Introduction of single mutation changes arylmalonate decarboxylase to racemase

Article abstract of DOI:10.1039/b607211a

The introduction of only one mutation based on the estimated reaction mechanism endowed arylmalonate decarboxylase with a racemase activity, which catalyses racemisation of α-arylpropionates. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b607211a

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Introduction of single mutation changes arylmalonate decarboxylase to
racemase{
Yosuke Terao, Kenji Miyamoto and Hiromichi Ohta*
Received (in Cambridge, UK) 22nd May 2006, Accepted 25th July 2006
First published as an Advance Article on the web 3rd August 2006
DOI: 10.1039/b607211a
The introduction of only one mutation based on the estimated
reaction mechanism endowed arylmalonate decarboxylase with
a racemase activity, which catalyses racemisation of a-arylpro-
pionates.
Scheme 1 Asymmetric decarboxylation.
Owing to the recent development of biotechnology and informatics
as well as databases, changing the function of enzymes has become
the intermediate enolate form of a-arylpropionates from only one
possible via site-directed mutagenesis and directed evolution.1–4
side of the enantiomeric face to give (R)-products.20,21 AMDase
Most of the changes are in the range of the same type of reaction,
has some homology with some racemases and isomerases, such as
such as changing the substrate- and enantio-selectivity, increasing
glutamate racemase,22 aspartate racemase,23 and others
the activity and thermal stability.5 On the other hand, a small
(Fig. 1).24,25 These enzymes are known to be categorized into
number of mutations directed to promiscuous enzymes have also
cofactor-independent racemase and isomerase families.14 The
been reported recently.6–8 In these cases, a mutated lipase and
common points between AMDase and these enzymes are as
some acylases have been demonstrated to catalyse the aldol
follows: the intermediates are the enolate form of the products and
reaction, Michael addition, and Markovnikov addition of
the essential amino acid residue in the active site is Cys188, which
N-heterocycles to vinyl esters. In nature also, some enzymes have
delivers a proton to the intermediate enolate. On the other hand,
catalytic promiscuity with regard to different types of substrates.9
the marked difference between these enzymes is that while
Such divergent evolution is considered to be at least one of the
racemases have another Cys located in the opposite side of the
ways in which nature has achieved a wide variety of enzymes that
enantiomeric face of the intermediates, AMDase has no
catalyse a number of biotransformations.10 Especially, the inter-
corresponding Cys around this region. Thus, racemases can
play of functions and structures of enzymes belonging to the
racemise the substrates via a two-base mechanism as revealed by
enolase superfamily has been extensively studied by Gerlt’s and
Rayment’s groups.11–14 Some enzymes are sterically superimpo-
analysis of the crystal structure and the site-directed mutagen-
esis,2,26–28 while AMDase gives the optically active decarboxylated
sable with each other, nonetheless they catalyse distinct reactions.
products.
Because the intermediate of the reaction catalysed by our enzyme
Based on the homology alignment we have prepared a double
described in this paper is estimated to be the enolate of carboxylic
mutant AMDase (G74C/C188S) and confirmed that it gave the
acid similarly to the case of the enolate superfamily, we expected
opposite enantiomer of arylpropionate compared to that obtained
that a simple mutation might endow the enzyme with new catalytic
with the wild type enzyme.29
activity. In such a trial, consideration of the reaction mechanism
Now we would like to examine a further challenging idea. The
above mentioned reactivity of G74C/C188S mutant led us to
suppose that G74C single mutant might exhibit racemisation
activity towards arylpropionates, in addition to its original
decarboxylase activity.
and well-designed mutagenesis will be essential. Here, we would
like to demonstrate that introduction of a single mutation changed
the decarboxylase of malonates to the racemase of a-substituted
monobasic acids.
We have been investigating a novel and unique enzyme,
arylmalonate decarboxylase (AMDase, E.C. 4.1.1.76, originating
from Alcaligenesis bronchisepticus KU1201), which catalyses
enantioselective decarboxylation of a-aryl-a-methylmalonates to
give optically active a-arylpropionates of high enantiomeric excess
in high yields (Scheme 1).15,16 The gene encoding AMDase has
already been cloned, overexpressed in E. coli, and the enzyme has
been purified.17–19 The essential amino acid in the active site of
AMDase is revealed to be Cys188 which is considered to protonate
Although decarboxylation and racemisation are entirely differ-
ent reactions, the key intermediate is the same type of species, i.e.,
the enolate form of an a-substituted carboxylic acid. Encouraged
by this fact, we introduced a G74C mutation into a wild type
AMDase gene by site-directed mutagenesis. The E. coli JM109 was
transformed with the plasmid containing G74C mutation and
Department of Biosciences and Informatics, Keio University, 3-14-1
Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan.
E-mail: hohta@bio.keio.ac.jp; Fax: +81-45-566-1551;
Tel: +81-45-566-1703
{ Electronic supplementary information (ESI) available: Experimental
section. See DOI: 10.1039/b607211a
Fig. 1 Amino acid homology between some racemases and AMDase.
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incubated in LB medium. The overexpressed G74C AMDase was
purified in the same way as wild type enzyme.19
catalysed by the same site of wild type and mutant enzymes.
Carboxylic acid derivatives, such as alcohol (9), amide (10), nitrile
(11), and ester (12), were totally inactive, indicating that the
presence of a free carboxyl group is indispensable for G74C
AMDase. The presence of an a-aryl group is also demonstrated to
be essential for the substrates. It is worth noting that a-ethyl
compound (6) was also racemised although the reactivity was
relatively small (Table 1, entry 6). This is the only example that
exhibited racemisation activity in spite of the inactivity of the
corresponding malonate to decarboxylation reaction.
First, we examined the racemisation reaction of a-(2-naphthyl)-
propionic acid (2). The corresponding malonate is one of the best-
accepted substrates of the decarboxylation reaction of wild type
AMDase. Enantiopure 2 was prepared by our unique enzymatic
decarboxylation reaction from the corresponding arylmalonate.
Wild type AMDase gave (R)-2 with high enantiomeric excess, and
(S)-2 was obtained with the aid of G74C/C188S mutant which was
prepared recently.29
G74C mutant AMDase retained its original decarboxylase
activity, and gave racemic arylpropionates when arylmalonates
were employed as the substrates. There are two possible
explanations for the formation of racemic products. One is
racemisation of originally resulting optically active products and
the other is the direct formation of racemic monobasic acids. To
distinguish between these two mechanisms, the kinetic parameters
of decarboxylation and racemisation were measured. It was
revealed that the catalytic efficiency (kcat/Km) of racemisation of
naphthylpropionate (2) is smaller (0.56 s21 mM21) than that of
decarboxylation (0.96 s21 mM21). In addition, the decarboxylated
product at the very initial stage of the reaction was confirmed to be
racemic. These results indicate that G74C mutant decarboxylates
the substrates non-selectively, delivering a proton from both sides
of the intermediate enolate. Thus, introduction of one acidic amino
acid residue brought about the protonation activity from both
sides of the intermediate and gave the enzyme the racemisation
activity. To the best of our knowledge, this is the first report of
changing a decarboxylase to a racemase by the introduction of
only one mutation based on the reaction mechanism.
The racemization reaction was performed as follows. To a
solution of (R)-2 or (S)-2 in Tris-HCl buffer (100 mM, pH 8.5) was
added purified G74C mutant AMDase and the mixture was
incubated at 37 uC for 14 h. After quenching the reaction with 2 M
HCl, the product was extracted and its ee was measured by HPLC.
As expected, G74C mutant AMDase gave the racemic product
regardless of the configuration of the starting materials, while the
control reaction without the enzyme resulted in no change in ee.
Moreover, non-mutated wild type AMDase exhibited no racemase
activity. These results indicate that the introduction of only one
mutation, i.e., cysteine instead of glycine74, changed the
decarboxylation enzyme to a racemase. The presence of two
active cysteine residues probably enabled the two-base mechanism
to work similarly to glutamate racemase.26–28
Next, we examined the substrate specificity of racemase activity
using several compounds as shown in Table 1. In general, good
substrates for decarboxylation reaction were also good substrates
for racemisation. The most active group consisted of arylpropio-
nates, such as a-phenyl- (1), a-(2-naphthyl)- (2), and a-(2-
thienyl)propionic acid (3), which were followed by mandelic acid
(5) and a-phenylbutyric acid (6). On the other hand, the rate
of racemisation of phenylglycine (4) was very slow. The order of
reactivity of these compounds is consistent with that of
decarboxylation reactions of the corresponding malonates cata-
lysed by the wild type AMDase. On the other hand, carboxylic
acids which had larger a-substituents such as iso-propyl (7) or
n-propyl (8) were inactive similarly to the case of decarboxylation.
This is considered to be due to the limitation of the size of the
active site pocket. The similarity of substrate specificity between
decarboxylation and racemisation indicates that both reactions are
This research was supported in part by the Ministry of
Education, Culture, Sports, Science and Technology, Grant-in-
Aid for the 21st Century Center of Excellence (COE) Program
entitled ‘‘Understanding and Control of Life’s Function via
Systems Biology (Keio University)’’. Financial support from
Takeda Science Foundation is also gratefully acknowledged.
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kcat/Km
(s21/mM)
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activity
Substrate
Ar
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1
2
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4
5
6
7
8
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n-Pr
Me
Me
Me
Me
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CH2OH
CONH2
CN
0.077
0.56
0.27
0.0038
0.011
0.018
—
—
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720
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0
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