Directed evolution of arylmalonate decarboxylase
5
and M159 are in a catalytically important locus, sug-
gesting that the amino acids in this locus could be tar-
gets for further mutagenesis to improve the level of
activity.
been structurally determined.23) The structures of
AMDase (PDB: 3IXL) and MI (PDB: 2XEC) were
well aligned, where P132 of MI corresponding to A125
of AMDase would be unlikely to disrupt the structural
conformation. To investigate the effect of the A125P
single mutation on enzymatic activity, an additional
variant, G74C/A125P/V156L/M159L/C188G (CLGL-
P), was prepared. The four AMDase variants were puri-
fied, and their activity toward 1a was measured
(Table 2). All variants showed excellent (S)-enantiose-
lectivity (>99% e.e.). CLGL-MP showed an equivalent
level of specific activity (3.5 U/mg) to that of CLG-L
(3.5 U/mg). On the other hand, CLGL-I, CLGL-P, and
CLGL-IP showed activity levels of 5.0, 4.6, and 7.1 U/
mg, respectively. In the kinetic analysis of CLGL-IP
against 1a, the variant showed 2.2-fold improvement in
kcat value with a slight increase in Km value, resulting
in a 2.1-fold improvement of catalytic efficiency
(kcat/Km), compared with CLG-L (Table 3). These
results suggest that the structural alteration of the
hydrophobic pocket by V43I mutation and indirect
modification of the hydrophobic pocket and/or dioxyan-
ion hole by A125P induced a better configuration of
the active site for the decarboxylation of the pro-R car-
boxyl residue of 1a. Consequently, the most active (S)-
selective variant, CLGL-IP, achieved 9,500-fold greater
Kcat/Km values compared with the ancestral (S)-selec-
tive AMDase variant CS (Table 3).
SSM targeting the region around V156 and M159 of
(S)-selective AMDase
The second-round SSM was performed on a gene
encoding CLG-L to further improve the activity. We
selected V43 and A125 as mutagenesis targets (Fig. 3).
V43 is one of the residues forming the hydrophobic
pocket and is situated close to both V156 and M159 at
distances of 4.1 and 3.8 Å, respectively, in the crystal
structure of the CS variant. Also, V43 has been
reported to interact with the methyl group of 1a.15) On
the other hand, A125 is located close to M159 (3.7 Å)
and behind V43 at a distance from the side of substrate
in the three-dimensional structure of CS. A125 is adja-
cent to Y126, one of the residues that form the
dioxyanion hole that stabilizes the pro-S carboxyl resi-
due of the substrate (Fig. 2). Thus, we expected that
mutagenesis toward A125 would indirectly optimize
the configurations of M159 and/or Y126 into catalyti-
cally suitable forms. These two target residues, V43
and A125, were simultaneously mutated using the
degenerated codon DTK (donating Leu, Ile, Val, Met,
and Phe) and NNK (donating all amino acids), respec-
tively. To cover >95% variety of the mutant gene
library coding V43X1/A125X2 (X1 = L, I, V, M and F/
X2 = all amino acids), the size of the required library
was estimated to be 574. In the activity screening of
672 E. coli transformants, three variants, V43M/G74C/
A125P/V156L/M159L/C188G (CLGL-MP), V43I/
G74C/V156L/M159L/C188G (CLGL-I), and V43I/
Substrate specificity of the improved (S)-selective
AMDase variants
The specific activities of CLG and its variants
toward α-(6-methoxynaphtyl)-α-methylmalonate (1b)
and α-(4-isobutylphenyl)-α-methylmalonate (1c), pre-
cursors of the analgesic compounds naproxen (2b) and
ibuprofen (2c), respectively, and phenylmalonate (1d)
were determined (Table 5). All (S)-selective variants
showed rigorous (S)-enantioselectivity toward 1b and
1c. Using the most active CLGL-IP variant, the decar-
boxylation reaction proceeded in >95% yield. CLGL-IP
showed the highest level of activity toward all sub-
strates: 2.3- to 6.8-fold compared with CLG, among
the (S)-selective AMDase variants tested. It is worth
discussing the effects of the substitutions of the aryl
group of the substrate on the specific activity of each
variant. CLG-I and CLG-L showed equivalent activity
G74C/A125P/V156L/M159L/C188G
(CLGL-IP),
showed increased activities compared with the template
CLG-L. Interestingly, A125P variant produced by the
replacement of an α-helical element A125 to proline
was included in two of the three variants (CLGL-MP
and CLGL-IP), despite the possibility that such a sub-
stitution could inhibit the formation of the α-helix
structure. Some amino acid racemases, which are
highly similar to AMDase on the basis of amino acid
sequence, harbor a proline at the corresponding posi-
tion to A125 of AMDase (Table 4). Among these pro-
teins, maleate cis-trans isomerase (MI) has already
Table 4. Amino acid sequence alignment between AMDase and Asp/Glu superfamilies.
Enzyme
Species
Sequence
AMDasea
AMDasea
AMDasea
Asp/GluRb
Asp/GluRb
Asp/Glu/hydRc
MId
Bordetella bronchiseptica KU1201 (This study)
Achromobacter sp. KU 1311
Enterobacter cloacae KU1313
Alkaliphilus metalliredigens QYMF
Caldivirga maquilingensis IC-167
Ilyobacter polytropus DSM 2926
Pyrococcus furiosus DSM 3638
Thermococcus sibiricus MM 739
Thermococcus litoralis DSM 5473
Halogeometricum borinquense DSM 11551
ALATAYIDDV
ALATAYIDDV
ALATAYIDDV
AVATPYIDEV
ALATPYTNDV
LVVTPYIDEL
VVVTPYIDEI
LVVTPYTDEI
LVVTPYTDEI
GVVTPYTEEL
MId
MId
MId
aArylmalonate decarboxylase.
bAspartate/glutamate racemase.
cAspartate/glutamate/hydantoin racemase.
dMaleate cis-trans isomerase.