Journal of Biotechnology 161 (2012) 235241

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

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 rst 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 specic 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 avoprotein 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 ne chemical industry,
the development of efcient 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: wuzhl@cib.ac.cn (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
insufcient 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 uorescens 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 identication 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-specic oxygenase subunit of the SMO from Pseudomonas putida S12 (PDB ID:

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H. Lin et al. / Journal of Biotechnology 161 (2012) 235241

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. uorescens (PDB
ID: 1CC4) provides insight into the putative substrate and avinbinding pockets, which has assisted the search for mutants with
altered substrate specicity 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 4346 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 afnity, because residues 4346 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 avin 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 efciency 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
SigmaAldrich (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 purication.
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

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 conrmed 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 Terric Broth (200 ml) containing 50 g kanamycin/ml in a 500 ml ask 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

H. Lin et al. / Journal of Biotechnology 161 (2012) 235241

237

oxygenase subunit was puried using Pronity IMAC Ni-Charged

resin (Bio-Rad, Hercules, CA, USA) as described previously (Lin et al.,
2010). Then, the puried 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.
Specic epoxidation activities were measured using whole cells
following the literatures (Bae et al., 2008; Park et al., 2006). Briey,
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 dened as the activity that produces 1 mol
of oxide per min.
GC analysis was performed on a Fuli 9790 II system
connected to a ame ionization detector using column BP5
(30 m 0.22 mm ID 0.25 m lm 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 gure was generated using the program Autodock
and displayed using the program Pymol.

the target sites for rational design. In addition, unlike the residues
4346, 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 modied 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
reect 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 4346 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 4346 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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H. Lin et al. / Journal of Biotechnology 161 (2012) 235241

Fig. 4. Specic 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).
Specic 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
conrmed 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 specic 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 specic 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.

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 efcient 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-sufcient one-component
SMO from R. opacus 1CP.

H. Lin et al. / Journal of Biotechnology 161 (2012) 235241

239

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.

identied 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-sufcient 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
structurefunctional 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 signicantly
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 reect 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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H. Lin et al. / Journal of Biotechnology 161 (2012) 235241

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 benet the intermolecular interaction causing higher activity in the
mutants Y73V and S96A. However, the double mutant combining
the benecial 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
benecial 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 exible 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 exibility 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 exibility 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 signicantly 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 identied via a rational design approach to enhance
the catalytic efciency 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 structuralsequence relationship of
SMOs and demonstrated that structure-based rational design was
an efcient 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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