4354
J. Am. Chem. Soc. 2001, 123, 4354-4355
Creation of an Enantioselective Hydrolase by
Engineered Substrate-Assisted Catalysis
Anders Magnusson, Karl Hult, and Mats Holmquist*
Department of Biotechnology
Royal Institute of Technology
SE-100 44 Stockholm, Sweden
ReceiVed January 30, 2001
We have engineered a hydrolase scaffold to perform enantio-
selective substrate-assisted catalysis. First, a transition-state
stabilizing residue in the enzyme active site was removed by site-
directed mutagenesis, causing reduced activity. The catalytic
activity was then partially and enantioselectively restored by a
substrate containing the missing catalytic functional group. By
this approach we converted the wild-type lipase with very poor
enantioselectivity (E ) 1.6) into an enzyme with remarkably
improved enantioselectivity (E ) 22) toward ethyl 2-hydroxy-
propanoate. Enzymes offer one of the best routes to enantiomeri-
cally pure building blocks for the synthesis of bioactive com-
pounds such as pharmaceuticals, pheromones, fragrances and fine
Figure 1. Active-site close-up of Candida antarctica lipase B. (A)
Transition-state stabilization in wild-type enzyme. (B) Substrate-assisted
transition state stabilization in a Thr40Val mutant.
at the 2-position in the acyl moiety of the substrate (Figure 1).
The steric requirements of the active site suggested that one
enantiomer of the substrate would be much more reactive
compared to the other.
We targeted Thr40 in CALB by site-directed mutagenesis to
create enzyme variants with a compromised oxyanion hole. Wild-
type lipase and the Thr40Val and Thr40Ala mutants were
13
1
produced in recombinant yeast Pichia pastoris in high yield and
purity.14 The produced lipases were purified by means of
hydrophobic interaction chromatography followed by gel filtra-
chemicals. Hydrolases such as lipases, esterases, and proteases
are commonly used catalysts for the preparation of optically active
2
carboxylic acids, alcohols, amino acids, and amines. Yet, it has
tion.14 Specificity constants (kcat/K
M
) for wild-type and mutated
remained a challenging task to rationally tailor or improve the
substrate specificities of these enzymes. To date, only a few
examples of improvements in enantioselectivities of lipases/
lipase were determined toward ethyl propanoate and ethyl
butanoate (Table 1). The enzymes did not show saturation kinetics
within the solubility range of the investigated substrates. The
Thr40Val and Thr40Ala mutants showed 3 orders of magnitude
3
esterases have been achieved using site-directed mutagenesis,
directed evolution, or chemical modification.
4
5
M
lower kcat/K values than wild-type enzyme, demonstrating the
Here we describe a new means to create enantioselective
hydrolases via engineered substrate-assisted catalysis (SAC).
crucial role of Thr40 in catalysis (Table 1). The lowered kcat/K
M
6
values correspond to increased activation energies by 15-19 kJ/
mol. These data agree well with the loss of one stabilizing
hydrogen bond to the transition state.15 In a few hydrolases the
importance of a side chain involved in oxyanion hole stabilization
has been investigated by site-directed mutagenesis and enzyme
Examples of SAC include naturally occurring enzymes as well
7
8
as engineered serine proteases and glycosidases. Our goal was
to engineer an enzyme to performe SAC toward 2- or 3-hydroxy-
acid esters. As a target enzyme lipase B was identified from
Candida antarctica (CALB) which has found numerous synthetic
1
6
9
kinetics. Such mutants of the serine protease subtilisin, the
applications. Our hypothesis was that the hydroxyl group in such
cysteine protease papain,17 and the esterase cutinase showed
18
substrates could substitute for an active-site residue, replaced by
reduced kcat/K
stabilization by 10-16 kJ/mol. In all cases the effect was
attributed to a large decrease in kcat and a minor change in K . It
M
values corresponding to reduced transition-state
site-directed mutagenesis in the enzyme. The catalytic machinery
of CALB consists of a Ser-His-Asp triad.10 The transition state
of the catalyzed ester hydrolysis reaction is an oxyanion (Figure
M
was thus concluded that the role of the oxyanion hole is to stabilize
the transistion state and not to facilitate binding of the substrate.
Having established the essential role of Thr40 in transition-
state stabilization in CALB, we now investigated if a hydroxyl
group positioned in the substrate could substitute for the side chain
of Thr40 replaced by site-directed mutagenesis in the mutant
lipases. The wild-type lipase showed very low substrate specific-
1
). The oxyanion hole is a spatial arrangement of hydrogen-bond
donors in the active site that is the major factor for lowering the
free energy of the transition state.11 The 3D-structures of CALB/
inhibitor-complexes suggest that the oxyanion is stabilized by two
backbone amide hydrogen atoms and the side-chain hydroxyl
group of Thr40.12 Our hypothesis was that the Thr40 side-chain
hydroxyl group of the enzyme could be replaced with one placed
19
ity for either enantiomer of ethyl 2-hydroxypropanoate compared
(
1) Faber, K. Biotransformations in Organic Chemistry, 4th ed.; Springer:
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(
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(
(
(
(
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2
(19) Substrate specificity is here defined by the ratio between the specificity
(
M
constants (kcat/K ) of the enzyme for competing substrates.
1
0.1021/ja015604x CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/12/2001