the contrary. Since the putative substrate binding cavity is located at the bottom of the FAD binding site (Ukaegbu et al. 2004. Applied Microbiology and Biotechnology 85.T.. L. Lin. formerly known as the Third World Organization for Women in Science.. Kantz. Z. the (S. K. Based on the fact that no reversal of stereoselectivity was observed for the Y73V mutant with the substrates styrene. N.G. 102–116. In addition.... Y. 2S)-1-phenylcyclohexene oxide with medium enantioselectivity (71%ee).. A. Mechanism of flavin transfer and oxygen activation by the two-component flavoenzyme styrene monooxygenase. Construction and characterization of a recombinant whole-cell biocatalyst of Escherichia coli expressing styrene monooxygenase under the control of arabinose promoter.-L. N.. Galli. 6A should be slightly preferred by the wild-type enzyme. smaller side chains at either position 73 or 96 may benefit the intermolecular interaction causing higher activity in the mutants Y73V and S96A. 1603–1606. and its enzymatic activity toward styrene remained similar to that of the mutant S96A (data not shown).240 H. Z.. and S96A were identified via a rational design approach to enhance the catalytic efficiency of SMO. Nguyen.. 2vinylprydine or trans-␤-methyl styrene.). These residues are located in the putative active pocket of the enzyme and could possibly interact with the phenyl ring of the native substrate. 1998.T. Ronco.E. Unlike styrene or styrene derivatives which displayed distinct preference to one single orientation in automated docking. 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. Wang. H.. 1976. E. Wu. Characterization of styrene catabolic pathway in Pseudomonas fluorescens ST. D387–D392. Jeong. Ahmed.. Bernasconi. Journal of Molecular Catalysis B: Enzymatic 67. L. J... 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. 2005. Wu. G. which contains a cyclohexenyl group that adopts a half-chair conformation with C2 symmetry.P. In the majority of cases in this study.A. Di Gennaro. Asymmetric epoxidation of styrene derivatives by styrene monooxygenase from Pseudomonas sp. Lee... Enzymic hydration of [18O]epoxides. Gassner.. Künzli... Therefore. 2009. E. Acknowledgements This work was supported by the National Natural Science Foundation of China (20802073 and 21072183). Colmegna. References Bae. Tetrahedron: Asymmetry 22. Panke et al. Gursky. G. L. Park. The biotransformation results indicated that the conformer in Fig.. and thus result in major deleterious effects on the catalytic reaction. Sello.. 2610–2612.. F. 2-vinylprydine and trans-␤-methyl styrene remained the same as the wild-type. 2011c. In vitro evolution of styrene monooxygenase from Pseudomonas putida CA-3 for improved epoxide synthesis. Feenstra. Liu. O’Connor.J. 1967. Di Gennaro. Therefore. 815–827. Arnold. and the Organization for Women in Science for the Developing World (to A. 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. 6A and B). Qiao. the active site of the Y73V mutant should not have changed significantly as its selectivity toward styrene.and enantio-selective epoxidation of secondary allylic alcohols catalyzed by styrene monooxygenase. International Biodeterioration & Biodegradation 54. Therefore. Bestetti. Gassner. U. further reduction of the size of the side chain at position 73 would have little beneficial effect. Liu.E. K.. Current Opinion in Chemical Biology 5. Y73V.. Y73F.-B. G. F. Bernasconi. S.M. Role of nucleophilic mechanisms. 2005. A.A. Z.. 6 were estimated by the program with very similar energy and docking scores. and often produced enantiopure oxide with >99% ee (Lin et al.. 523–532.. Tetrahedron: Asymmetry 15. 6A and B. we hypothesized that this reversal of stereoselectivity might be due to the structural flexibility of this particular substrate.A. 2010). 77–89. 2000. F. Schwede..W. Chemical Communications 47. Kholiq.P. Moreover. Journal of Molecular Catalysis B: Enzymatic 72. the size of the pocket may have expanded due to the reduced volume of the side chain. T. 183–187. I. G. 2010... Lin et al. which may also facilitate the reversal of enantiomeric preference. W. S. ... Galli. 137–143.... Orsini.M. D. Tetrahedron Letters 41.. leading to the formation of (1S... 2011b. Green Chemistry 12. Wu.. Colmegna. Pelizzoni... 69–76.. 2005).. 134–137.. 2010. H. Schmid.. G. 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.... H. 1952–1955. Bestetti. R. styrene. K. However. Orsini. Galli. K. Therefore. Park et al. The result indicates that the S96A mutation might have created enough space for the proper orientation of the ␤-sheets. Enantioselective substrate binding in a monooxygenase protein model by molecular dynamics and docking. Raj. In conclusion.. G. R. Lin. 1-phenylcyclohexene. M. H. Biophysical Journal 91. M. displayed reversed enantiomeric preference toward the substrate 1-phenylcyclohexene while retaining the same enantioselectivity toward other substrates. J. Y73V.. M. G. Nucleic Acids Research 37. S. Liu. Intensification and economic and ecological assessment of a biocatalytic oxyfunctionalization process. M.A.. 9157–9161. the mutants from directed evolution were also reported to produce (S)-enantiomers (Gursky et al. P. Sello. Colmegna. J. the symmetric cyclohexenyl group in 1-phenylcyclohexene apparently resulted in much reduced stereoselectivity as the two highest scoring conformers in Fig. Sello. The SWISS-MODEL Repository and associated resources. 2004. Highly diastereo. Tischler et al. 236–241. 995–1004.J. A. Y. LQ26 catalyzes the asymmetric epoxidation of both conjugated and unconjugated alkenes. G. Lin. P. Nikodinovic-Runic. Pohl.-L..A. Hofstetter.N.. 2000. S. van Hellemond et al. Chin. Hanzlik. Archives of Biochemistry and Biophysics 442. Kantz. 2010. Scott.. One of the mutants.and ␤substituents... Edelman. Biotechnology and Bioprocess Engineering 13. 6A and B). Wiley. 2009... Biocatalysis as an alternative for the production of chiral epoxides: a comparative review. Di Gennaro et al. Reagents for Organic Synthesis.-L..... Bornscheuer. F.-Y. Bacterial monooxygenase mediated preparation of nonracemic chiral oxiranes: study of the effects of substituent nature and position. A. Styrene monooxygenase from Pseudomonas sp.

C. van Hellemond. 2032–2043. J. M. M. Ukaegbu. Witholt. 5619–5623. Characterization of styrene catabolic genes of Pseudomonas putida SN1 and construction of a recombinant Escherichia coli containing styrene monooxygenase gene for the production of (S)-styrene oxide.H. 1678–1688.. Rothlisberger. B. Schlomann. Janssen. Journal of Biotechnology 139. E. Discovery of an epoxide forming monooxygenase from the metagenome.. Ward.. Kaschabek. Imae. Lee. S. 1998. Gassner. Lee.. G.. H.S. J.G. M.. Bae. 2001. Journal of Biotechnology 118. S.. T. Lee. W. Kaschabek. D... Journal of Computational Chemistry 19. K. A... J. Schmid. Bornscheuer.H. 2009. H.. 2004.D. (Ed. S. Identification of amino acid residues involved in 4-chloroindole 3-hydroxylation by cytochrome P450 2A6 using screening of random libraries..R. H. Held... U. 1998. Witholt.. J.S... Wu. Witholt. Panke. 1639–1662. Advanced Synthesis and Catalysis 343. Journal of Bacteriology 191... pp.. Hofstetter.J. Han. D. strain VLB120. and perspectives for biotechnological applications. R.. 2009. W. Liu.J. B.. 2006.. Witholt. Applied and Environment Microbiology 73. Eulberg. 2005. Z. J.. Lin... Kaiser. 241 Schmid. Park. Toda...L.. Discovery of a novel styrene monooxygenase originating from the metagenome.W.G.. Park. Groning. S... B. O’Connor.. Otto... Protein engineering of carboxyl esterases by rational design and directed evolution.R. In: Arnold. 12–18. So M. Schmidt. Identification of a novel self-sufficient styrene Monooxygenase from Rhodococcus opacus 1CP. H.B. E. 5220–5227..A. Olson. K. 1162–1171. Towards a biocatalyst for (S)styrene oxide production: characterization of the styrene degradation pathway of Pseudomonas sp. vol. Yoo. K. Feiten.... Wubbolts. S131.T. van Berkel. 2006.T.G. Rational design of styrene monooxygenase mutants with altered substrate preference... S.. F. Hollmann. R.E. Qaed..Y. Wang. Microbial degradation of styrene: biochemistry.. Applied Microbiology and Biotechnology. D. A...-G. 1999. M. Journal of Chemical Engineering 22.. ..J. A... / Journal of Biotechnology 161 (2012) 235–241 Mooney... Schlomann. Lakner... ST-5 and ST-10 for synthesizing enantiopure (S)-epoxides.. F.S... Komio.H. D. Matse. Structure and ligand binding properties of the epoxidase component of styrene monooxygenase. 5292–5302. Wubbolts.. Schmid. A..B. Applied and Environmental Microbiology 65.F. de Lorenzo. Halliday. Z.. Fraaije. Pilot-scale production of (S)-styrene oxide from styrene by recombinant Escherichia coli synthesizing styrene monooxygenase. Han. Lin et al. Korean. F. Hofstetter. B.C. Park. E. A..E.M.. Beaton. S. Panke. E. Rational evolutionary design: the theory of in vitro protein evolution.). Kauffman. Z. A.W. 732–737. U. Hart.S.H.. 33–41. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function.G. Biotechnology Letters 33. A. M.. Biochemical characterization of StyAB from Pseudomonas sp strain VLB120 as a two-component flavin-diffusible monooxygenase. D.W.. Guengerich. Applied Microbiology and Biotechnology 72. D. Protein and Peptide Letters 16.. Evolutionary Protein Design. Wubbolts.A... B.. G. 2010. M.B. van Berkel. Goodsell. D.G. 611–616. 4996–5009. Degradation of styrene by a new isolate Pseudomonas putida SN1. Biotechnology and Bioengineering 80. M. Wu. R.. M... Voigt. A. S. 2002.. Panke..G. 79–160. Y. Bottcher.. Rosenzweig. Journal of Microbiology and Biotechnology 16..W. D. Kantz. V. M. Park. Journal of Bacteriology 192. Biochemistry 49. Advances in Protein Chemistry. Integrated biocatalytic synthesis on gram scale: the highly enantio selective preparation of chiral oxiranes with styrene monooxygenase. M. Engineering of a stable whole-cell biocatalyst capable of (S)-styrene oxide formation for continuous two-liquid-phase applications. M. 1007/s00253-011-3849-3.R.H. doi:10.. Kermer. StyA1 and StyA2B from Rhodococcus opacus 1CP: a multifunctional styrene monooxygenase system. 5832–5839. K. Witholt. 2012. 1–10. Tischler. J. Itoh. Janssen.. Journal of Bacteriology 186.. P. Schmid. A. Tang. 418–424. Zhang. 2005.K.. 2011..H..A. Tischler. N. S. Morris. M. Lee. Z. 2009.. W. S.. S. A.Y. van Hellemond.J.. 1032–1040.H.-L. 2001.P. Ruth. Applied and Environment Microbiology 64..W. S. 2007. molecular genetics. Fraaije. Chen. 2010. S. Expression and characterization of styrene monooxygenases of Rhodococcus sp.. 55.