Substitutions of coenzyme-binding, nonpolar residues improve the low-temperature activity of thermophilic dehydrogenases

Article abstract of DOI:10.1021/bi200925f

Although enzymes of thermophilic organisms are often very resistant to thermal denaturation, they are usually less active than their mesophilic or psychrophilic homologues at moderate or low temperatures. To explore the structural features that would improve the activity of a thermophilic enzyme at less than optimal temperatures, we randomly mutated the DNA of single-site mutants of the thermostable Thermus thermophilus 3-isopropylmalate dehydrogenase that already had improved low-temperature activity and selected for additional improved low-temperature activity. A mutant (Ile279 → Val) with improved low-temperature activity contained a residue that directly interacts with the adenine of the coenzyme NAD⁺, suggesting that modulation of the coenzyme-binding pocket's volume can enhance low-temperature activity. This idea was further supported by a saturation mutagenesis study of the two codons of two other residues that interact with the adenine. Furthermore, a similar type of amino acid substitution also improved the catalytic efficiency of another thermophilic dehydrogenase, T. thermophilus lactate dehydrogenase. Steady-state kinetic experiments showed that the mutations all favorably affected the catalytic turnover numbers. Thermal stability measurements demonstrated that the mutants remain very resistant to heat. Calculation of the energetic contributions to catalysis indicated that the increased turnover numbers are the result of destabilized enzyme-substrate-coenzyme complexes. Therefore, small changes in the side chain volumes of coenzyme-binding residues improved the catalytic efficiencies of two thermophilic dehydrogenases while preserving their high thermal stabilities and may be a way to improve low-temperature activities of dehydrogenases in general.

Full text of DOI:10.1021/bi200925f

Article
pubs.acs.org/biochemistry
Substitutions of Coenzyme-Binding, Nonpolar Residues Improve the
Low-Temperature Activity of Thermophilic Dehydrogenases
Sayaka Hayashi, Satoshi Akanuma, Wakana Onuki, Chihiro Tokunaga, and Akihiko Yamagishi*
Department of Molecular Biology, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392,
Japan
S
* Supporting Information
ABSTRACT: Although enzymes of thermophilic organisms
are often very resistant to thermal denaturation, they are
usually less active than their mesophilic or psychrophilic
homologues at moderate or low temperatures. To explore the
structural features that would improve the activity of a
thermophilic enzyme at less than optimal temperatures, we
randomly mutated the DNA of single-site mutants of the
thermostable Thermus thermophilus 3-isopropylmalate dehy-
drogenase that already had improved low-temperature activity
and selected for additional improved low-temperature activity.
A mutant (Ile279 → Val) with improved low-temperature activity contained a residue that directly interacts with the adenine of
the coenzyme NAD+, suggesting that modulation of the coenzyme-binding pocket’s volume can enhance low-temperature
activity. This idea was further supported by a saturation mutagenesis study of the two codons of two other residues that interact
with the adenine. Furthermore, a similar type of amino acid substitution also improved the catalytic efficiency of another
thermophilic dehydrogenase, T. thermophilus lactate dehydrogenase. Steady-state kinetic experiments showed that the mutations
all favorably affected the catalytic turnover numbers. Thermal stability measurements demonstrated that the mutants remain very
resistant to heat. Calculation of the energetic contributions to catalysis indicated that the increased turnover numbers are the
result of destabilized enzyme−substrate−coenzyme complexes. Therefore, small changes in the side chain volumes of coenzyme-
binding residues improved the catalytic efficiencies of two thermophilic dehydrogenases while preserving their high thermal
stabilities and may be a way to improve low-temperature activities of dehydrogenases in general.
nderstanding how homologous proteins from different
organisms adapt their functions and tertiary structures to
psychrophilic proteins are a consequence of conformational
flexibility in regions involved in catalysis.14−16 However, the
structural origin(s) of such flexibility has not been delineated.
Moreover, structural comparisons of psychrophilic and
mesophilic or mesophilic and thermophilic homologous
proteins have not elucidated a structural feature(s) that can
be correlated with low-temperature adaptation of enzyme
activity.16
Characterization of mutated enzymes with increased low-
temperature activities can be used to identify the structural
properties that improve catalytic activity at lower than optimal
temperatures and could be applied to industrial applications
that use thermophilic enzymes. Because robust biocatalysts are
necessary for most industrial applications, thermophilic
enzymes are attractive tools.5,17−19 However, a major drawback
to their use is the fact that they are generally much less active at
moderate temperatures even though their thermal stability is an
asset. Therefore, improving the low-temperature activity of
thermophilic enzymes offers an economically beneficial tool
for industrial processes.20 Many attempts have been made to
U
different environments is an important aspect of function−
structure studies. Structural comparisons among thermophilic
and mesophilic proteins have suggested that various factors,
e.g., a reduced number of neutral polar residues, improved
hydrophobic interactions and packing density in the core, an
increased number of ion pairs and ion pair networks on the
protein surface, an increased number of subunits in an
oligomeric protein, and an increased number of ion pairs or
improved hydrophobic interactions between subunits, might
be responsible for the increased stability of thermophilic
proteins.1−10 However, we still do not have a comprehensive
understanding of structure−stability relationships because no
single structural feature modulates the stabilities of thermo-
philic proteins and because the contribution by each factor is
usually small and often strongly depends on its structural
context. A similar uncertainty exists concerning how proteins
physically adapt to lower temperatures.
Increasing numbers of low-temperature-adapted proteins are
being isolated from psychrophilic organisms, and these proteins
are generally less thermally stable and have greater catalytic
activities at low temperatures than their mesophilic and
thermophilic homologues.11−13 Several researchers have
proposed that the physical and catalytic properties of
Received: June 15, 2011
Revised: September 1, 2011
Published: September 6, 2011
© 2011 American Chemical Society
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improve the low-temperature activities of mesophilic and thermo-
philic enzymes.21−28 Early studies clearly demonstrated that one
or a small number of mutations can improve a protein’s
low-temperature activity. However, catalytic adaptation to
lower temperatures is often the consequence of many different
types of subtle conformational changes, and no predictive rules,
based on physical and/or chemical guidelines, have been
established.
30 times); and step 3, 20 min at 72 °C. For five arbitrarily
selected mutated genes, a total of 21 base substitutions were
found, which suggested that, on average, 4.2 base substitutions
were present in each gene. The mutated genes were digested with
XbaI and EcoRI and then each ligated into a pUC19 plasmid. The
ligates were each amplified in Escherichia coli JM109, and then
the plasmids were recovered (denoted library 1).
Selection. leuB-deficient E. coli strain OM17, which was
constructed from E. coli JM105 via deletion of its chromosomal
leuB,29 was transformed with library 1. The transformed cells were
plated onto agar containing M9 minimal medium and
50 μg/mL ampicillin. After 2 days at 25 °C, colonies that
contained a library 1 plasmid and grew faster than OM17 colonies
that contained the corresponding parent plasmid were selected.
Site-Directed Mutagenesis. Site-directed mutagenesis of
leuB was conducted using the splicing-by-overlap-extension
PCR method.36 For amplification, the PCR mixture contained
1× PCR buffer for KOD-plus polymerization (Toyobo), 1 mM
MgSO4, dNTPs (0.2 mM each), synthetic oligonucleotides
(0.2 μM each), and 1.0 unit of KOD-plus DNA polymerase.
The time−temperature program was as follows: step 1, 95 °C
for 3 min; step 2, 95 °C for 30 s; step 3, 55 °C for 30 s; step 4,
68 °C for 1 min (steps 2−4 were repeated 25 times). After
amplification, the PCR product was digested with NdeI and
BamHI (New England Biolabs) and then cloned into the
NdeI−BamHI site of pET21c. The genes encoding LDH A75G
and V99I were constructed in a similar manner.
Saturation Mutagenesis and Selection. The codons for
L254 and G255 in wild-type leuB were replaced with NNS
(N = A, C, G, or T; S = C or G) using the splicing-by-overlap-
extension PCR method. The PCR conditions were the same as
those used for site-directed mutagenesis (see above). The
amplified, mutated genes were digested with XbaI and EcoRI
and then each ligated into a pUC19 plasmid. The ligates were
then amplified in E. coli JM109, after which the plasmids were
recovered (denoted library 2). For selection, leuB-deficient
E. coli OM17 was transformed with library 2 and then plated
onto agar containing M9 minimal medium, 50 μg/mL
ampicillin, and 5 μM isopropyl thio-β-D-galactopyranoside.
The plates were incubated at 25 °C, and colonies that grew
faster than the OM17 strain harboring the T. thermophilus wild-
type leuB gene were selected after 2 days.
In our laboratory, efforts have been made to improve the
low-temperature catalytic activity of 3-isopropylmalate dehy-
drogenase (IPMDH) from the extreme thermophile Thermus
thermophilus.29−32 The enzyme is the product of leuB and
participates in leucine biosynthesis. The catalytic properties and
thermal stability of T. thermophilus IPMDH are well-
characterized.33 Its three-dimensional crystal structure has
been determined at 2.2 Å resolution34 and shows the enzyme
to be a homodimer, with subunits of 345 amino acid residues. A
number of T. thermophilus IPMDH mutants that are catalyti-
cally more active than the wild-type enzyme at 30 or 40 °C
have been isolated from a library composed of randomly
mutated IPMDH genes.29,30 These mutants had an improved
NAD+
Km
value for the coenzyme, nicotinamide adenine
dinucleotide (NAD+), or an increased kcat value. The mutations
are distributed throughout its tertiary structure, and therefore,
no conclusions concerning structural features that modulate
low-temperature activity could be made.
For the study reported herein, to identify a general rule(s)
that can be used to guide the design of low-temperature-
adapted mutants of thermostable enzymes, we randomly
mutated five single-site T. thermophilus IPMDH mutants that
had been shown to have improved low-temperature activity and
then selected for mutants with additionally improved low-
temperature activity. The results suggested that a small change
in the side chain volume of a residue with a nonpolar side chain
(I279) that interacts with the adenine of NAD+ was responsible
for the additional improvement in the low-temperature activity.
This hypothesis was supported by saturation mutagenesis of the
codons of two residues that also interact with the adenine.
Moreover, a similar mutation improved the catalytic activity of
the thermophilic T. thermophilus lactate dehydrogenase (LDH)
at 25 °C. Thus, modulation of the coenzyme-binding site
volume may be a generic means of controlling the low-
temperature activity of thermophilic dehydrogenases.
Enzyme Purification. To prepare and purify the T.
thermophilus wild-type and mutant IPMDHs, we cultivated
E. coli MA153 inoculums each harboring one of the expres-
sion plasmids in LB medium supplemented with ampicillin
(150 μg/mL). After overnight cultivation at 37 °C, cells were
harvested and disrupted by sonication. The soluble fractions
were each isolated after centrifugation at 60000g for 20 min.
The supernatants were individually heated at 70 °C for 20 min
and centrifuged at 60000g for 20 min. To purify the enzymes,
the supernatants were each successively chromatographed
through HiTrapQ (GE Healthcare Bioscience) and butyl-
Toyopearl (Tosoh). To prepare and purify the T. thermophilus
wild-type and mutant LDHs, we individually cultivated E. coli
Rosetta2 (DE3) inoculums each harboring one of the expres-
sion plasmids in LB medium supplemented with ampicillin
(150 μg/mL). After overnight cultivation at 37 °C, cells were
harvested and disrupted by sonication. The soluble fractions
were each isolated after centrifugation at 60000g for 20 min. The
supernatants were individually heated at 75 °C for 15 min and
centrifuged at 60000g for 20 min. To purify each enzyme, its
respective supernatant was successively chromatographed through
In this report, the mutants denoted R85C and I279V are
those in which Arg85 was replaced with a cysteine and Ile279
was replaced with a valine, respectively. Mutants that contain
two or more mutations are denoted with a slash (e.g., R85C/
I279V).
EXPERIMENTAL PROCEDURES
■
Random Mutagenesis by Error-Prone Polymerase
Chain Reaction (PCR). The genes encoding the
T. thermophilus IPMDH mutants that contained a G12 → S,
K21 → T, R85 → C, S248 → T, or A335 → V substitution
were each cloned into the XbaI−EcoRI site of a pUC19 plasmid
and then used as a template mixture for a slightly modified
version of error-prone PCR35 that also used primers T7P (5′-
TAATACGACTCACTATAGGG-3′) and T7T (5′-CTAGT-
TATTGCTCAGCGGT-3′), Gene Taq DNA polymerase
(Nippon Gene), and 0.3 mM MnCl2. The time−temperature
program was as follows: step 1, 1 min at 95 °C; step 2, 30 s at
95 °C, 1 min at 53 °C, and 5 min at 72 °C (step 2 was repeated
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HiTrapQ (GE Healthcare Bioscience), butyl-Toyopearl (Tosoh),
and ResourceQ (GE Healthcare Bioscience). The homogeneities
of the enzymes were assessed using sodium dodecyl sulfate−
polyacrylamide gel electrophoresis and found to be >95%.
Analytical Methods. Protein concentrations were deter-
mined using the A280 values of protein-containing solutions as
described by Pace and colleagues,37 who followed the procedure
of Gill and von Hippel.38 Extinction coefficients of 30400 M−1 cm−1
(IPMDH) and 25900 M−1 cm−1 (LDH) were used.
RESULTS
■
Random Mutagenesis by Error-Prone PCR Followed
by Genetic Selection. We previously used random muta-
genesis and phenotypic selection to isolate five T. thermophilus
IPMDHs that each contained a single-site amino acid
substitution that improved the enzyme’s low-temperature
activity.30 These IPMDH mutants each contained one of the
following mutations: G12 → S, K21 → T, R85 → C, S248 →
T, or A335 → V. The G12 → S, K21 → T, and A335 → V
mutations moderately improved the Km value for NAD+ and the
kcat value at 30 °C. The R85 → C and S248 → T mutations
increased the kcat value 6.9- and 5.7-fold at 30 °C but improved
IPMDH activities were determined by measuring the absor-
bance at 340 nm, which corresponded to production of NADH,
a product of catalysis.33 The assay buffer consisted of 50 mM
HEPES (pH 8.0), 100 mM KCl, 5 mM MgCl2, 0.2 mM D-3-
isopropylmalate (D-3-IPM, the substrate), and 5.0 mM NAD+.
k
cat only 1.4- and 1.3-fold at 60 °C, respectively.30 The presence
of one mutation sometimes negatively impacted the positive
effect induced by a second mutation. Indeed, our previous
study showed that the presence of several beneficial mutations
in the same mutant, each of which had improved the catalytic
efficiency of the corresponding single-site mutant, did not
additively enhance the catalytic activity of the multisite
mutant.31 Therefore, we did not synthesize a mutant that
contained all five mutations. Instead, to identify additional
beneficial mutations, the genes containing the G12 → S, K21 →
T, R85 → C, S248 → T, or A335 → V mutation were
pooled, and the mixture was then subjected to error-prone PCR.
The resulting randomly mutated IPMDH genes were then
each cloned into a pUC19 plasmid, and the ligates were
each transformed into E. coli JM109. The plasmids were
recovered from 2.8 × 104 colonies (library 1). Because the
IPMDH gene is 1035 nucleotides in length and the mutation
rate was 4.2 base substitutions per gene, library 1 probably
covered all possible nucleotide substitutions within the IPMDH
gene. Library 1 was used for the subsequent selection
experiment despite the relatively small total number of mutated
genes. leuB-deficient E. coli strain OM17, which was
constructed from E. coli JM105 via deletion of its chromosomal
leuB,29 was transformed with library 1 and was grown at 25 °C
on M9 minimal medium agar. Five colonies that grew faster
than those carrying a plasmid containing one of the original five
mutated genes were selected after a 24 h incubation at 25 °C.
The faster-growing colonies were assumed to each contain a
mutated parent gene that encoded an IPMDH mutant with
further improved activity. Plasmid DNAs were recovered from
the selected E. coli OM17 transformants. The isolated plasmids
were then transformed into E. coli OM17 inoculums that were
cultured on agar containing M9 minimal medium at 25 °C.
Only one transformant grew more rapidly than E. coli OM17,
which contained the parent gene encoding R85C. The plasmid
DNA was isolated from the former colony, and the gene was
sequenced and found to contain four mutations. One of the
mutations corresponded to R85C, and the other three
mutations corresponded to K76 → E, V168 → E, and I279 → V
substitutions.
One enzyme unit equaled 1 μmol of NADH formed per minute.33
D-3-IPM
The values of Km
and kcat were determined using steady-
state kinetic data with assay buffers of 50 mM HEPES (pH 8.0),
100 mM KCl, 5 mM MgCl2, 5 mM NAD+, and 2−30 μM
NAD+
D-3-IPM. To determine the Km
value, the coenzyme
concentration was varied between 10 and 2500 μM, while the
D-3-IPM concentration was fixed at 0.2 mM. To determine the
kinetic parameters of LDHs, the decrease in absorbance at
340 nm was measured. The assay buffer consisted of 50 mM
HEPES (pH 7.5), 3.5 mM MgCl2, 2.0 mM sodium pyruvate, and
10−200 μM NADH. The kinetic constants were obtained by
nonlinear least-squares fitting of the steady-state velocity data to
the Michaelis−Menten equation using the Enzyme Kinetics
module of SigmaPlot (Systat Software, Richmond, CA).
To determine the dissociation constants (Kd) for the
NADH−IPMDH complexes, fluorescence titration experiments
were performed at 25 °C with 2 mL of enzyme (0.5 μM) in 50 mM
HEPES (pH 8.0), with 5 mM MgCl2. Aliquots (1−4 μL) of
an NADH solution (2 mM) were individually added to a
protein solution, and then the mixture was stirred for 1 min
to ensure equilibration prior to the collection of data. The
decrease in the intrinsic tryptophan fluorescence intensity at
340 nm was measured after each NADH addition (excitation at
295 nm). The fluorescence data were corrected for the inner-
filter effect of NADH by monitoring the fluorescence emission
of N-acetyltryptophanamide.39 The fluorescence intensities and
protein concentrations were also corrected for dilution effects.
The Kd values were calculated by nonlinear least-squares fitting
of the corrected fluorescence data to a ligand binding equation
that assumed a single binding site for NAD(H) per protein
subunit40 using SigmaPlot. Kd values for binding of NAD+ to
the wild-type and mutant LDHs were obtained indirectly from
competition experiments. The quenching of the intrinsic
tryptophan fluorescence upon incremental addition of NADH
was measured at 25 °C with 2 mL of protein (1.0 μM) in
50 mM HEPES (pH 7.5) and 0−10 μM NAD. After the
fluorescence data had been corrected for the inner-filter
effect of NADH and for dilution, KdNADH and KdNADH
Purification of the IPMDH Mutants and Activity
Assays. To assess the individual contribution of the three
amino acid substitutions, K76 → E, V168 → E, and I279 → V,
to the improved activity of the four-mutation mutant, we
synthesized three IPMDH genes that contained codons
encoding the R85 → C substitution and a K76 → E, V168 →
E, or I279 → V substitution using site-directed mutagenesis.
To measure the in vitro activities of the IPMDH variants, the
mutated genes were first expressed in E. coli MA153, which
lacked chromosomal leuB.23 Then, the IPMDH mutants were
(apparent)
values were calculated by nonlinear least-squares fitting to the
+
ligand binding equation described above.40 KdNAD values were
then calculated using the following equation:41
Protein thermal denaturations were monitored using the
changes in ellipticity at 222 nm, as described previously.23
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Table 1. Kinetic Constants for Wild-Type and Mutant T. thermophilus IPMDHs at 30 °C
NAD+
D-3-IPM
NAD+
a
a
a
D-3-IPM
Km
(μM)
Km
(μM)
kcat (s−1
)
kcat/Km
(s−1 μM−1
)
kcat/Km
(s−1 μM−1
)
wild type
R85C
0.7 ± 0.4
27 ± 3
45 ± 5
7.1 ± 0.9
200 ± 20
630 ± 30
1.1 ± 0.0
5.4 ± 0.2
6.9 ± 0.1
1.5
0.15
0.20
0.15
0.027
0.011
R85C/I279V
a
Km and kcat were determined using steady-state kinetic data obtained at 30 °C with an assay solution of 50 mM HEPES (pH 8.0), 100 mM KCl,
5 mM MgCl2, and various concentrations of D-3-IPM and NAD+. The values and standard errors of the kinetic constants were obtained by nonlinear
least-squares fitting of the steady-state velocity data to the Michaelis−Menten equation.
Table 2. Kinetic Constants for Wild-Type and Mutant T. thermophilus IPMDHs
a
a
a
D-3-IPM
NAD+
D-3-IPM
NAD+
enzyme
Km
(μM)
Km
(μM)
kcat (s−1
25 °C
)
kcat/Km
(s−1 μM−1
)
kcat/Km
(s−1 μM−1
)
b
b
wild type
L254V
G255A
I279V
nd
6.3 ± 0.8
0.57 ± 0.01
3.0 ± 0.1
3.6 ± 0.1
1.5 ± 0.0
40 °C
nd
nd
nd
nd
0.092
b
b
b
b
b
b
nd
110 ± 10
210 ± 20
33 ± 2
0.027
0.017
0.044
nd
nd
wild type
L254V
G255A
I279V
1.8 ± 0.3
2.0 ± 0.4
3.0 ± 0.7
2.0 ± 0.3
34 ± 5
220 ± 20
240 ± 20
67 ± 6
2.7 ± 0.1
9.2 ± 0.3
7.9 ± 0.3
5.9 ± 0.1
70 °C
1.5
4.7
2.7
3.0
0.078
0.042
0.032
0.087
wild type
L254V
G255A
I279V
2.3 ± 0.5
2.0 ± 0.6
3.7 ± 0.7
3.7 ± 0.6
250 ± 20
620 ± 50
1200 ± 200
820 ± 20
51 ± 1
52 ± 2
47 ± 4
82 ± 1
22
0.21
25
13
22
0.083
0.041
0.10
a
Km and kcat were determined using steady-state kinetic data obtained at 25, 40, or 70 °C with an assay solution of 50 mM HEPES (pH 8.0), 100 mM
KCl, 5 mM MgCl2, and various concentrations of D-3-IPM and NAD+. The values and standard errors of the kinetic constants were obtained by
nonlinear least-squares fitting of the steady-state velocity data to the Michaelis−Menten equation. Km values for D-3-IPM at 25 °C could not be
determined because appropriately designed steady-state kinetic experiments would have included reactions that used very low D-3-IPM
concentrations, and at such concentrations, the reactions would have been over quickly, precluding accurate velocity measurements.
b
purified and their specific activities determined at 30 °C. Only
R85C/I279V had an improved specific activity (11 ± 0 units/mg)
in comparison with that of R85C (9.5 ± 0.3 units/mg) at
30 °C. The specific activities of the other variants (5.8 ± 0.1
units/mg for R85C/K76E and 7.4 ± 0.2 units/mg for R85C/
V168E) were smaller than that of R85C. To compare the
kinetic parameters of R85C/I279V with those of the wild-type
enzyme and R85C, we determined their Km and kcat values using
steady-state kinetic data obtained at 30 °C (Table 1). For
R85C/I279V, its Km values for both D-3-IPM and NAD+ are
larger than those for wild-type IPMDH and R85C, but it has a
kcat value larger than those of wild-type IPMDH and R85C.
Thus, the I279 → V substitution improved the low-temperature
activity of R85C by increasing the turnover number by ∼28%.
It is not known if the increase in kcat by the I279 → V sub-
stitution positively affected the growth rate of the K76E/R85C/
Figure 1. Active site of T. thermophilus IPMDH with NAD+ bound.43
V168E/I279V-containing E. coli OM17 transformant on the
The four nonpolar residues that interact with the adenine of NAD+ are
identified. This figure was drawn using PyMOL (http://www.pymol.
org).
selective medium. Possibly, the faster growth of the auxotrophic
E. coli host was a result of a change in the expression level of the
mutated IPMDH. However, more importantly, introduction of
the I279 → V substitution into wild-type IPMDH resulted in
2.5- and 2.2-fold increases in kcat at 25 and 40 °C, respectively
(see below and Table 2).
Saturation Substitution of Residues That Interact
with the Adenine of NAD. Miyazaki and Oshima suggested
that Ile279 is the residue that is mainly responsible for IPMDH
coenzyme specificity.42 In the T. thermophilus IPMDH crystal
structure,43 Ile279 is located in the NAD+-binding pocket and
directly interacts with the adenine of NAD+ (Figure 1). Val15
also interacts with the adenine. We previously found that the
V15 → I substitution improved the turnover number by 4.2-
fold at 40 °C.29 We therefore hypothesized that, in general,
mutation of a residue that interacts with the adenine of NAD+
would enhance the low-temperature activity of T. thermophilus
IPMDH. Leu254 and Gly255 also interact with the adenine
(Figure 1). We therefore subjected the wild-type IPMDH gene
to saturation mutagenesis at the codons corresponding to
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positions 254 and 255 using the NNS oligonucleotides (see
Experimental Procedures). The mutated genes (library 2) were
then individually cloned into pUC19 plasmids, and the ligates
were each amplified in E. coli JM109. For selection, leuB-
deficient E. coli OM17 was transformed with library 2 and then
incubated on agar containing M9 minimal medium. Colonies
that grew faster than the OM17 strain harboring the
T. thermophilus wild-type leuB-containing plasmid were selected
2 days later. Plasmids were recovered from the selected colonies,
and the mutated genes were sequenced. As summarized in
Table 3, 15 of 26 isolated mutated genes contained either the
Table 3. Number of Transformed and Selected Colonies
That Contained a Mutated IPMDH Gene with a Single or
Double Amino Acid Substitution That Were Identified as
Fast-Growing Colonies
amino acid substitution
no. of selected colonies
L254V
9
3
L254M
L254P
1
G255A
6
G255S
3
G255Y
1
L254I/G255A
L254I/G255S
L254M/G255S
total
1
1
1
Figure 2. Specific activities of wild-type and mutant T. thermophilus
IPMDHs. The assay solution consisted of 50 mM HEPES (pH 8.0),
100 mM KCl, 5 mM MgCl2, 0.2 mM D-3-IPM, 5 mM NAD+, and
2 μg/mL (25−40 °C) or 0.2 μg/mL (50−70 °C) protein. Each value
is the average of three replicas. The specific activities of all mutants
are greater than that of wild-type IPMDH below 40 °C (inset).
(B) Arrhenius plots for the specific activities of wild-type and mutant
IPMDHs. Activation energies (Ea) were calculated from the slopes of
the plots: wild type, 93 ± 3 kJ/mol; L254V, 62 ± 2 kJ/mol; G255A,
52 ± 3 kJ/mol; I279V, 79 ± 3 kJ/mol. Symbols: (●) T. thermophilus
IPMDH, (▲) L254V, (△) G255A, and (□) I279V.
26
L254 → V or the G255 → A mutation. Mutations cor-
responding to L254 → M and G255 → S substitutions were
also found in several of the selected clones (Table 3).
Specific Activities as a Function of Temperature. To
assess the effects of the mutations of residues involved in NAD+
binding in vitro, L254V, G255A, I279V, and wild-type IPMDH
were expressed in E. coli MA153 and purified to homogeneity.
Figure 2A depicts their specific activities as a function of
temperature, which were measured using almost saturating
amounts of D-3-IPM (0.2 mM) and NAD+ (5.0 mM). For all
IPMDHs, their specific activities increased as the temperature
increased, and the specific activities of the mutants are 2.4−6.1-
fold greater than that of wild-type IPMDH at 25 °C. At 70 °C,
L254V and I279V have specific activities similar to that of wild-
type IPMDH, whereas G255A exhibited a slightly decreased
specific activity. Activation energies (Ea) for the specific
activities were calculated from the slopes of Arrhenius plots
for temperatures between 25 and 70 °C (Figure 2B). Although
I279V had an only slightly reduced Ea value (79 kJ/mol)
compared with that of wild-type IPMDH (93 kJ/mol), L254V
and G255A had substantially reduced Ea values (62 and 52 kJ/mol,
respectively). Thus, the improvements in the low-temperature
activities for this thermophilic enzyme are at least partially
related to the smaller Ea values, which is a phenomenon that has
also often been found for naturally occurring, cold-adapted
enzymes.44−46
had significantly improved kcat values at 25 °C. G255A has the most
NAD+
unfavorable Km
but also has the most improved kcat value at
25 °C. A similar relationship was found for the kcat values at 40 °C,
although the extent to which the kcat values of the mutants increased
was smaller than at 25 °C. Moreover, at 70 °C, only the kcat value
for I279V was greater than that of wild-type IPMDH. Therefore,
the mutations adapted the catalytic activity of T. thermophilus
IPMDH to lower temperatures. All mutated IPMDHs have
NAD+
decreased kcat/Km
values at 25 °C. Thus, the low-temperature
adaptation of the mutated IPMDHs reflects an increased value
for the turnover number accompanied by anincrease in the
NAD+
Km
value.
Dissociation Constants for NADH. The Kd values for the
reaction product NADH were determined by measuring the
decrease in the intrinsic tryptophan fluorescence of the wild-
type and mutant IPMDHs by titration with NADH. As
summarized in Table 4, the KdNADH values for the two mutants,
L254V and G255A, are similar to that of wild-type IPMDH.
Therefore, the affinity of NADH for L254V and G255A
probably does not affect the increased kcat values found for
these mutants. In contrast, a significantly increased KdNADH
value was observed for I279V, suggesting that the weakened
interaction between NADH and I279V could contribute to the
enhanced catalytic efficiency of I279V.
Kinetic Parameters. The kinetic parameters of wild-type
IPMDH, L254V, G255A, and I279V were calculated using
steady-state kinetic data obtained at 25, 40, and 70 °C (Table 2).
The KmD-3-IPM values for the three mutants at 40 and 70 °C were,
within the margin of error, the same as or slightly larger than that
of wild-type IPMDH. Although the Km
NAD+
values for the mutants
were significantly larger than that of wild-type IPMDH, the mutants
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Table 4. Kd Values for Dissociation of NAD(H) from Wild-
Type and Mutant T. thermophilus Dehydrogenases at 25 °C
+
a
b
KdNADH (μM)
IPMDH
46 ± 3
47 ± 1
31 ± 0
310 ± 50
LDH
KdNAD (μM)
c
wild type
L254V
G255A
I279V
nd
c
nd
c
nd
c
nd
wild type
A75G
1.1 ± 0.0
1.0 ± 0.0
0.70 ± 0.04
0.50 ± 0.03
a
Kd values for NADH dissociation were determined using fluorescence
titration data obtained at 25 °C. The values and standard errors were
obtained by nonlinear least-squares fitting of the fluorescence titration
data to the equation ΔF = ΔFmax[NADH]/(Kd + [NADH]), where ΔF
is the corrected change in enzyme fluorescence upon addition of
NADH and ΔFmax is the maximum change in fluorescence intensity.
b
Kd values for dissociation of NAD+ from wild-type and mutant LDHs
were determined indirectly by competition experiments.41 Not
c
determined.
Thermal Stability. Generally, thermophilic enzymes are
stable but poorly active at low temperatures. In contrast, most
cold-adapted enzymes found in nature are less stable but
substantially more active at low temperatures. Apparently, the
increased flexibility caused by destabilization of the catalytic site
or the tertiary structure contributes to the greater catalytic
efficiency of a psychrophilic enzyme at low temperatures.12,16
This activity−stability trade-off is also found for mutants that
evolved in vitro that develop enhanced low-temperature
activities.25−27 However, certain cold-active enzymes are also
very stable.15,47,48 Therefore, an inverse relationship between
thermal stability and activity at low temperatures is not always
found. To determine if the mutant IPMDHs are as resistant to
thermal denaturation as wild-type IPMDH, temperature-
induced unfolding of all IPMDHs was monitored by circular
dichroism spectroscopy because the ellipticity at 222 nm is a
measure of protein secondary structure content. The
denaturation curves of all IPMDHs are similar (Figure 3A).
Therefore, the NAD+-binding-residue mutations improved the
catalytic activities without affecting the thermal stabilities of the
mutants, which had been found previously29,30 and has been
found for low-temperature-adapted mutants of thermostable
subtilase,22 xylose isomerase,24 and indoleglycerolphosphate
synthase.28
Figure 3. Thermal melting profiles for wild-type and mutant T.
thermophilus IPMDHs and wild-type and mutant T. thermophilus
LDHs. Ellipticities were monitored at 222 nm. The scan rate was
1.0 °C/min. The solutions consisted of 0.25 mg/mL protein, 20 mM
potassium phosphate (pH 7.6), and 0.5 mM EDTA: (A) (●) T.
thermophilus IPMDH, (○) L254V, (△) G255A, and (▲) I279V and
(B) (●) T. thermophilus LDH, (△) A75G, and (□) V99I.
decarboxylates and dehydrogenates its substrate simultane-
ously, whereas LDH catalyzes only a dehydrogenation.34
Two aliphatic residues, Ala75 and Val99, interact with the
adenine of NAD+ in LDH (Figure 4). We therefore produced
Improvement of the Low-Temperature Activity of
T. thermophilus LDH. Because a single example is not sufficient
to demonstrate that, in general, the low-temperature activities
of thermophilic dehydrogenases can be improved by mutating
residues that interact with the adenine of NAD+, we examined
the low-temperature activity of thermophilic NADH-dependent
dehydrogenase after slightly changing the side chain volume of
a nonpolar residue that interacts with the adenine of NADH.
We chose T. thermophilus lactate dehydrogenase (LDH) to test
the generality of our hypothesis, as its NAD+-complexed crystal
structure has been determined.49 In addition, LDH differs
structurally and functionally from IPMDH. LDH is a
homotetramer, and each subunit has the typical NAD+-binding
Rossmann fold; T. thermophilus IPMDH is a homodimer, and
each polypeptide chain folds into two domains with parallel α/
β motifs that are not Rossmann folds.34 Furthermore, IPMDH
Figure 4. Active site of T. thermophilus LDH with bound NAD+.49 Two
of the nonpolar residues that interact with the adenine of NAD+ are
identified. This figure was drawn using PyMOL (http://www.pymol.org).
the LDH mutants, A75G and V99I, by site-directed muta-
genesis. These variants were expressed in E. coli and purified to
homogeneity. Their kinetic parameters were determined from
steady-state kinetic data obtained at 25 and 40 °C using NADH
as the cofactor, as were those of wild-type LDH. The kcat values
for A75G are 1.9- and 2.7-fold larger at 25 and 40 °C,
NADH
respectively, than that of wild-type LDH, and the Km
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Table 5. Kinetic Constants for Wild-Type and Mutant T. thermophilus LDHs
a
a
Km
(μM)
kcat (s−1
)
kcat/Km
25 °C
(s−1 μM−1
)
NADH
NADH
enzyme
25 °C
40 °C
25 °C
40 °C
40 °C
wild type
A75G
31 ± 3
54 ± 8
33 ± 5
160 ± 20
240 ± 20
100 ± 10
0.20 ± 0.01
0.38 ± 0.02
0.20 ± 0.01
6.3 ± 0.4
17 ± 1
4.0 ± 0.2
0.0063
0.0071
0.0061
0.039
0.072
0.039
V99I
a
Km and kcat were determined using steady-state kinetic data obtained at 25 and 40 °C with an assay solution of 50 mM HEPES (pH 7.5), 3.5 mM
MgCl2, 2 mM sodium pyruvate, and various concentrations of NADH. The values and standard errors of the kinetic constants were obtained by
nonlinear least-squares fitting of the steady-state velocity data to the Michaelis−Menten equation.
values for A75G increased, but by a factor of <2, at the two
temperatures (Table 5). A75G also has increased kcat/Km
mutants are greater than that of wild-type IPMDH (Table 2).
Because the affinity of NAD+ likely correlates with that of
NADH, we hypothesized that mutations that destabilize the
enzyme−substrate−NAD+ complex also destabilize the enzyme−
product−NADH complex and thus would facilitate the release
of NADH, which should enhance catalysis. Indeed, the KdNADH
value for I279V is significantly greater than that of wild-type
IPMDH (Table 4). However, the KdNADH values for the other
two mutants, L254V and G255A, are similar to that of wild-type
IPMDH and therefore do not support the hypothesis that the
increases in kcat for the mutant enzymes are consequences of
facilitated NADH release.
NADH
values at the two temperatures in comparison with those of
wild-type LDH. Thus, the low-temperature activity of
T. thermophilus LDH was also improved by removing a methyl
from a nonpolar side chain that interacts with the adenine of
NAD(H). Conversely, the kinetic parameters of V99I were
similar to those of wild-type LDH. As given in Table 4, the
+
KdNAD value for A75G (0.50 ± 0.03 μM), determined by
competitive fluorescence titration, is slightly better than that of
the wild-type enzyme (0.70 ± 0.04 μM). Therefore, the
improved kcat value for A75G is probably not a consequence of
the improved release of NAD+.
The rate-limiting step for the reduction of oxaloacetate
catalyzed by Thermus aquaticus malate dehydrogenase also
involves the release of NAD+.39 The catalytic rate for this
thermophilic malate dehydrogenase was improved by replace-
ment of an acidic residue that forms hydrogen bonds with the
2′- and 3′-hydroxyls of the adenosine ribose.39 This mutation
increases the rate of NAD+ release and thereby improves its
catalytic turnover number. Although LDH and malate
dehydrogenase have similar protein folds and catalyze
analogous reactions, the increase in the kcat value for the
reaction catalyzed by the LDH mutant A75G seemed not to be
We also assessed the thermal stabilities of the wild-type and
mutant LDHs. The thermal melting curves of the mutants are
very similar to that of wild-type LDH (Figure 3B). Therefore,
as is true for the IPMDH mutants, small changes in side chain
volumes of the residues that interact with NAD+ improved the
low-temperature activity of T. thermophilus LDH without
compromising its thermal stability.
DISCUSSION
■
a consequence of weakened LDH−NAD+ binding that would
We describe here the selective mutagenesis of the thermostable
3-isopropylmalate dehydrogenase (IPMDH) and lactate
dehydrogenase (LDH) from the extreme thermophile
T. thermophilus that improved their low-temperature activities.
We initially found that an amino acid substitution that affects
coenzyme binding improves the low-temperature activity of
T. thermophilus IPMDH. The saturation mutagenesis experiment
supported the concept that a small change in the side chain
volume of a nonpolar residue that interacts with the adenine of
NAD+ increases the turnover number. The low-temperature
activity of T. thermophilus LDH was also enhanced by a similar
amino acid substitution. In contrast to the substitution of a
polar residue for a nonpolar residue, the substitution of a
nonpolar residue for another nonpolar residue should maintain
the electrostatic environment around the active site and not
perturb the catalytic mechanism. The thermostabilities of the
mutants were almost identical to those of the wild-type
enzymes. Therefore, a small change in the side chain volume of
a nonpolar residue that interacts with the adenine of NAD+ may
generally improve the low-temperature activities of thermo-
philic dehydrogenases without affecting their stabilities.
+
facilitate the release of NAD+ because the KdNAD value for
A75G is slightly better than that for wild-type LDH (Table 4).
A free energy profile for a dehydrogenase-catalyzed reaction
is illustrated in Figure 5. The energetic contributions of the
Figure 5. Gibbs free energy profile along the reaction coordinate. The
dashed line shows the effect of a mutation that decreases the change in
free energy upon NAD(H) binding. Abbreviations: E, enzyme; S,
substrate; C, coenzyme; P, product; C*, product coenzyme; ESC,
ground-state enzyme−substrate−coenzyme ternary complex; (ESC)⧧,
transition-state ternary complex.
The kinetic mechanism of E. coli isocitrate dehydrogenase
(ICDH) has been characterized by others and includes the
release of product and NADPH as the rate-limiting catalytic
step.50 In addition to their similar tertiary structures, ICDH and
T. thermophilus IPMDH catalyze similar oxidative decarbox-
ylations. Therefore, similar kinetic mechanisms might be
amino acid substitutions that improve enzymatic activity can be
evaluated using the experimentally determined steady-state
kinetic parameters for the forward reaction;16 the change in free
expected for ICDH and IPMDH. Our steady-state kinetic
NAD+
experiments showed that the Km
values for the IPMDH
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Table 6. Thermodynamic Parameters of the Enzymatic Reactions of Wild-Type and Mutant T. thermophilus Dehydrogenases at
25 °C
a
b
c
b
d
b
⧧
ΔG⧧ (kJ/mol)
ΔΔG⧧ (kJ/mol)
ΔGm (kJ/mol)
ΔΔGm (kJ/mol)
ΔGT (kJ/mol)
ΔΔGT (kJ/mol)
⧧
enzyme
IPMDH
−29.0
−22.0
−20.5
−25.0
LDH
wild type
L254V
G255A
I279V
74.4
70.2
69.9
72.1
−
−
45.3
48.2
49.3
47.0
−
−4.2
−4.5
−2.3
7.0
8.5
4.0
2.9
4.0
1.7
wild type
A75G
77.0
75.4
77.0
−
−1.6
0
−25.1
−23.8
−25.0
−
1.3
51.8
51.5
51.9
−
−0.3
0.1
V99I
0.1
a
ΔG⧧ is the activation free energy calculated from the experimentally determined kcat value according to the equation ΔG⧧ = −RT ln(kcath/kBT),
where R is the gas constant (8.31 J K−1 mol−1), T is the temperature in kelvin, h is Planck's constant (6.63 × 10−34 J s−1), and kB is Boltzmann's
b
constant (1.38 × 10−23 J K−1). ΔΔG⧧, ΔΔGm, and ΔΔGT are the differences between the parameters for the mutant enzyme and the wild type.
c
d
NAD(H)
⧧
ΔGm is the change in free energy upon NAD(H) binding calculated from the Km
value. ΔGT is the difference in free energy between the
initial state and transition state, calculated using the relationship ΔGT = ΔG⧧ + ΔGm.
⧧
energy upon NAD(H) binding, ΔGm, is calculated from
the KmNAD(H) value, and the activation free energy in the forward
direction, ΔG⧧, which is the free energy difference between the
ground-state and transition-state (enzyme−substrate−coen-
zyme) complexes, is calculated using kcat. The calculated values
for ΔG⧧, ΔGm, and the difference in free energy between the
of the active site in the cold-adapted LDH-A4 affected the
values of Ea and Km.45 The smaller Ea value was ascribed to
weakened binding of the substrate, which also increased the Km
value.53 Psychrophilic enzymes also typically have larger kcat
values at a given temperature than their mesophilic and
thermophilic homologues.16 The IPMDH mutants that have
⧧
initial and transition states, ΔGT , are listed in Table 6. The
improved kcat values at the low temperatures also have smaller Ea
NAD+
amino acid substitutions that improved the IPMDH and LDH
values (Figure 2B) and increased Km
values (Table 2). A
⧧
⧧
kcat values affected ΔGm and ΔGT . An increased ΔGT value
smaller Ea value decreases the temperature dependency of
was calculated for the mutated IPMDHs, whereas a marginally
thereaction rate, and thus, a similar reaction rate can be
smaller ΔGT value was calculated for LDH A75G. Moreover,
maintained as the temperature is lowered.53 The LDH
⧧
NADH
the ΔGm values of the mutants are substantially smaller than those
A75G also showed simultaneous increases in Km
kcat (Table 5).
and
of the corresponding wild-type enzymes. Therefore, the increased
k
cat values (decreased ΔG⧧ values) found for the mutant enzymes
Nevertheless, the amino acid substitutions found in this study
do not parallel those found for enzymes that have adapted to
lower temperatures during evolution. Gly255 and Ile279 in
IPMDH and Ala75 in LDH are strongly conserved in the
homologous thermophilic, mesophilic, and psychrophilic
enzymes. In addition, residue 254 of IPMDH is apparently
always a Leu, Ile, or Met. Residues 255, 279, and 254 directly
contact the adenine of NAD(H), and therefore when they were
mutated, the Km values increased (Table 2). Because NAD(H)
is used in many different reactions, kcat/KmNAD(H), rather than
kcat, likely regulates enzymatic activity in vivo, which would
explain why the mutations found in this study are not found in
nature as adaptive mutations that increased the low-temper-
ature activities of the mesophilic and psychrophilic enzymes.
For in vitro applications involving enzymes, the kinetic
parameter that controls the catalytic rate depends on
the substrate (and coenzyme) concentrations.54 In many
industrial applications, substrate and coenzyme are present
at nearly saturating concentrations. Therefore, in these
situations, the catalytic power of an enzyme is determined by
its kcat value.
We also examined the physical relationships between
adenine-binding residues and adenine for four other
thermophilic dehydrogenases: ICDH from T. thermophilus,
glyceraldehyde-3-phosphate dehydrogenase from T. thermophi-
lus, alcohol dehydrogenase from Sulfolobus tokodaii, and
glycerol-3-phosphate dehydrogenase from Thermotoga maritima
(Figure S1 of the Supporting Information). A common feature
among the four dehydrogenases is a glycine is present near the
adenine of the bound coenzyme. Similar to residue 255 of
IPMDH, these glycine residues are strongly conserved among
the respective enzymes and, therefore, would be a mutagenesis
are caused by the destabilization of the enzyme−substrate−
coenzyme complexes rather than by the stabilization of the
transition states; i.e., a reduction in the binding affinity of the
coenzyme mostly affects kcat.
It has been argued that the catalytic efficiency of a cold-
adapted enzyme is reflected in the flexibility of segment(s)
directly and indirectly involved in catalysis. Localized flexibility
can be increased by reducing the strength of a hydrogen bond15
or by removing a salt bridge.51 Similarly, because glycine lacks a
side chain, substitution with a glycine increases the flexibility of
the peptide backbone, which would account for the catalytic
behavior of LDH A75G. However, the IPMDH mutant, L254V,
which has a β-branched chain residue substituted for a γ-
branched residue, would seem to have a reduced flexibility
because a β-branched side chain is conformationally more
constrained than a γ-branched chain.52 The other IPMDH
mutant, G255A, which has an additional methyl group, also
possibly reduces the flexibility of the surrounding regions.
Therefore, given the less negative values of the free energy
changes associated with NAD+ binding (Table 6), these
mutants likely have improved catalytic turnover numbers as a
consequence of less than optimal hydrophobic packing of the
adenine in the Michaelis complex.
The mutants with improved low-temperature activities
obtained in this study have thermodynamic and kinetic
properties similar to those of naturally occurring cold-adapted
enzymes.16,44−46 Several thermodynamic and kinetic features
are common among naturally occurring cold-adapted enzymes,
including smaller Ea and greater Km values compared to those of
their more thermostable homologues.44−46 Fields et al.
reported that substitutions of a residue located in the vicinity
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target for improving the low-temperature activity of these
thermophilic dehydrogenases. The four coenzyme-binding sites
also contain several nonpolar residues, most of which are not
well conserved among their corresponding homologues, and no
remarkable difference in amino acid type is found between the
mesophilic and thermophilic homologues of these dehydro-
genases. Only for the position corresponding to residue 337 of
S. tokodaii alcohol dehydrogenase is a glycine found in most of
the mesophilic homologues, whereas the thermophilic homo-
logues contain residues other than glycine at this position
(Figure S2 of the Supporting Information). Perhaps, therefore,
the highly conserved glycine in the mesophilic alcohol
dehydrogenases causes an increase in flexibility at the
coenzyme-binding site, which would account for enhanced
catalysis at low temperatures. The second shell of adenine-
binding residues might also be important for the flexibility
of the coenzyme-binding sites. However, no distinct trend
in the amino acid type in the second shell that would be
responsible for the difference in low-temperature activity
between thermophilic and mesophilic homologues was
apparent when we compared the mesophilic and thermophilic
sequences.
Because we do not have detailed structural information for
the mutants, the relationships between structure and activity
cannot be explored at the molecular level. X-ray crystallography
and nuclear magnetic resonance are powerful methods that are
routinely used to characterize the structure of a protein at the
atomic level. These structural biological approaches will allow
us to identify the specific structural requirement(s) responsible
for the enhanced low-temperature catalytic efficiencies. Efforts
to do so will be made in the future.
For the work reported here, we clearly demonstrated that
single amino acid substitutions each enhanced the catalytic
turnover numbers of the two thermophilic dehydrogenases
examined. However, even the mutant (IPMDH G255A) that
has the most improved turnover number had an only 6.2-fold
increase in its kcat value. In contrast, the kcat of the mesophilic
E. coli IPMDH is 57-fold greater than that of T. thermophilus
IPMDH at 30 °C.30 It is usually assumed that a small change in
an enzyme’s structure should not have a large effect on its
properties.55 The presence of many beneficial substitutions is
expected to produce a mutant enzyme with a dramatically
increased catalytic efficiency when the effects of the
substitutions are additive. Future efforts along this line will be
directed at T. thermophilus IPMDH and LDH.
ABBREVIATIONS
■
IPMDH, 3-isopropylmalate dehydrogenase; NAD+, nicotina-
mide adenine dinucleotide; LDH, lactate dehydrogenase; D-3-
IPM, D-3-isopropylmalate; ICDH, isocitrate dehydrogenase.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Coenzyme-binding sites of four thermophilic dehydrogenases
with NAD(P)+ bound and a portion of the multiple-sequence
alignment of the alcohol dehydrogenase sequences. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Department of Molecular Biology, Tokyo University of
Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji,
Tokyo 192-0392, Japan. Telephone: +81-426-76-7139. Fax:
+81-426-76-7145. E-mail: yamagish@toyaku.ac.jp.
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