Enhancement of the enzymatic activity of escherichia coli acetyl esterase by a double mutation obtained by random mutagenesis

Article abstract of DOI:10.1271/bbb.120430

A double mutant of Escherichia coli acetyl esterase (EcAE) with enhanced enzymatic activity was obtained by random mutagenesis using error-prone PCR and screening for enzymatic activity by observing halo formation on a tributyrin plate. The mutant contain

Full text of DOI:10.1271/bbb.120430

Biosci. Biotechnol. Biochem., 76 (11), 2082–2088, 2012
Enhancement of the Enzymatic Activity of Escherichia coli Acetyl Esterase
by a Double Mutation Obtained by Random Mutagenesis
y
1;
Ryuichi KOBAYASHI,1 Nobutaka HIRANO,1 Shigenori KANAYA,2 and Mitsuru HARUKI
1Department of Chemical Biology and Applied Chemistry, College of Engineering, Nihon University,
Koriyama, Fukushima 963-8642, Japan
2Department of Material and Life Science, Graduate School of Engineering, Osaka University,
2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Received June 6, 2012; Accepted August 20, 2012; Online Publication, November 7, 2012
[doi:10.1271/bbb.120430]
A double mutant of Escherichia coli acetyl esterase
(EcAE) with enhanced enzymatic activity was obtained
by random mutagenesis using error-prone PCR and
screening for enzymatic activity by observing halo
formation on a tributyrin plate. The mutant contained
Leu97Phe (L97F) and Leu209Phe (L209F) mutations.
Single mutants L97F and L209F were also constructed
and analyzed for kinetic parameters, as well as double
mutant L97F/L209F. Kinetic analysis using p-nitro-
phenyl butyrate as substrate indicated that the kcat
values of L97F and L97F/L209F were larger than that of
the wild-type enzyme, by 8.3-fold and 12-fold respec-
tively, whereas no significant change was observed in the
kcat value of L209F. The Km values of L209F and L97F/
L209F were smaller than that of the wild-type enzyme,
by 2.9-fold and 2.4-fold respectively, whereas no signifi-
cant change was observed in the Km value of L97F. These
results indicate that a combination of an increase in kcat
values due to the L97F mutation and a decrease in
Km value due to the L209F mutation renders the kcat/Km
value of the double mutant enzyme 29-fold higher than
that of the wild-type enzyme.
EcAE is encoded by the aes (ybaC) gene, and is
composed of 319 amino acid residues.3,4) It has been
identified as a member of the hormone-sensitive lipase
(HSL) family.5) The biochemical properties and thermo-
stabity of EcAE have also been extensively studied.
EcAE exists in a monomeric form and hydrolyzes
substrates with an acyl chain length of less than 8.5)
A
site-directed mutagenesis study has identified Ser165,
Asp262, and His292 as the catalytic triad of the
enzyme.6) It has been found that EcAE has an unusual
resistance to thermal denaturation, displaying a denatu-
ration temperature of 68 ꢀC7) and an optimal temperature
at 65 ꢀC with p-nitrophenyl butanoate as substrate.8)
Mutant T74A with increased thermostability was dis-
covered using a random mutagenesis approach.8) We
have obtained mutant R48S with enhanced enzymatic
activity, and we proposed that the R48S mutation not
only facilitates the release of the fatty acid product due
to a loss of interaction between the positive charge of
Arg and the negative charge of the fatty acid product,
but also relieves the steric hindrance caused by the
alcohol moiety of the substrate.2)
In this study, we aimed to obtain further mutations that
enhance the enzymatic activity of EcAE by random
mutagenesis followed by screening based on larger halo
formation. A double mutation, Leu97Phe/Leu209Phe
(L97F/L209F), was found by the screening, and single
mutations L97F and L209F were individually introduced
into EcAE. Kinetic analyses of the mutant enzymes harbor-
ing these double and single mutations suggested that the
increase in kcat value and the decrease in Km value
observed for the double mutant are to be attributed to the
L97F mutation and the L209F mutation respectively.
Key words: Escherichia coli acetyl esterase; enzymatic
activity; screening; random mutagenesis
Enhancement of the enzymatic activities of esterases
and lipases, which catalyze the hydrolysis and trans-
esterification of fatty acid esters, is of great significance,
because these enzymes are most frequently used in
organic synthesis as biocatalysts.1) Random mutagenesis
followed by functional screening is employed in
exploring mutations that enhance enzymatic activity.
Lipases and esterases can easily be screened for hydro-
lytic activity by observing halo formation on a tributyrin
plate. We subjected Escherichia coli acetyl esterase
(EcAE) to such screening, and found that the Arg48Ser
(R48S) mutation enhanced the enzymatic activity of the
enzyme, possibly by improving both catalytic efficiency
and substrate binding.2) Hence accumulation of such
information by screening of further mutations should be
helpful in the development of general methods of
improving the enzymatic activity of lipase/esterase
family enzymes.
Materials and Methods
Cells and plasmids. Plasmid pDR319, which contains wild-type
ybaC genes under the control of the tac promoter and was used in both
screening and overexpression, was constructed previously.5) Compe-
tent cells of E. coli JM109 (recA1, endA1, gyrA96, thi, hsdR17,
SupE44, relA1, ꢀꢁ, ꢀ(lac-ProAB)/F0, traD36, ProAþBþ, lacIqZꢀM15)
were obtained from Toyobo (Osaka, Japan). The cells were grown in
Luria-Bertani (LB) medium9) containing 100 mg/L of ampicillin.
Restriction enzymes, Taq DNA polymerase, and T4 DNA ligase were
from New England Biolabs (Ipswich, MA).
y
To whom correspondence should be addressed. Tel: +81-24-956-8794; Fax: +81-24-956-8862; E-mail: haruki@chem.ce.nihon-u.ac.jp
Abbreviations: EcAE, Escherichia coli acetyl esterase; StAE, Salmonella typhimurium acetyl esterase; HSL, hormone-sensitive lipase; EST2,
Alicyclobacillus acidocaldarius esterase 2

Enhancement of Enzymatic Activity of E. coli Acetyl Esterase
2083
Mutagenesis and screening. To avoid the R48S mutation, which
was found in our previous study,2) a random mutation was introduced
in the region following Arg48 as below. A XhoI site was silently
introduced into the sequence encompassing amino acid residues 46–47
of the ybaC gene in pDR319 with a QuikChange II Site-Directed
Mutagenesis kit (Stratagene, La Jolla, CA) using a 50-primer, 50-
CGACAGTATTACACGCTCGAGCGCCGATTCTGGAATGC-30, and
a 30-primer, 50-GCATTCCAGAATCGGCGCTCGAGCGTGTAATA-
CTGTCG-30 to construct pDR319XhoI. In these sequences, the
position of the XhoI site is underlined and the nucleotides encoding
Arg48 are shown in italics. Random mutagenesis of the ybaC gene was
carried out by PCR with modifications to increase the frequency of
misincorporation of nucleotides,10) with 1 mM dNTP, 10 mM MgCl2,
0.25 U Taq DNA polymerase, and a 0.25 nM template. The ybaC gene
in pDR319 was amplified using a 50-primer, 50-CGCGCCTCGAG-
CGCCGATTCTGGAATGC-30, and a 30-primer, 50-CCGTCGACTT-
AAAGCTGAGCCGTAAAGAAC-30. In these sequences, the positions
of the XhoI and SalI sites are underlined. After digestion of the PCR
product with XhoI and SalI, the resulting 1-kbp DNA fragment was
ligated to the large XhoI-SalI fragment of pDR319XhoI. The resulting
pDR319XhoI derivatives were used to transform E. coli JM109.
Colonies were grown on a plate of an LTB-agar medium (LB
supplemented with 1%v/v tributyrin, 0.1%v/v Tween 80, 100 mg/L of
ampicillin, 1 mM isopropyl ꢁ-D-thiogalactopyranoside, and 1.5% agar)
at 37 ꢀC. Plasmid DNAs were isolated from colonies that gave larger
haloes, and were used in further sequencing. The DNA sequences were
determined by the dideoxy-chain termination method with fluorescent
dye terminators using an ABI PRISMꢀ 310 DNA sequencer (Life
Technologies Japan, Tokyo). Leu97Phe (L97F) and Leu209Phe
(L209F) mutations were introduced individually into the ybaC gene
in pDR319XhoI with a QuikChange II Site-Directed Mutagenesis kit
(Stratagene, La Jolla, CA) using following primers: for the L97F
mutation, a 50-primer, 50-CATGGAGGCGGTTTTATTTTCGGCAA-
TCTCGATAC-30, and a 30-primer, 50-GTATCGAGATTGCCGAA-
AATAAAACCGCCTCCATG-30, and for the L209F mutation, a 50-
primer, 50-GTGACTCGTCGTCTGTTTGGCGGTGTCTGGGATGG-30,
and a 30-primer, 50-CCATCCCAGACACCGCCAAACAGACGAC-
GAGTCAC-30. In these sequences, the nucleotides encoding Phe97
and Phe209 are underlined.
residual activities were determined using p-nitrophenyl butyrate as
substrate.
Protein concentration. The protein concentration of EcAE was
determined from the UV absorption. We used an A
0:1%
280
value of 1.60,
determined by amino acid analysis of the wild-type protein,5) and a
molecular weight of 36,000, calculated from the protein sequence.
Circular dichroism. Far-UV CD spectra (200–260 nm) and near-UV
CD spectra (250–320 nm) were measured on a J-805 Spectropolarim-
eter (Japan Spectroscopic, Tokyo, Japan). Spectra were obtained at
25 ꢀC using solutions containing the protein at 0.2 mg/mL and 1 mg/
mL in 10 mM Tris–HCl buffer (pH 7.5) containing 1 mM EDTA in cells
with optical paths of 2 mm and 10 mm for the far-UV and near-UV CD
spectra respectively. The mean residue ellipticity, [ꢂ], which has a unit
of degꢂcm2ꢂdmolꢁ1, was calculated using an average amino acid
molecular weight of 110.
Structural model of EcAE. A model of the three-dimensional (3-D)
structure of EcAE was constructed by comparative protein modeling
with the program SwissModel12–14) with the acetyl esterase from
Salmonella typhimurium (PDB accession no. 3GA7) as template,2) and
was visualized with program MolFeat (FiatLux, Tokyo, Japan).
Results and Discussion
Screening of EcAE mutants with enhanced enzymatic
activity
In our previous study, we identified the Arg48Ser
(R48S) mutation that enhances the enzymatic activity of
EcAE by random mutagenesis, followed by screening on
a tributyrin plate.2) In the present study, random muta-
genesis was performed in the region following Arg48 by
the error-prone PCR method to obtain activity-enhanc-
ing mutations other than the R48S mutation. When
E. coli JM109 was transformed with plasmid
pDR319XhoI derivatives encoding the randomly muta-
genized ybaC gene, about 103 colonies were observed,
and one colony formed a larger halo. We determined the
sequence of the ybaC gene borne on the plasmid isolated
from the colony. The nucleotides encoding Leu97
(CTC) and Leu209 (TTG) were changed to those
encoding Phe97 (TTC) and Phe209 (TTT) respectively,
resulting in amino acid replacement of Leu to Phe
(L97F/L209F). The single mutations, L97F and L209F,
were also individually introduced into the ybaC gene.
Preparation of mutant enzymes. E. coli JM109 transformants
with plasmid pDR319XhoI derivatives were cultivated, and the
overproduced mutant enzymes were purified from the cells, as
described previously for the wild-type enzyme.5) The purity of the
mutant enzymes was analyzed by SDS–PAGE,11) followed by
staining with Coomassie Brilliant Blue R (Sigma-Aldrich Japan,
Tokyo).
Assay for enzymatic activity. The enzymatic activity for the
hydrolysis of p-nitrophenyl butyrate was determined at 30 ꢀC over
15 min in 100 mL of a 20 mM phosphate buffer (pH 7.1) containing
5%v/v acetonitrile. The reaction was terminated by the addition of
SDS to a final concentration of 0.2%. The amount of p-nitrophenol
produced by the reaction was determined from the absorbance with a
Characterization of EcAE mutants obtained from the
screening
Mutant enzymes L97F, L209F, and L97F/L209F
were overproduced in E. coli cells and purified to give a
single band on the SDS–PAGE (Fig. 1). Upon purifica-
tion, the host-derived wild-type enzyme was copurified
with the mutant enzymes. However, we found previ-
ously that the active-site mutants (S165A and H292A),
prepared by the purification procedure described in this
report, showed little enzymatic activity (less than 0.01%
of that of the wild-type enzyme),6) indicating that the
amount of the wild-type enzyme copurifying the mutant
enzymes is negligible. This is consistent with the fact
that the basal level of EcAE expression is very low.3) In
this study, the yields of the mutant enzymes (about
30 mg from 1-L of culture) were high enough that the
host-derived wild-type enzyme was negligible.
ꢁ1
molar absorption coefficient value of 14,200 M ꢂcmꢁ1 at 412 nm. One
unit of enzymatic activity was defined as activity that produces 1 mmol
of p-nitrophenol per min at 30 ꢀC.
Specific activity was defined as enzymatic activity per mg of
protein. In the kinetic analyses, the substrate concentration was varied
from 0.050 mM to 5.0 mM, so that at least the highest and lowest
concentrations of the substrate were larger and smaller than the Km.
The concentration of the enzyme was 360 ng/mL, 60 ng/mL, 350 ng/
mL, and 50 ng/mL for the wild type, L97F, L209F, and L97F/L209F
respectively. Hydrolysis of the substrate with the enzyme followed
Michaelis-Menten kinetics, and the kinetic parameters, Km and Vmax
,
were determined by nonlinear regression analysis of a plot of velocity
versus substrate concentration using GraphPad Prism software (Graph-
Pad Software, La Jolla, CA). All the fits were good, with R-squared
values greater than 98%.
The thermostabilities of the EcAE wild type and mutant enzymes
were determined by measuring residual activity after heating to 55 ꢀC.
The enzymes (50 ng/mL) were incubated in a 20-mM phosphate buffer
(pH 7.1) to 55 ꢀC for designated durations and cooled on ice, and the
The kinetic parameters of the mutant enzymes for
the hydrolysis of p-nitrophenyl butyrate are shown in

2084
R. KOBAYASHI et al.
Table 1. The Km value of L97F/L209F was smaller than
that of the wild-type enzyme by 2.4-fold, while the kcat
value of L97F/L209F was larger than that of the wild-
type enzyme by 12-fold. These results indicate that
enzymatic activity is enhanced by the improvement of
both catalytic efficiency and substrate binding affinity.
To dissect the contribution of each constituent mutation
to the enhancement of enzymatic activity, mutant
enzymes L97F and L209F were analyzed for kinetic
parameters. The kcat value of L97F was larger than that
of the wild-type enzyme by 8.3-fold, whereas there was
no significant change in the kcat value of L209F,
suggesting that the increase in the kcat value for L97F/
L209F is to be attributed to the L97F mutation. In
contrast, there was no significant change in the Km value
of L97F, whereas the Km value of L209F was smaller
than that of the wild-type enzyme by 2.9-fold, suggest-
ing that the decrease in the Km value for L97F/L209F is
to be attributed to the L209F mutation.
SwissModel server12–14) based on the 3-D structure of
the acetyl esterase from Salmonella typhimurium (StAE)
(PDB accession no. 3GA7). Among the HSL family
enzymes whose 3-D structures have been determined to
date, StAE has the highest sequence identity, of 75.2%,
to EcAE. This value was considered to be high enough
to assume that the 3-D structures of these two proteins
are highly similar to each other.15) Moreover, the amino
acid sequences of EcAE and StAE around Leu97 and
Leu209 are also highly homologous, with no insertions
or deletions, making StAE the most suitable template for
constructing a 3-D model of EcAE to detremine the
reason for the enhancement of catalytic activity by the
L97F and L209F mutations.
In the EcAE model, Leu97 is located in a loop
containing an HGGG motif (His91–Gly94), which is
involved in the formation of the oxyanion hole16)
(Fig. 2). It has been suggested that the oxyanion hole
contributes to the catalytic efficiency of acetylcholine
esterase by stabilizing the tetrahedral intermediate.17)
The 3-D structure of Alicyclobacillus acidocaldarius
esterase 2 (EST2), which belongs to the HSL family,
complexed with HEPES (PDB accession no. 1EVQ), has
been determined.18) This complex is an analog of the
second tetrahedral intermediate for the deacylation step.
It has been found that the deacylation step is the rate-
limiting step of EST2 for substrates with shorter acyl
chains.19) In this complex, the O2 atom of the sulfonyl
group of the HEPES adduct in the oxyanion hole forms
hydrogen bonds with the NH groups of Gly83 and Gly84
An interpretation of the effects of the mutations on
enzymatic activity
To interpret the effects of the mutations on enzymatic
activity, a 3-D model of EcAE was constructed using the
2
3
4
M
1
kDa
111
66
Table 1. Kinetic Parameters of EcAE Wild Type and Mutant
Enzymes for the Hydrolysis of p-Nitrophenyl Butyrate
45
35
Hydrolysis of p-nitrophenyl butyrate with the enzyme was carried
out at 30 ꢀC over 15 min in 100 mL of 20 mM phosphate buffer (pH 7.1)
containing 5%v/v acetonitrile. Kinetic parameters were determined
using GraphPad Prism (GraphPad Software, La Jolla, CA). Data are
expressed as mean values of four replicate experiments with the
corresponding standard errors.
25
Fig. 1. SDS–PAGE of Purified Wild Type and Mutant Enzymes.
Amounts corresponding to 1.4 mg of the wild-type enzyme
(lane 1) and mutant enzymes L97F, L209F, and L97F/L209F (lanes
2, 3, and 4 respectively) were loaded onto a 12% SDS–PAGE and
stained with Coomassie Brilliant Blue R. A molecular weight
marker kit (Fermentas, Glen Burnie, MD) containing ꢁ-galactosi-
dase, bovine serum albumin, ovalbumin, lactate dehydrogenase,
Bsp98I, and ꢁ-lactoglobulin was used (lane M).
Km
relative
kcat
(sꢁ1
relative
kcat/Km
Protein
(ꢃ10ꢁ5 M) Km
)
kcat (ꢃ105 sꢁ1
M
ꢁ1
)
Wild-type
L97F
L209F
17 ꢄ 4:1 1.0
26 ꢄ 3:8 1.5
5:7 ꢄ 1:2 0.34
29 ꢄ 3:0
240 ꢄ 15
37 ꢄ 2:9
1.0
8.3
1.3
1.7
9.2
6.5
L97F/L209F 6:9 ꢄ 1:4 0.41 340 ꢄ 28
12
49
α2-helix
α2-helix
Leu97
Leu97
Gln40
Ser165
His292
Asp262
Gln40
Ser165
His292
HGGG
HGGG
Asp262
loop Gly210-Thr217
Phe261
loop Gly210-Thr217
Phe261
Leu209
Leu209
Fig. 2. Stereo View of the Structural Model of Wild-Type E. coli Acetyl Esterase.
The structure was drawn with program MolFeat. The ꢃ2-helix and the HGGG motif are shown in black. Loop Gly210–Leu217 and the acyl-
binding pocket are shown in gray. The side chains of Leu97, Gln40, Leu209, Phe261 and active-site residues (Ser165, Asp262, and His292) are
indicated by ball and stick.

Enhancement of Enzymatic Activity of E. coli Acetyl Esterase
2085
HEPES
HEPES
Asp262
Asp262
(Asp252)
(Asp252)
His292
His292
(His282)
Gly94
Gly94
(His282)
(Gly84)
(Gly84)
Ser165
(Ser155)
Ser165
(Ser155)
Gly93
(Gly83)
Gly93
(Gly83)
HGGG
HGGG
Fig. 3. Superposition of the Catalytic Site Region of the EcAE Model and the EST2-HEPES Complex.
The structure was drawn with program MolFeat. The side chains of the active site residues and the main chain and side chains of the residues
in the loop containing the HGGG motif (His91–Leu97 for EcAE and His81–Val87 for EST2) are indicated by ball and stick. The residues of
EcAE and EST2 are shown in black and gray respectively. The active site residues and glycine residues in the oxyanion hole are numbered (in
parenthesis for EST2). The HEPES molecule covalently bound to Ser155 is shown by space-filling. The hydrogen bonds formed between the O2
atom of the sulfonyl group of the Hepes adduct and the NH groups of Gly83 and Gly84 (Gly93 and Gly94 in EcAE respectively) are shown by
dotted lines.
HGGG
Gly94
HGGG
Gly94
Leu97
Ser165
Asp262
Gln40
Gln40
Leu97
Ser165
Asp262
Gly93
Gly93
His292
His292
Tyr44
Tyr44
α2-helix
α2-helix
Fig. 4. Stereo View of the Structure around Leu97.
The structure was drawn with program MolFeat. The ꢃ2-helix and the HGGG motif are shown in black. The side chains of Leu97, Gln40,
Tyr44, Ser165, Asp262, and His292 and the main-chain nitrogen atoms of Gly93 and Gly94 are indicated by ball and stick. The surfaces of the
side chains of Leu97, Gln40, and Tyr44 are indicated by dot surface.
(Fig. 3). The geometry of the catalytic triad residues
(Ser165/Asp262/His292 and Ser155/Asp252/His282
for EcAE and EST2 respectively) and the HGGG motif
is nearly identical as between the EcAE model and
EST2 (Fig. 3). Therefore, Gly93 and Gly94 in EcAE,
which correspond to Gly83 and Gly84 in EST2,
respectively, are thought to form hydrogen bonds with
the oxyanion of the second tetrahedral intermediate. The
side chain of Leu97 is in contact with the side chains of
Gln40 and Tyr44 in the ꢃ2-helix (Ile37–Arg49) (Fig. 4).
Replacement of Leu97 with Phe should cause a steric
hindrance with the side chain of Gln40 and strengthen
the hydrophobic interaction with the side chains of
Tyr44. As a consequence, the loop containing Leu97 and
the HGGG motif can shift to a position favorable for
stabilizing the second tetrahedral intermediate, thereby
improving catalytic efficiency. A possible mechanism
for this stabilization is that the NH groups of Gly93 or
Gly94 come closer to the oxyanion of the tetrahedral
intermediate and form a stronger hydrogen bond.
binding pocket.18) Leu209 is replaced by Phe in StAE
too. In its 3-D structure, the side chain of Leu209 is in
contact with the side chain of Phe261 (Fig. 5A, B). Such
structural features are thought to be similar for the
L209F mutant of EcAE. When Phe is located at position
209, the C" and Cꢄ atoms of Phe209 make hydrophobic
contact with the side chain of Phe261 (Fig. 5B). This
contact is missing when Leu is located at position 209
(Fig. 5A). Therefore, it is possible that the position of
Leu209 is altered by replacement of Leu209 with Phe,
resulting in a shift of loop Gly210–Leu217. In the EST2-
HEPES complex, Leu206 (Leu216 in EcAE) in the
corresponding loop is located in the vicinity of the N1
atom of the piperazine ring and two carbon atoms
between the piperazine ring and the sulfonyl group of
HEPES (Fig. 6). These atoms fit well into the narrow
hydrophobic tunnel consisting of Gly83, Gly84, Trp85,
Ala156, Leu206, Phe214, and Leu254.18) In the EcAE
model, these residues correspond to Gly93, Gly94,
Phe95, Ala166, Leu216, Tyr224, and Leu264 respec-
tively, and the geometry of the hydrophobic tunnel is
well conserved between EST2 and the EcAE model
(Fig. 6). It is likely that the butyryl group of the second
Leu209 is located in the vicinity of loop Gly210–
Leu217 (Fig. 2). This loop corresponds to loop Gln200–
Thr207 in EST2, which has been identified as an acyl-

2086
R. KOBAYASHI et al.
A
Phe261
Ser165
Phe261
Ser165
His292
Asp262
His292
Asp262
Leu209
Leu209
B
Phe261
Phe261
Ser165
Ser165
His292
Asp262
His292
Asp262
Phe209
Phe209
Fig. 5. Stereo View of the Structures around Leu209 of EcAE and Phe209 of S. typhimurium Acetyl Esterase.
Structure model around Leu209 of EcAE (A) and Phe209 of S. typhimurium acetyl esterase (3GA7) (B) were drawn with program MolFeat.
Loop Gly210–Thr217 in (A) is shown in black. The side chains of Leu/Phe209, Phe261, Ser165, Asp262, and His292 are indicated by ball and
stick. Note that the numbering system is common to EcAE and S. typhimurium acetyl esterase.
6HUꢈꢆꢇ
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Fig. 6. Superposition of the Acyl-Binding Pocket of the EcAE Model and the EST2-HEPES Complex.
The structure was drawn with program MolFeat. The side chains of the active site residues and the residues involved in the acyl-binding
pocket are indicated by ball and stick. Loop Gly210–Leu217 in EcAE and loop Gln200–Thr207 in EST2 are shown by ribbon model. The
residues of EcAE and EST2 are shown in black and gray respectively. The numbers of the EcAE residues are shown with corresponding numbers
for the EST2 residues in parenthesis. The HEPES molecule covalently bound to Ser155 is shown by space-filling.
tetrahedral intermediate fits into the hydrophobic tunnel
in a way similar to HEPES when p-nitrophenyl butyrate
is used as substrate, because the butyryl group of the
second tetrahedral intermediate corresponds to the part
of HEPES from the sulfonyl group to the N1 atom of the
piperazine ring. It is possible that the interaction of the
acyl-binding pocket with the substrate is principally
conserved also for the first and second tetrahedral
intermediates in acetylcholine esterase.17,20) Hence it is
possible that Leu216 and the butyryl group of the
substrate are located in close proximity in EcAE, and
come closer due to the L209F mutation, resulting in
increased substrate binding affinity. The Km value
depends on the catalytic rate constant as well as the
apparent substrate binding affinity. Since the L209F
mutation caused no significant change in the kcat value, it
is likely that the decrease in the Km value due to the
L209F mutation is a result of an increase in substrate
binding affinity.
Thermostability
The stabilities of the EcAE wild type and the mutant
enzymes against irreversible heat inactivation were
determined at pH 7.1 by measuring residual activities
after heating for various durations at 55 ꢀC (Fig. 7). The
wild-type enzyme and L209F showed similar thermo-
stabilities with a half-life of about 40 min. In contrast,
L97F and L97F/L209F showed higher thermostabilities
than the wild-type enzyme, with a half-life of about
60 min. Leu97 is considered to make hydrophobic
contact with the side chain of Tyr44 located in the ꢃ2-
helix as well as the side chain of Gln40 (Fig. 4).
Therefore, Phe at position 97, rather than Leu, is
expected to form tighter hydrophobic interaction with
the side chains of Tyr44 and Gln40, thereby increasing
thermostability. Among thermophilic esterases belong-
ing to the HSL family, the residue corresponding to
Leu97 in EcAE is phenylalanine for esterase (EstE1)
found from a metagenomic library (accession no.:

Enhancement of Enzymatic Activity of E. coli Acetyl Esterase
2087
thermostability of these esterases, but detailed thermo-
dynamic and structural investigations, as well as
mutagenic studies, are necessary for a resolution as to
the effects of the mutations on enzymatic activity and
thermostability.
100
50
(
)
WT
(−−−)
L209F(- - - -)
CD spectra
L97F
The far-UV CD spectra of the mutant enzymes were
nearly identical to that of the wild-type enzyme
(Fig. 8A), indicating that the mutant enzyme is not
markedly changed in its secondary structure. The near-
UV CD spectra of the mutant enzymes deviated slightly
from that of the wild-type enzyme, especially in the 260-
to 280-nm range (Fig. 8B). The bands of the phenyl-
alanine, tyrosine, and tryptophan residues overlap in this
region. The spectrum of the wild-type enzyme gave a
trough with a minimum [ꢂ] value of about ꢁ100 around
270 nm. This trough became shallower for L97F and
L209F, with a minimum [ꢂ] values of about ꢁ40 and
about ꢁ60 respectively. These changes might have
reflected both the addition of a phenylalanine residue
and conformational changes around the aromatic resi-
dues caused by the L97F and L209F mutations. We
hypothesize that the L97F and L209F mutations affect
hydrophobic interactions with Tyr44 and Phe261 re-
spectively. In addition, Phe95 and Trp214, which are
located in the same loops as Leu97 and Leu209
respectively, can shift due to these mutations. Thus
there is a possibility that these effects contribute to the
spectral changes, but they are indistinguishable from
those due to the addition of a phenylalanine residue. For
L97F/L209F, the trough around 270 nm also became
shallower, with a minimum [ꢂ] value of about ꢁ70,
comparable to that for L209F. Thus the spectral changes
due to the L97F and L209F mutations were not additive.
This suggests that the simultaneous introduction of the
two mutations causes additional changes in environ-
ments around aromatic residues other than those caused
by a single mutation, but no additional changes were
observed in kinetic parameter and thermostability when
the two mutations were introduced simultaneously.
Hence such changes should not seriously affect enzy-
matic activity or thermostability.
L97F/L209F (
)
-------
10
0
20
40
60
Incubation time (min)
Fig. 7. Stability against Irreversible Heat Inactivation of the EcAE
Wild Type and Mutant Enzymes.
Semi-log plots of residual activity versus incubation time are
shown. The wild-type ( ), L97F ( ), L209F ( ), and L97F/L209F
(
) enzymes (50 ng/mL) were incubated in a 20 mM phosphate
buffer (pH 7.1) at 55 ꢀC for the designated durations and cooled on
ice, and the residual activities were determined using p-nitrophenyl
butyrate as substrate. Error bars indicate standard deviations from
the average values for four experiments. Lines were obtained by
linear regression of the data.
A
Wavelength (nm)
B
Acknowledgment
We thank Professor Isao Saito for helpful discussion
and encouragement. This work is supported in part by a
Grant to Promote Advanced Scientific Research and by a
Matching Fund Subsidy for Private Universities from
the Ministry of Education, Culture, Sports, Science, and
Technology of Japan.
Wavelength (nm)
Fig. 8. CD Spectra of the Wild Type and Mutant Enzymes.
The far-UV (A) and near-UV (B) CD spectra of mutant enzymes
L97F, L209F, and L97F/L209F and the wild-type enzyme are
shown by thin solid line, thin broken line, thick solid line, and thick
broken line respectively. All spectra were measured as described in
‘‘Materials and Methods.’’
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