Substrate specificity of an esterase from the archaeon Sulfolobus tokodaii bearing a GGG(A)X motif

Article abstract of DOI:10.3109/10242422.2015.1095679

A GGG(A)X-type esterase (Est0071) from an archaeon catalyzes asymmetric hydrolysis of prochiral bulky malonic diesters in good enantioselectivity. The selectivity of Est0071 was for the opposite enantiomer to that previously shown for pig liver esterase, and the resulting enantiomeric excess of the products was higher. Est0071 could also catalyze the hydrolysis of various acetates of secondary alcohols, and showed moderate enantioselectivity in these reactions.

Full text of DOI:10.3109/10242422.2015.1095679

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Substrate specificity of an esterase from the
archaeon Sulfolobus tokodaii bearing a GGG(A)X
motif
Reina Wada, Masanaru Ozaki, Takashi Kumon, Hiromichi Ohta & Kenji
Miyamoto
To cite this article: Reina Wada, Masanaru Ozaki, Takashi Kumon, Hiromichi Ohta &
Kenji Miyamoto (2015) Substrate specificity of an esterase from the archaeon Sulfolobus
tokodaii bearing a GGG(A)X motif, Biocatalysis and Biotransformation, 33:3, 188-190, DOI:
10.3109/10242422.2015.1095679
To link to this article: http://dx.doi.org/10.3109/10242422.2015.1095679
Published online: 29 Oct 2015.
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Date: 05 February 2016, At: 09:11

Biocatalysis and Biotransformation, 2015; 33: 188–190
SHORT COMMUNICATION
Substrate specificity of an esterase from the archaeon Sulfolobus
tokodaii bearing a GGG(A)X motif
REINA WADA, MASANARU OZAKI, TAKASHI KUMON, HIROMICHI OHTA
& KENJI MIYAMOTO
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama, Japan
Abstract
A GGG(A)X-type esterase (Est0071) from an archaeon catalyzes asymmetric hydrolysis of prochiral bulky malonic diesters
in good enantioselectivity. The selectivity of Est0071 was for the opposite enantiomer to that previously shown for pig liver
esterase, and the resulting enantiomeric excess of the products was higher. Est0071 could also catalyze the hydrolysis of
various acetates of secondary alcohols, and showed moderate enantioselectivity in these reactions.
Keywords: Esterase, GGG(A)X-motif, hydrolysis, malonic diester, Sulfolobus tokodaii
Introduction
residues and exhibited extremely thermostability
with optimum activity observed at 480 ꢀC.
Recently, we have developed a thermally driven
domino reaction for the synthesis of (S)-a-arylpro-
pionates (profens) using Est0071 (Wada et al. 2013).
However, the substrate specificity of the enzyme was
not determined. Est0071 showed moderate identity
with some esterases (e.g. lipase from Candida rugosa
(CRL), p-nitrobenzylesterase from Bacillus subtilis
(BsubpNBE)) which belong to a group of a/b-hydro-
lases containing a GGG(A)X motif, which is
located in the active center of the hydrolases and is
involved in the formation of the oxyanion hole. This
motif has been reported to determine the activity of
esterases or lipases toward bulky molecules such as
tertiary alcohols (Henke et al. 2002; Kourist et al.
2007).
Enzymatic transformations of synthetic substrates
are playing an increasingly important role in syn-
thetic organic chemistry, especially in asymmetric
synthesis. Optically active compounds are important
intermediates for the synthesis of pharmaceuticals
and naturally occurring compounds. Chiral carbon
centers are found in many of these compounds. The
attractive routes for synthesis of these compounds
are hydrolytic desymmetrization of prochiral malon-
ate diesters into their chiral monoesters or kinetic
resolution of the racemic esters by hydrolytic
enzymes (e.g. lipase and esterase). The optically
active products may serve as chiral building blocks in
the synthesis of more complex compounds of
biological and pharmacological interest (Schneider
et al. 1984; Toone et al. 1991; Canet et al. 1992;
Pig liver esterase (PLE) is commercially obtained
by extraction from animal tissue and is composed of
isozymes (a, b, and g). The g-isoenzyme of pig liver
esterase (g-PLE) contains a GGGX motif, and
cloning and functional expression of g-isoenzymes
in Pichia pastris and E. coli has been successful
´
Breznik et al. 1997; Marıa et al. 2005; Wallert et al.
2005; Iosub et al. 2010).
In this study, we focused on an esterase from the
thermostable archaeon Sulfolobus tokodaii strain 7.
The putative esterase gene (ST0071) was selected
by in silico screening from the total genome of the
strain and the characterized esterase expressed in
Escherichia coli (Suzuki et al. 2004). The enzyme,
called Est0071, was comprised of 303 amino acid
(Bru¨sehaber et al. 2007).
recombinant isozymes of PLE revealed significant
differences in kinetic resolution reactions
A
comparison of
Correspondence: Kenji Miyamoto, Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi,
Yokohama 223-8522, Japan. Tel: +81 45 566 1786. E-mail: kmiyamoto@bio.keio.ac.jp
(Received 26 February 2015; revised 1 April 2015; accepted 16 September 2015)
ISSN 1024-2422 print/ISSN 1029-2446 online ß 2015 Taylor & Francis
DOI: 10.3109/10242422.2015.1095679

Substrate specificity of an esterase 189
(Musidlowska-Persson et al. 2002). There have been
many reports on pig liver esterase (PLE) catalyzed
hydrolysis of prochiral bulky malonate diesters to
optically active (R)-malonate monoesters. In con-
trast, there is no report of an esterase exhibiting
selectivity opposite to that of PLE. Thus, we
examined whether Est0071 could catalyze the
hydrolysis of prochiral malonate diesters.
Figure 1. Est0071-catalyzed hydrolysis of diethyl malonates.
Table 1. Asymmetric hydrolysis of prochiral malonate diesters.
Run Ar
R
Time Yield e.e.
(%)
Config.
(h)
(%)
Methods
1
2
3
4
5
6
Ph
Ph
Ph
Me
Et
H
36
Me 120
Me 264
48
240
72
96
94
79
77
81
99
96
26
0
499
94
83
S
S
–
S
S
R
Preparation and enzyme assay of Est0071
p-methylphenyl Me
2-naphthyl
Benzyl
The gene encoding ST0071 was cloned, over-
expressed in E. coli, and the His-tagged fusion
enzyme was purified (Suzuki et al. 2004). The
esterase activity against p-nitrophenyl esters was
determined by measuring the amount of resulting
p-nitrophenol via enzymatic hydrolysis. One unit (U)
of the esterase activity was defined as the amount of
enzyme required to liberate 1 mmol of p-nitrophenol
per minute. The specific activity of Est0071 was 165
U/mg protein at 30 ꢀC.
Figure 2. Enantioselective hydrolysis of various acetates.
Enzyme-catalyzed hydrolysis
The reaction of various esters with Est0071 was
performed with 10 mM concentration of the sub-
strate and 450 U of the enzyme in 200 mM HEPES
buffer (pH 7) for malonic diesters or 200 mM Tris–
HCl (pH8) for acetates of secondary alcohols in a
total volume of 10 ml. After stirring at 30 ꢀC for 36–
264 h (malonic diesters) or 2 h (acetates), the
reaction was stopped by the addition of 2 M HCl
(2 ml) and the mixture was extracted with diethyl
ether. After removal of the solvent in vacuo, the
enantiomeric excess of the products was then
analyzed with HPLC.
in Table 1. The product derived from the substrate
with phenyl and methyl groups (Run 1) was obtained
in high yield and high excess of the S-enantiomer.
However, when the aryl group was benzyl (Run 6),
the configuration of the product was R. When the R
group was changed to ethyl (Run 2) or hydrogen
(Run 3), the enatiomeric excesses was low (ethyl:
26% e.e., hydrogen: 0% e.e.). This indicated that the
R
group was significant for enatioselectivity.
Furthermore, the pocket, which the alkyl group fits
within, in the active site of this enzyme, may not be
so large. When the aryl group was p-methylphenyl
(Run 4) or 2-naphthyl group (Run 5), the reaction
rates were slower than for the other substrates,
indicating that the enzyme has a large hydrophobic
pocket in the active site. Furthermore, the selectivity
of Est0071 was opposite that of PLE.
HPLC system
High-performance liquid chromatography (HPLC)
was carried out on an LC-2010A HT system
(Shimadzu, Kyoto, Japan) equipped with a chiral
column OD (4.6 mm ꢁ 250 mm, Daicel Corporation,
Japan). The separation of enantiomer peaks was
achieved with a flow of 0.5 ml/min at 260 nm, by
using a mobile phase composed of hexane–i-PrOH–
trifluoroacetic acid (93:7:1.5).
The results of hydrolysis of various secondary
alcohol acetates with Est0071 (Figure 2) are sum-
marized in Table 2. The enzyme showed moderate
enantioselectivity toward 1-phenylethyl acetate
(Table 2, Run 1, E ¼ 11) in contrast to the naturally
occurring PLE mixture (E ¼ 7) (Hummel et al.
2007). Interestingly, the preferences of Est0071
and PLE were the same in the kinetic resolution of
this compound. When the R group was changed to
ethyl (Run 2) or propyl (Run 3), the enatioselectivity
gradually decreased.
Results and discussion
Est0071 catalyzed the hydrolysis of prochiral bulky
diesters to give half ester with high selectivity and
yield (Figure 1), and the results are summarized

190 R. Wada et al.
Table 2. Enantioselectivities of Est0071 in the kinetic resolution of acetate of various secondary alcohols.
Run
R1
R2
Time (h)
Conversion (%)
Acetate
e.e. (%)
Alcohol
e.e. (%)
E value
1
2
3
4
Ph
Ph
Ph
PhCH2
CH3
2
2
2
2
40
32
8.6
12
49
39
5
74
81
55
50
11
15
3.6
3.2
CH2CH3
(CH2)2CH3
CH3
7
function relationships. Angew Chem Int Ed Engl 41:3211–
3213.
Conclusions
Hummel A, Bru¨sehaber E, Bo¨ttcher D, Trauthwein H,
Doderer K, Bornscheuer UT. 2007. Isoenzymes of pig-liver
esterase reveal striking differences in enantioselectivities. Angew
Chem Int Ed Engl 46:8492–8494.
Iosub V, Haberl AR, Leung J, Tang M. 2010. Enantioselective
synthesis of r-quaternary amino acid derivatives by sequen-
tial enzymatic desymmetrization and curtius rearrangement
of r, r-disubstituted malonate diesters. J Org Chem 75:1612–
1619.
Est0071 was shown to hydrolyze a wide range of
substrates with high chemical yield. Additionally, this
esterase showed high enantioselectivity compared
with that of PLE. Highly enantioselective hydrolysis
of diethyl 2-phenyl-2-methylmalonate (Table 1,
Run 1) to the (S)-monoester by this esterase was
successful. Moreover, Est0071 catalyzed the
hydrolysis of various acetates of secondary alcohols
(Table 2). Thus, Est0071 is a useful tool for the
synthesis of pharmaceuticals and naturally occurring
compounds with a chiral center.
Kourist R, Krishna S, Patel JS, Bartnek F, Hitchman TS,
Weiner DP, Bornscheuer UT. 2007. Identification of
a
metagenome-derived esterase with high enantioselectivity in
the kinetic resolution of arylaliphatic tertiary alcohols. Org
Biomol Chem 5:3310–3313.
´
Marıa D, Kossmann B, Potgrave N, Buchholz S, Trauthwein H,
May O, Gro¨ger H. 2005. Improved process for the enantiose-
lective hydrolysis of prochiral diethyl malonates catalyzed by pig
liver esterase. Synlett 11:1746–17480.
Musidlowska-Persson A, Bornscheuer UT. 2002. Substrate spe-
cificity of the g-isoenzyme of recombinant pig liveresterase
towards acetates of secondary alcohols. J Mol Catal B Enzym
19–20:129–133.
Schneider M, Engel N, Boensmann H. 1984. Enzymatic synthesis
of chiral building blocks from prochiral substrates: enantiose-
lective synthesis of monoalkyl malonates. Angew Chem Int Ed
Engl 23:66.
Suzuki Y, Miyamoto K, Ohta H. 2004. A novel thermostable
esterase from the thermoacidophilic archaeon Sulfolobus tokodaii
strain 7. FEMS Microbiol Lett 236:97–102.
Toone EJ, Jones JB. 1991. Enzymes in organic synthesis 50.
Probing the dimensions of the large hydrophobic binding
regionof the active site of pig liver esterase using
substituted aryl malonate substrates. Tetrahedron Asyn
2:104–1052.
Wada R, Kumon T, Kourist R, Ohta H, Uemura D, Yoshida S,
Miyamoto K. 2013. Thermally driven asymmetric domino
reaction catalyzed by a thermostable esterase and its variants.
Tetrahedron Lett 54:1921–1923.
Declaration of interest
The authors report no conflicts of interest. The
authors alone are responsible for the content and
writing of this article.
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