Rational control of enantioselectivity of lipase by site-directed mutagenesis based on the mechanism

Article abstract of DOI:10.1039/b508244g

The enantioselectivity of a Burkholderia cepacia lipase toward secondary alcohols could be both increased and decreased rationally by introducing only a single mutation on the basis of the mechanism proposed previously. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b508244g

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Rational control of enantioselectivity of lipase by site-directed
mutagenesis based on the mechanism{
Tadashi Ema,* Toshiyuki Fujii, Misa Ozaki, Toshinobu Korenaga and Takashi Sakai*
Received (in Cambridge, UK) 10th June 2005, Accepted 22nd July 2005
First published as an Advance Article on the web 12th August 2005
DOI: 10.1039/b508244g
substituent (R2) of the slower-reacting enantiomer is increased by
The enantioselectivity of a Burkholderia cepacia lipase toward
means of site-directed mutagenesis (Fig. 1a), the enantioselectivity
will increase (and vice versa). The I287F mutation, which increases
the steric bulkiness (Fig. 1c) as compared with the wild-type
enzyme (Fig. 1b), will suppress the reaction of the slower-reacting
enantiomer and improve the enantioselectivity. On the other hand,
the I287A mutation, which decreases the steric hindrance (Fig. 1d),
will facilitate the reaction of the slower-reacting enantiomer and
diminish the enantioselectivity.
secondary alcohols could be both increased and decreased
rationally by introducing only a single mutation on the basis of
the mechanism proposed previously.
Despite the remarkable advances in enzyme science and technol-
ogy,1,2 the ‘‘rational control’’ of enantioselectivity of an enzyme
still remains difficult because of the complexity of the enzymatic
reaction. Although directed evolution, using random mutagenesis
combined with a high-throughput screening system, can improve
the enantioselectivity of an enzyme without knowledge of the
structure and mechanism, a huge number of mutants need to be
screened.3–5 On the other hand, once the mechanistic aspect has
been clarified, site-directed mutagenesis based on rational design
can also be effective for changing the enantioselectivity.6–9
Although it is desirable that the random and rational approaches
should be complementary to each other to create a biocatalyst
showing higher performance, in reality, the latter seems to be more
difficult and inefficient than the former because of unknown
factors in biocatalysis. In this context, a semi-rational and semi-
random approach has also been examined.10,11 Obviously, the
mechanistic basis of biocatalysis needs to be strengthened
further.12 Here we report for the first time that the enantioselec-
tivity of a Burkholderia cepacia lipase toward secondary alcohols
could be both increased and decreased rationally and easily by
mutating only one amino acid residue in the proximity of the
active site on the basis of a well-defined mechanism.
The gene encoding the mature lipase was PCR-cloned from the
genomic DNA of Burkholderia cepacia NBRC 14595 and over-
expressed in E. coli.17 The denatured lipase was subjected to the
in vitro refolding in the presence of an activator and 8 M urea.18
After the refolded lipase was purified to homogeneity by
hydrophobic chromatography and ion-exchange chromatography,
Previously, we have proposed a stereo-sensing mechanism of
lipases toward secondary alcohols as shown in the transition-state
model (Fig. 1a, an expanded version is shown).13 Enantioselectivity
results principally from the conformational requirements and
repulsive interactions in the transition state, and no attractive
interactions between the enzyme’s pockets and the substrate’s
polar/nonpolar substituents are involved. This mechanism has
been proved by kinetic and thermodynamic analyses,13,14 by
Fig. 1 (a) Transition-state model to rationalize the enantioselectivity in
the lipase-catalyzed kinetic resolution of secondary alcohol: an expanded
version of the model (residue 287 is added to the original version). (i) The
C–O bond of the substrate takes the gauche conformation with respect to
the breaking C–O bond, which is due to the stereoelectronic effect. (ii) The
hydrogen atom attached to the stereocenter in the substrate is syn-oriented
toward the carbonyl oxygen atom. Enantioselectivity is explained by the
conformational requirements and repulsive interactions and/or strains
caused in the transition state. The catalytic triad residues, the ester being
produced, and the mutation site are shown in green, blue, and red,
respectively. The amino acid numbers for Burkholderia cepacia lipase are
shown. (b)–(d) Space-filling representations of (b) the wild-type enzyme,
(c) the I287F mutant, and (d) the I287A mutant. The catalytic triad
residues and residue 287 are shown in green and red, respectively. These
structures are viewed from the right side of Fig. 1a. The crystal structure of
a Burkholderia cepacia lipase (PDB code 1OIL) having 96% sequence
identity with that used in this study was used after the thirteen different
residues had been replaced. The structures were drawn with SYBYL 6.4
(Tripos Inc.).
using
a gigantic secondary alcohol, 5-[4-(1-hydroxyethyl)-
phenyl]-10,15,20-triphenylporphyrin,15 and by highly enantioselec-
tive reactions at high temperatures up to 120 uC.16
Enantioselectivity results from the suppression mechanism work-
ing on the slower-reacting enantiomer in the transition state.
Therefore, if the steric repulsion between the enzyme and the larger
Department of Applied Chemistry, Faculty of Engineering, Okayama
University, Tsushima, Okayama, 700-8530, Japan.
E-mail: ema@cc.okayama-u.ac.jp; Fax: +81-86-251-8092;
Tel: +81-86-251-8091
{ Electronic supplementary information (ESI) available: Construction of
plasmids, site-directed mutagenesis, expression and purification of lipase
and kinetic resolution. See http://dx.doi.org/10.1039/b508244g
4650 | Chem. Commun., 2005, 4650–4651
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the effectiveness of the single amino acid substitution at
position 287.
Biocatalysts are behind artificial catalysts, such as chiral ligand–
metal complexes and chiral organocatalysts, in rational design
approaches, in the latter of which enantioselectivity can be tuned
rationally by altering the structures of catalysts.22,23 Here we have
succeeded in controlling (both increasing and decreasing) the
enantioselectivity of the lipase rationally by mutating only one
amino acid residue on the basis of the mechanism, which is
rational enough as compared with the alteration of artificial
catalysts. Further work is currently under way to evolve the
enzyme rationally.
Scheme 1
Table 1 Enantioselectivity of the wild-type lipase and mutantsa
Ee (%)
Entry Lipase
Alcohol Time/h cb
TTNc (R)-2 (S)-1 E valued
This work was supported by a Grant-in-Aid for Scientific
Research from Japan Society for the Promotion of Science (JSPS).
We are grateful to the SC-NMR Laboratory of Okayama
University for the measurement of NMR spectra.
1
2
3
4
5
6
7
8
9
10
a
Wild-type 1a
4.5
7
9
3.5
12.5
9
28
16
14
42
0.473 7800 94.1 84.4 88
0.457 7500 96.8 81.5 156
0.438 7200 95.8 74.6 105
0.484 8000 88.4 82.8 42
I287F
I287L
I287M
I287A
1a
1a
1a
1a
0.491 8100 52.6 50.8
5
Wild-type 1b
0.460 7600 90.0 76.8 44
0.443 3700 96.3 76.5 123
0.477 7900 90.9 82.8 54
0.411 6800 95.2 66.3 81
0.349 5800 87.5 46.9 24
Notes and references
I287F
I287L
I287M
I287A
1b
1b
1b
1b
1 Enzyme Catalysis in Organic Synthesis, ed. K. Drauz and H. Waldmann,
Wiley-VCH, Weinheim, 2nd edn, 2002.
2 U. T. Bornscheuer and R. J. Kazlauskas, Hydrolases in Organic
Synthesis, Wiley-VCH, Weinheim, 1999.
3 M. T. Reetz, Tetrahedron, 2002, 58, 6595–6602.
4 G. P. Horsman, A. M. F. Liu, E. Henke, U. T. Bornscheuer and
R. J. Kazlauskas, Chem. Eur. J., 2003, 9, 1933–1939.
5 R. Carr, M. Alexeeva, M. J. Dawson, V. Gotor-Ferna´ndez,
C. E. Humphrey and N. J. Turner, ChemBioChem, 2005, 6, 637–639.
6 F. Manetti, D. Mileto, F. Corelli, S. Soro, C. Palocci, E. Cernia,
I. D’Acquarica, M. Lotti, L. Alberghina and M. Botta, Biochim.
Biophys. Acta, 2000, 1543, 146–158.
Conditions: lipase (100 mg except for entry 7 (200 mg), 1% (w/w)
enzyme/Toyonite-200M), 1 (0.50 mmol), vinyl acetate (1.0 mmol),
˚
molecular sieves 3 A (three pieces), dry i-Pr2O (5.0 mL), 30 uC.
b
c
Conversion calculated from c 5 ee(1)/(ee(1) + ee(2)). TTN is
the total number of moles of the product formed per mole of
the enzyme. Calculated from TTN 5 0.5 6 c 6 33000 (molecular
weight of the lipase). Calculated from E 5 ln[1 2 c(1 + ee(2))]/
ln[1 2 c(1 2 ee(2))].
d
7 D. Rotticci, J. C. Rotticci-Mulder, S. Denman, T. Norin and K. Hult,
ChemBioChem, 2001, 2, 766–770.
8 C. Gentner, R. D. Schmid and J. Pleiss, Coll. Surf. B: Biointerfaces,
2002, 26, 57–66.
9 Y. Ijima, K. Matoishi, Y. Terao, N. Doi, H. Yanagawa and H. Ohta,
Chem. Commun., 2005, 877–879.
10 Y. Koga, K. Kato, H. Nakano and T. Yamane, J. Mol. Biol., 2003, 331,
585–592.
it was immobilized on the porous ceramic called Toyonite-200M.16
Site-directed mutagenesis was introduced by the overlap-extension
PCR method.19 The lipase-catalyzed kinetic resolutions of 1 were
conducted with vinyl acetate in dry i-Pr2O at 30 uC (Scheme 1).
The results are listed in Table 1. The enantioselectivities were
compared by using the E value.20
11 S. Park, K. L. Morley, G. P. Horsman, M. Holmquist, K. Hult and
R. J. Kazlauskas, Chem. Biol., 2005, 12, 45–54.
12 T. Ema, Curr. Org. Chem., 2004, 8, 1009–1025.
13 T. Ema, J. Kobayashi, S. Maeno, T. Sakai and M. Utaka, Bull. Chem.
Soc. Jpn., 1998, 71, 443–453.
14 T. Ema, K. Yamaguchi, Y. Wakasa, A. Yabe, R. Okada,
M. Fukumoto, F. Yano, T. Korenaga, M. Utaka and T. Sakai,
J. Mol. Catal. B: Enzym., 2003, 22, 181–192.
15 T. Ema, M. Jittani, K. Furuie, M. Utaka and T. Sakai, J. Org. Chem.,
2002, 67, 2144–2151.
16 T. Ema, M. Kageyama, T. Korenaga and T. Sakai, Tetrahedron:
Asymmetry, 2003, 14, 3943–3947.
17 S. Aoyama and N. Yoshida, Eur. Pat. 0657535A2, 1995.
18 D. T. Quyen, C. Schmidt-Dannert and R. D. Schmid, Appl. Environ.
Microbiol., 1999, 65, 787–794.
19 S. N. Ho, H. D. Hunt, R. M. Horton, J. K. Pullen and L. R. Pease,
Gene, 1989, 77, 51–59.
20 C.-S. Chen, Y. Fujimoto, G. Girdaukas and C. J. Sih, J. Am. Chem.
Soc., 1982, 104, 7294–7299.
As shown in Table 1, the mutants catalyzed the transesterifica-
tion of 1 more slowly than the wild-type enzyme did in most cases.
The mutation at position 287 seems to perturb the catalytic action
to some degree. Nevertheless, the total turnover numbers (TTN) of
the mutants were high enough, ranging from 3700 to 8100
(Table 1), which are comparable to those for the wild-type enzyme
(TTN 7600 or 7800) and that reported for a commercially
available lipase (TTN 5000).21 Importantly, the E value increases
as the amino acid residue at position 287 is more bulky: Phe . Leu
# Met # Ile . Ala. This trend is consistent with the prediction
described above. The E values for the I287F mutant toward 1a
and 1b were 1.8- and 2.8-fold higher, respectively, than the
corresponding values for the wild-type enzyme. On the other hand,
the E values for the I287A mutant toward 1a and 1b were 17.6-
and 1.8-fold lower, respectively, than the corresponding values for
the wild-type enzyme. The E values for the I287F mutant toward
1a and 1b were 31- and 5-fold higher, respectively, than those for
the I287A mutant. The 31-fold difference in E value amounts to
the energetic difference of 22.0 kcal mol21, according to the
equation DF–ADR–SDG{ 5 –RTlnEF/EA, which clearly represents
21 T. Ema, S. Maeno, Y. Takaya, T. Sakai and M. Utaka, J. Org. Chem.,
1996, 61, 8610–8616.
22 R. E. Gawley and J. Aube´, Principles of Asymmetric Synthesis, Elsevier,
Oxford, 1996.
23 A. Berkessel and H. Gro¨ger, Asymmetric Organocatalysis, Wiley-VCH,
Weinheim, 2005.
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