From the Department of Biochemistry, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan
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ABSTRACT |
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Directed evolution was used to change the
substrate specificity of aspartate aminotransferase. A mutant enzyme
with 17 amino acid substitutions was generated that shows a 2.1 × 106-fold increase in the catalytic efficiency
(kcat/Km) for a non-native
substrate, valine. The absorption spectrum of the bound coenzyme,
pyridoxal 5'-phosphate, is also changed significantly by the mutations.
Interestingly, only one of the 17 residues appears to be able to
contact the substrate, and none of them interact with the coenzyme. The
three-dimensional structure of the mutant enzyme complexed with a
valine analog, isovalerate (determined to 2.4-Å resolution by x-ray
crystallography), provides insights into how the mutations affect
substrate binding. The active site is remodeled; the subunit interface
is altered, and the enzyme domain that encloses the substrate is
shifted by the mutations. The present results demonstrate clearly the
importance of the cumulative effects of residues remote from the active
site and represent a new line of approach to the redesign of enzyme activity.
Despite the impressive ability of natural enzymes to catalyze a
broad array of reactions and utilize diverse substrates, attempts to
make even minor modifications in enzyme activity or substrate specificity have proven to be difficult. Efforts to change the properties of existing enzymes (1-4) have highlighted a limitation to
enzyme design; in most cases, we can consider only amino acid residues
that constitute the active site. Enzymes, however, exert their
functions not only through the chemical properties of the side chains
of the amino acid residues that contact substrates and cofactors.
Residues distant from an active site may be important in holding the
catalytic residues in their required orientations. Charge distribution
throughout a whole enzyme molecule may facilitate substrate binding by
electrostatically guiding the substrate into the active site. These
ideas are consistent with the fact that enzymes are macromolecules
composed of hundreds of amino acid residues. It has been difficult to
demonstrate by protein engineering the importance of residues that are
remote from the active site because the number of these residues is
large and the contribution of each residue may be modest. It is also
beyond our present understanding to predict a priori the
effects of the mutations of remote residues on enzymatic activity
through changes in the complex architecture of tertiary and/or
quaternary structure. Although random mutagenesis and directed
evolution have recently proven to be useful in addressing such problems
in the rational redesign of enzymes (5-10), creating enzymes with the
desired activity remains challenging.
Aspartate aminotransferase
(AspAT)1 is a homodimeric
enzyme and catalyzes amino group transfer between acidic amino acids, aspartate and glutamate, and their corresponding 2-oxo acids (11). Each
subunit has a pyridoxal 5'-phosphate (PLP) molecule at the active
center. The structure (12, 13) and reaction mechanism (14) of AspAT
have been studied extensively. AspAT from Escherichia coli
shows moderate activity for aromatic amino acids. The activity for
To alter the substrate specificity of AspAT toward Here we describe the further improvement in both the catalytic
efficiency and the Km value of AV5A-7 for valine. The affinity of a new mutant AspAT for valine is high enough to allow
crystallization of a complex between the mutant enzyme and a valine
analog. The crystal structure shows that the mutations in residues
distant from the active site cause significant changes in the higher
order structure of the enzyme, which influence substrate and cofactor binding.
Directed Evolution--
An auxotrophic E. coli
strain, YJ103 ( Expression, Purification, and Activity Measurement of Mutant
AspATs--
The coding region of the mutant AspATs was subcloned into
pUC18. The mutant enzymes were expressed in E. coli TY103
(20), which is deficient in the AspAT gene, and purified as described (21). The activity for each substrate was measured at 25 °C by
spectrophotometrically monitoring the single turnover reaction using an
Applied Photophysics stopped-flow apparatus (model SX.17MV) as
described (15). The buffer system was 50 mM Hepes, pH 8.0, containing 0.1 M KCl and 10 µM EDTA.
Crystallography--
The crystals of ATB17 complexed with
isovalerate were grown by the sitting drop vapor diffusion method.
Three microliter drops containing 37 mg/ml protein were mixed with 1 µl of 0.2 M sodium isovalerate and 3 µl of the
reservoir solution containing 1.6 M ammonium sulfate and
0.1 M Na·Hepes, pH 7.5. The drops were equilibrated
against 0.5 ml of the reservoir solution at 20 °C. An x-ray data set
was collected with a Rigaku R-AXIS IIc image plate detector mounted on
a Rigaku RU-200 rotating anode generator operated at 40 kV and 100 mA
with monochromatized CuK Creation of a Mutant AspAT with Higher Affinity for
Valine--
Sequence analysis of the mutant AspATs obtained from the
previous selection showed that 5 of the 13 substitutions found in AV5A-7, Asn34
In the present study, we used two different strategies to find
additional beneficial mutations. In one approach, we added each of the
20 unique substitutions found in four selected mutant AspATs, AV5A-1,
AV5B-1, AV5B-4, and AV5B-5 (10), to AV5A-7 one mutation at a time and
assayed each new mutant for 2-oxovaline activity (data not shown). Two
substitutions, Ser361 Characterization of the Mutant AspAT--
Compared with AV5A-7,
the kcat/Km values of ATB17
for branched-chain substrates are increased, whereas those for acidic
2-oxo acid substrates are decreased (Table
I). In particular, the
Km value for valine is decreased 76-fold to 5.5 mM. The kcat/Km
value of ATB17 for valine or 2-oxovaline is increased >2.1 × 106 or 6.7 × 105-fold, respectively, and
that for isoleucine or 2-oxoisoleucine is increased >6.0 × 104 or 5.4 × 105-fold, respectively,
compared with that of the wild-type AspAT. ATB17 retains significant
activity for acidic substrates. Although this activity could have been
easily eliminated by a single mutation at Arg292 (see below
and also Refs. 16 and 17), none of the mutants examined had such
mutations. Probably Arg292 was maintained simply because no
selection pressure was applied to minimize the activity for acidic
substrates or because acidic amino acids may serve as the amino group
donors to 2-oxovaline in E. coli cells growing under the
selection conditions.
During the purification of ATB17, we noticed that the color of the
fractions containing the enzyme was orange, rather than yellow like the
wild-type AspAT and AV5A-7. Thus, the absorption spectra of the
purified AspATs were measured (Fig. 1B). AspAT has
absorption bands in the region 300-500 nm, which derive from the bound
PLP molecule and are influenced significantly by amino acid residues
interacting with PLP (11, 26, 27). The wild-type AspAT and AV5A-7
exhibit two major bands around 360 and 430 nm, whereas in ATB17 the
latter band is red-shifted to 450 nm and has a broad shoulder above 500 nm. The differences in the relative intensity of the two bands between
the wild-type and mutant AspATs show that the pKa of
the imine nitrogen of the Schiff base formed between PLP and
Lys258 (26, 27) is decreased in both mutant AspATs. These
changes in the absorption spectra imply that the electronic
distribution within the
Despite such drastic changes in the substrate specificity and
absorption spectrum, only one of the mutated residues in ATB17 appears
to be located at a position contacting the substrate, and none of them
interact directly with PLP. The higher affinity of ATB17 for valine
allowed us to crystallize ATB17 in the presence of an amino-free valine
analog, isovalerate, to further investigate the effects of the mutations.
Effects of the Mutations on the Tertiary and Quaternary Structure
of AspAT--
The x-ray crystal structure of ATB17 complexed with
isovalerate was solved at 2.4-Å resolution (Table
II) and was compared with the wild-type
AspAT complexed with an aspartate analog, maleate (Fig.
2). Three features stand out in the gross
structure of ATB17. First, the spatial arrangement of the two subunits
of the dimer is altered. Second, the domain motion may be enhanced in
ATB17. Third, two clusters of mutated residues are observed, one shown in green (Leu-24, Asp-34, Met-37, and Asn-41) and the other shown in
red (Thr-353, Phe-361, Gly-363, Leu-387, and Leu-397) in Fig. 2. These
findings may provide clues to elucidate how the non-active site
mutations influence the substrate binding of AspAT and thus are further
described below.
The rearrangement of the two subunits in ATB17 is evident. When the
large domain (residues 49-325) of one subunit of ATB17 was
superimposed on that of the wild-type AspAT, the other subunits of the
two enzymes overlapped poorly (deviated about 1 Å in the core region
of the large domain) (Fig. 2). This difference in the subunit
arrangement likely affects substrate binding because the active site is
located at the subunit interface, although it is not known which
mutations caused this subunit rearrangement.
The structure of AspAT changes from an "open" to a "closed"
conformation when the substrate binds to the enzyme (12, 13). In the
ATB17-isovalerate structure, the domain closure is enhanced. The small
domain of ATB17 comes closer to the active site compared with the
corresponding domain of the wild-type AspAT. One of the clusters (Fig.
2, green), of which Met-37 is the only residue interacting
with isovalerate, is located at the lid of the active site which closes
against the bound substrate. The other cluster (Fig. 2, red)
is in the core of the small domain, except Leu-397, and may affect the
domain motion given that these residues are located at the domain
interface. Thus, these two clusters may influence in concert the domain motion.
Detailed analysis may help us to understand how the two clusters of
mutated residues cause the observed conformational changes. Fig.
3 shows that the enzyme structure is
changed especially around the two clusters. The
Leu20-Ile33 loop (the lid of the active site)
shifts toward the active site while maintaining the overall
configuration of the loop structure (Fig.
4B). The average deviation is
about 1.5 Å, but the deviation is larger around the tip of the loop
than around its bottom. This cluster of mutations is located at the
hinge-like region and thus may cause the shift of the loop. On the
other hand, the largest deviation in the whole molecule is observed at
the domain interface (Fig. 3, position 363, and Fig.
4C). The backbone of Gly363-Gly364
has flipped toward the solvent side. The Ser361 The Active Sites and Substrate-binding Modes of the Mutant and
Wild-type AspATs--
Trp140 is thought to adjust the tilt
of the PLP molecule during the course of catalysis by AspAT (11, 14).
In the ATB17-isovalerate structure, large conformational changes are
observed in the Pro138-Thr142 loop (Fig. 3 and
Fig. 4A). This loop contains two substitutions, Ser139
The wild-type AspAT has two arginine residues, Arg292 and
Arg386, that are essential for substrate binding.
Arg292 interacts with the side chain carboxylate group of
acidic substrates. The side chain of Arg292 of ATB17
protrudes into solvent, whereas that of the wild-type AspAT flips
toward the active site interacting with the carboxylate group of
maleate (Fig. 4A). The same shift in the position of Arg292 was observed previously in the crystal structure of
a mutant AspAT complexed with aromatic substrate analogs (28). It is therefore possible that the difference in the position of
Arg292 between the two enzymes is caused by the difference
in the bound substrate analogs, dicarboxylic maleate and monocarboxylic
isovalerate, rather than by the mutations of ATB17. Arg386
is also an important residue of which the side chain interacts with the
One of the side walls of the active site consists of the residues
belonging to the other subunit (Fig. 4A,
asterisks). Thus, the changes in the subunit interface in
ATB17 have caused deviations of these residues. This portion of the
active site also contains two substitutions, Ala293
Met37 constitutes the upper wall of the isovalerate binding
pocket and is the only mutated residue contacting isovalerate. The Ile37 Conclusions--
The isolation of a mutant AspAT with a
million-fold increase in the catalytic efficiency for a non-native
substrate shows that directed evolution is a powerful technique for
altering enzyme activity. Our present selection system could also be
used to produce an enzyme with transaminase activity from that with an
unrelated activity. The three-dimensional structure of a complex
between the mutant AspAT and a substrate analog suggests that
conformational changes in the enzyme are responsible for the alteration
in the substrate specificity. Significantly, only one of the 17 mutated residues contacts the substrate directly. Several of the mutations that
particularly enhance the activity for
Most attempts to redesign enzyme activity by mutating only active site
residues have met with limited success. It may turn out to be common
rather than exceptional that changing enzyme activity requires
remodeling the active site through changes in the backbone flexibility,
domain motion, or subunit arrangement. Results supporting this idea
were recently reported for a catalytic antibody (29, 30). These
findings emphasize the benefits of directed evolution over rational
design and would justify present and future intense efforts to develop
new strategies for directed evolution.
INTRODUCTION
Top
Abstract
Introduction
References
-branched amino acids, valine and isoleucine, is barely detectable and even lower than that for basic amino acids (15). Several studies
were previously done to increase the activity for basic or aromatic
amino acids (16-18). These results imply that the substrate specificity of AspAT like many other enzymes cannot be easily manipulated by mutating one or a few active site residues.
-branched
amino acids, we established an experimental system based on directed evolution where mutant AspATs with higher activity for branched-chain substrates, especially valine and 2-oxovaline, evolve
during successive rounds of selection (10). Briefly, the selection
system uses an auxotrophic E. coli strain, which is
deficient in the gene for branched-chain amino acid aminotransferase. The higher the activity of a plasmid-encoded mutant AspAT for 2-oxovaline is, the faster the auxotrophic E. coli carrying
the plasmid grows on a selection plate. After five rounds of the
selection, the catalytic efficiency
(kcat/Km) of mutant AspATs
for
-branched substrates was increased about 105-fold.
One of the mutant AspATs showing the highest activity (AV5A-7) was
analyzed in detail (10). The mutant had 13 amino acid substitutions, but interestingly, only one of the mutated residues seemed to interact
directly with the substrate based on the three-dimensional structure of
the wild-type AspAT. To elucidate the effects of these mutations on
substrate binding, we set out to determine the three-dimensional
structure of the mutant AspAT complexed with a
-branched substrate
analog. The affinity of AV5A-7 for valine was, however, still too low
(Km = 400 mM) to yield a crystal of such
an enzyme-inhibitor complex.
MATERIALS AND METHODS
ilvE::kan), the selection
medium, 5' and 3' primers used for the polymerase chain reaction amplification of the coding region of aspC, and the
construction of the expression plasmid were reported previously (10).
Briefly, the first round of selection was done as follows. The coding
region of the aspC gene containing the three mutations,
Ser139
Gly, Asn142
Thr, and
Asn297
Ser, was subjected to DNA shuffling (6, 9, 19),
and the mutant genes were ligated downstream of the promoter of the tetracycline resistance gene of pBR322. After a 45-h incubation at
37 °C, 116 colonies from a library of 5.6 × 107
colonies were picked up, and a mixture of the plasmids was prepared. As
for the second and third rounds of selection, the conditions were the
same as those for the first round except the incubation time: the
second round, a 40-h incubation, a library size of 7.9 × 107 colonies, and 136 colonies picked; the third round, a
28-h incubation, a library size of 2.8 × 107
colonies, and 48 colonies picked. Among the 48 clones, 9 clones exhibiting the highest activity for 2-oxovaline were chosen, and the
coding regions of the aspC genes were sequenced.
radiation at room temperature.
The oscillation images were processed and reduced using a data
processing software, Rigaku PROCESS (22). Refinement of the structure
began with the structure of the wild-type AspAT2 (12) as an initial
model using X-PLOR 3.851 (23) with parameters derived by Engh and Huber
(24). After conventional positional refinement, simulated annealing
using the slow cool protocol was performed. The models were improved by
conventional positional refinement and the isotropic
B-factor refinement, and manual rebuilding using Xfit (25)
on the omit map was calculated with the coefficients |Fo|
|Fc|. After
R-factors were adequately lowered, water molecules were
added to the models, and the structures were further refined.
RESULTS AND DISCUSSION
Asp, Ile37
Met,
Ser139
Gly, Asn142
Thr, and
Asn297
Ser, were conserved among all of the mutants
examined, and these substitutions were found to be functionally
important (10). The fact that one substitution (Val387
Leu) that significantly increased the activity for
-branched substrates was unique to AV5A-7 (10), however, implied that the
sequence space had not yet been fully searched. Despite this finding,
the initial selection system appeared to have reached its limit after
five rounds of selection, because further increases in the catalytic
efficiency for
-branched substrates did not benefit the growth of
the host E. coli cells under the most stringent selection conditions.
Phe and Ser363
Gly, were chosen in this manner. In a second strategy, directed evolution was once again employed. This time, however, the experiment was started from a mutant AspAT that had three substitutions, Ser139
Gly, Asn142
Thr, and
Asn297
Ser, to facilitate the evolution. After three
rounds of selection, mutant AspATs that showed similar activity for
2-oxovaline to that of AV5A-7 were obtained, and the coding regions of
9 mutant AspATs were sequenced. Each mutant AspAT had 4-9 additional
substitutions, and, again, Asn34
Asp and
Ile37
Met were conserved in all the mutants. After
adding each potential substitution one at a time to
AV5A-7/Ser361
Phe/Ser363
Gly, 3 substitutions, Ala11
Thr, Phe24
Leu,
and Ile353
Thr, were chosen. One of the 13 substitutions of AV5A-7, Glu7
Val, was mutated back to
the wild-type sequence because the substitution was found to decrease
the expression level of AspAT while not affecting the activity for
-branched substrates. The resulting mutant, ATB17, thus has 17 substitutions (Fig. 1A), 11 of
which are clearly functionally important. As for the other 6 substitutions, Lys41
Asn, Lys126
Arg,
Ala269
Thr, Ala293
Val,
Ser311
Gly, and Met397
Leu, of which
the contribution to the total effect was 10-20% in AV5A-7, the
importance of each substitution could not be determined (10).
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Fig. 1.
Analysis of protein sequences and absorption
spectra of AspATs. A, amino acid residues mutated in
AV5A-7 (10) and ATB17. B, absorption spectra of the
wild-type AspAT (solid line), AV5A-7 (dotted
line), and ATB17 (broken line). All the spectra were
measured in a 50 mM Pipes buffer, pH 6.5, at a protein
concentration of 19 µM.
Kinetic parameters of AspATs for branched and acidic substrates
-electron system of the bound PLP molecule
is changed significantly by the mutations, although the nature of these
changes is difficult to predict.
Crystallographic parameters
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Fig. 2.
Superimposition of the ATB17-isovalerate
complex (pink, large domain; gray, small domain) and the
wild-type AspAT-maleate complex (purple). The backbone
of one subunit of ATB17, of which the large domain (residues 49-325)
was superimposed on that of the wild-type AspAT (31), is indicated by a
thick line. The NH2- and COOH termini of the
subunit are indicated (N and C). The side chains
of the residues that were mutated in ATB17 are shown as follows: the
two clusters of the residues (see text) are in red and
green, and the other residues are in light blue.
The coenzyme, pyridoxal 5'-phosphate (yellow), and the bound
valine analog, isovalerate (dark blue), are also shown.
Maleate of the wild-type AspAT-maleate complex is omitted. Figs. 2 and
4 were produced with MOLSCRIPT and RASTER 3D (32-34).
Phe
substitution would cause a steric hindrance due to its phenyl side
chain. The Ser363
Gly substitution yields two
consecutive glycine residues of which the flexible backbone would flip
to release the unfavorable strain. The effects of these two
substitutions are, however, not fully cooperative because each
substitution independently increased the activity for 2-oxovaline when
added to AV5A-7. Other changes observed in Fig. 4C would
have occurred to readjust the packing of the side chains surrounding
the cluster of the mutated residues. All the above findings, including
changes in the dimer interface, likely influence the substrate
specificity of AspAT, but we cannot explain how these changes allow
AspAT to accommodate
-branched substrates.
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Fig. 3.
Deviations of the C atoms between the
ATB17-isovalerate complex and the wild-type AspAT-maleate
complex. The large domains of the two structures (269 C
atoms
for each) were superimposed with the root mean square deviation of
0.316 Å. The root mean square deviation for all the 396 C
atoms was
0.552 Å. Figs. 2 and 4 show the superimposed images obtained in this
manner. The numbers of the residues which showed large deviations are
indicated.
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Fig. 4.
Close-up views of the active site and the two
clusters of the mutated residues. The residues are colored as
shown in Fig. 2. The structure of the ATB17-isovalerate complex is
indicated by thick gray lines and that of the wild-type
AspAT-maleate complex is indicated by thin purple lines.
A, several water molecules are introduced into the active
site of ATB17 (light blue spheres). One water molecule
(WAT1) is located at almost the same position as a water
molecule observed in the wild-type AspAT (a purple sphere).
Arg292, Val293, and Ser297 belong
to the other subunit of the dimer (asterisks). B
and C, viewed from the same direction as in Fig. 2.
Gly and Asn142
Thr.
Trp140 is moved toward the "bottom" of the active site,
making more space in the substrate binding pocket. The indole ring of
Trp140 is stacked against the pyridine ring of PLP by the
hydroxyl group of Thr142, which is bulkier than the
hydrogen atom at the corresponding position of the side chain of the
original Asn142. The resulting tight interaction of the
election systems of the indole ring and PLP may in part explain the
spectral changes observed in ATB17. Interestingly, one of the evolved
mutants we reported previously (10), AV5B-4, had an isoleucine residue at position 142, which also has a bulky methyl group at C
of the
side chain. Thus, the PLP molecule may be required to be pushed back by
Trp140 for AspAT to bind
-branched substrates.
-carboxylate group of all amino acid substrates. The orientation of
the Arg386 side chain of ATB17 remains essentially
unaltered despite the large conformational changes adjacent to
Arg386 (Fig. 4, A and C).
Val
and Asn297
Ser. Although the functional importance of
the Ala293
Val substitution is not clear, the
Asn297
Ser substitution increased the activity of
AV5A-7 for 2-oxovaline (10). It was reported previously for the mutant
AspAT with increased activity for aromatic substrates that the
Asn297
Ser substitution eliminates a structural water
molecule which is held in place by the side chains of
Ser296 and Asn297 in the wild-type AspAT,
concomitantly introducing new water molecules (28). Malashkevich
et al. (28) proposed that this substitution makes space for
the binding of bulky aromatic substrates. In our case, however, the
side chain of isovalerate is much smaller than those of aromatic
substrates, and, indeed, a water molecule (Fig. 4A, WAT1) is
re-introduced at almost the same position, constituting the side wall
of the isovalerate-binding pocket together with the other newly
introduced water molecules. We thus cannot explain how the
Asn297
Ser substitution increases the activity for valine.
Met substitution would apparently benefit the
binding of
-branched substrates by removing the steric hindrance
between the
-methyl groups of the Ile37 side chain and
the bound substrate. To determine how important this substitution is,
we mutated Met37 of ATB17 back to isoleucine. The
kcat/Km values of this revertant for valine and 2-oxovaline were decreased 4- and 2-fold, respectively; the kcat, Km,
and kcat/Km values for valine
were 15 ± 1 s
1, 30 ± 5 mM, and
500 s
1 M
1, respectively, and
those for 2-oxovaline were 31 ± 2 s
1, 1.8 ± 0.3 mM, and 1.7 × 104 s
1
M
1, respectively. This shows that the
Ile37
Met substitution contributes only 5-10% to the
total effect of all the mutations of ATB17 (calculated as follows:
(1
(RT ln{(kcat/Km)ATB17/Met37
Ile/(kcat/Km)WT})/(RT ln{(kcat/Km)ATB17/(kcat/Km)WT})) × 100 (%)).
-branched substrates are
located >10 Å distance from the active site.
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ACKNOWLEDGEMENTS |
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We thank K. Hirotsu for help in the x-ray data collection and V. W. Cornish and D. R. Liu for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported by the Ministry of Education, Science, Sports, and Culture of Japan (to T. Y.) and the Japan Society for the Promotion of Science ("Research for the Future" Program) (to H. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (code 1yoo) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
To whom correspondence should be addressed: Dept. of Biochemistry,
Osaka Medical College, Takatsuki, Osaka 569-8686, Japan. Tel.:
81-726-83-1221 (ext. 2644); Fax: 81-726-84-6516; E-mail: med001{at}art.osaka-med.ac.jp.
The abbreviations used are: AspAT, Aspartate aminotransferase; PLP, pyridoxal 5'-phosphate; 2-oxovaline, 2-ketoisovaleric acid; Pipes, 1,4-piperazinediethanesulfonic acid.
2 The atomic coordinates for the crystal structure of the wild-type AspAT have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY, code 1ART (12).
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REFERENCES |
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