Improving the pyrophosphate-inosine phosphotransferase activity of Escherichia blattae acid phosphatase by sequential site-directed mutagenesis

Article abstract of DOI:10.1271/bbb.68.1046

Escherichia blattae acid phosphatase/phosphotransferase (EB-AP/PTase) exhibits C-5′-position selective pyrophosphate-nucleoside phosphotransferase activity in addition to its intrinsic phosphatase. Improvement of its phosphotransferase activity was investigated by sequential site-directed mutagenesis. By comparing the primary structures of higher 5′-inosinic acid (5′-IMP) productivity and lower 5′-IMP productivity acid phosphatase/phosphotransferase, candidate residues of substitution were selected. Then a total of 11 amino acid substitutions were made with sequential substitutions. As the number of substituted amino acid residues increased, the 5′-IMP productivity of the mutant enzyme increased, and the activity of the 11 mutant phosphotransferases of EB-AP/PTase reached the same level as that of Morganella morganii AP/PTase. This result shows that Leu63, Ala65, Glu66, Asn69, Ser71, Asp116, Thr135, and Glu136, whose relevance was not directly established by structural analysis alone, also plays an important role in the phosphotransferase activity of EB-AP/PTase.

Full text of DOI:10.1271/bbb.68.1046

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Improving the Pyrophosphate-inosine
Phosphotransferase Activity of Escherichia blattae
Acid Phosphatase by Sequential Site-directed
Mutagenesis
Yasuhiro MIHARAa, Kohki ISHIKAWAb, Ei-ichiro SUZUKIb & Yasuhisa ASANOc
a Applied Microbiology Laboratory, Fermentation and Biotechnology Laboratories,
Ajinomoto Co., Inc.
b Institute of Life Sciences, Ajinomoto Co., Inc.
c Biotechnology Research Center, Toyama Prefectural University
Published online: 22 May 2014.
To cite this article: Yasuhiro MIHARA, Kohki ISHIKAWA, Ei-ichiro SUZUKI & Yasuhisa ASANO (2004) Improving the
Pyrophosphate-inosine Phosphotransferase Activity of Escherichia blattae Acid Phosphatase by Sequential Site-directed
Mutagenesis, Bioscience, Biotechnology, and Biochemistry, 68:5, 1046-1050, DOI: 10.1271/bbb.68.1046
To link to this article: http://dx.doi.org/10.1271/bbb.68.1046
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Biosci. Biotechnol. Biochem., 68 (5), 1046–1050, 2004
Improving the Pyrophosphate-inosine Phosphotransferase Activity of
Escherichia blattae Acid Phosphatase by Sequential Site-directed Mutagenesis
y
3
Yasuhiro MIHARA,1; Kohki ISHIKAWA,2 Ei-ichiro SUZUKI,2 and Yasuhisa ASANO
1Applied Microbiology Laboratory, Fermentation and Biotechnology Laboratories, Ajinomoto Co., Inc.,
1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan
2Institute of Life Sciences, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan
3Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939-0398, Japan
Received October 10, 2003; Accepted January 27, 2004
Escherichia blattae acid phosphatase/phosphotrans-
ferase (EB-AP/PTase) exhibits C-50-position selective
pyrophosphate-nucleoside phosphotransferase activity
in addition to its intrinsic phosphatase. Improvement of
its phosphotransferase activity was investigated by
sequential site-directed mutagenesis. By comparing the
primary structures of higher 50-inosinic acid (50-IMP)
productivity and lower 50-IMP productivity acid phos-
phatase/phosphotransferase, candidate residues of sub-
stitution were selected. Then a total of 11 amino acid
substitutions were made with sequential substitutions.
As the number of substituted amino acid residues
increased, the 50-IMP productivity of the mutant
enzyme increased, and the activity of the 11 mutant
phosphotransferases of EB-AP/PTase reached the same
level as that of Morganella morganii AP/PTase. This
result shows that Leu63, Ala65, Glu66, Asn69, Ser71,
Asp116, Thr135, and Glu136, whose relevance was not
directly established by structural analysis alone, also
plays an important role in the phosphotransferase
activity of EB-AP/PTase.
As well as amino acid production, nucleotide pro-
duction is another key area of interest in biotechnology,
because 50-inosinic acid (50-IMP) and 50-guanylic acid
(50-GMP) have a characteristic taste and are used as a
flavor potentiator in various foods. Since purine nucleo-
sides such as inosine and guanosine can be efficiently
produced by fermentation,1,2) the progress of large-scale
50-nucleotide production depends largely on the im-
provement of the nucleoside-phosphorylation process.
Nowadays, there are two main phosphorylation meth-
ods. One is a chemical phosphorylation process that uses
phosphoryl chloride (POCl3),3) and the other is an
enzymatic phosphorylation process that uses inosine
kinase of E. coli.4,5) An enzymatic phosphorylation
process is used by Kyowa Hakko Kogyo, and IMP
production involves inosine fermentation by Coryne-
bacterium ammoniagenes coupled with energy-requiring
phosphorylation by guanosine/inosine kinase.
Recently, we have investigated a novel nucleoside
phosphorylation process. We found that Morganella
morganii acid phosphatase exhibits C-50-position selec-
tive pyrophosphate (PPi)-nucleoside phosphotransferase
activity in addition to its intrinsic phosphatase activity,
as shown in Scheme 1.6) Then its phosphotransferase
activity was improved by random mutagenesis, and a
primary nucleoside phosphorylation process using PPi as
Key words: Escherichia blattae; acid phosphatase;
pyrophosphate-inosine phosphotransferase;
50-inosinic acid
Scheme 1.
y
To whom correspondence should be addressed. Tel: +81-44-210-5946; Fax: +81-44-244-4757; E-mail: yasuhiro mihara@ajinomoto.com

Improved E. blattae Acid Phosphatase/Phosphotransferase
1047
a phosphate donor was established.7,8) Next we em-
barked on research to understand the structure activity
relationship of these enzymes and to obtain an even
more efficient enzyme with further improved its phos-
photransferase activity. We have isolated genes encod-
ing an acid phosphatase with regioselective phospho-
transferase activity (AP/PTase) from five genes of
enteric bacteria.9) Among five AP/PTases, we have
determined the crystal structure of Escherichia blattae
Each acid phosphatase was purified from harvested
cells of E. coli JM109 transformants harboring mutant
phoC by ammonium sulfate fractionation and ion-
exchange, and hydrophobic column chromatographies,
as described previously.10) The purity of the recovered
samples was checked by sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (14% polyacrylamide).
Enzyme assay. Phosphotransferase activity was as-
sayed in a standard reaction mixture containing
ꢀ
AP/PTase at 1.9 A, and selected the enzyme as the
target of rational mutagenesis.10) We designed several
mutations and introduced them into the G74D/I153T
mutant, which was generated using knowledge derived
from random mutagenesis of the M. morganii enzyme,
and succeeded in producing an industrially viable
mutant enzyme, S72F/G74D/I153T, with a further
reduced Km value for inosine.11)
The primary structures of E. blattae AP/PTase and
M. morganii AP/PTase were highly homologous, and
the G74D/I153T mutations were also effective in both
enzymes, but the productivity of the G74D/I153T
mutant of E. blattae was greatly inferior to that of the
G74D/I153T mutant of M. morganii. Although we have
investigated the improvement of AP/PTases as men-
tioned above, the reason for such a difference remains to
be understood. In the present study, we enhanced the
phosphotransferase activity of EB-NSAP by a sequential
site-directed mutagenesis. Through this attempt to
enhance phosphotransferase activity, the role of some
residues that are important for phosphotransferase
activity but whose relevance was not fully established
by structural analysis, was successfully elucidated.
100 ꢀmol of sodium acetate buffer (pH 5.0), 40 ꢀmol
of inosine, 100 ꢀmol of tetrasodium pyrophosphate, and
the enzyme solution in a total volume of 1 ml. The
reaction mixture was incubated for 10 m at 30 ꢀC and
then the reaction was stopped by adding 0.2 ml of 2 N
HCl. Quantitative determination of inosine and 50-IMP
was carried out by HPLC as described previously.8) One
unit of phosphotransferase activity was defined as the
amount of enzyme that produces 1 ꢀmol of 50-IMP per
minute under the assay conditions. 50-Nucleotidase
activity was assayed in a standard reaction mixture
containing 100 ꢀmol of sodium acetate buffer (pH 4.0),
10 ꢀmol of 50-IMP, and the enzyme solution in a total
volume of 1 ml. The reaction mixture was incubated for
5 m at 30 ꢀC. The reaction was stopped by adding 0.2 ml
of 2 N HCl, and then released inosine was measured by
HPLC. One unit of 50-nucleotidase activity was defined
as the amount of enzyme that produced 1 ꢀmol of
inosine per minute under the assay conditions. Kinetic
constants for inosine in the transphosphorylation reac-
tion and for 50-IMP in the dephosphorylation reaction
were measured at pH 4.0, at which 50-IMP synthesis was
performed. The initial velocities were determined under
the assay conditions described above, and the steady-
state kinetic constants were calculated by using a
Hanes–Woolf plot. Since the solubility of inosine was
limited, kinetic constants for inosine were determined
with a substrate concentration ranging from 0.5 to
80 mM.
Material and Methods
Strains, plasmids, and culture conditions. Escherichia
coli JM10912) was used as the host strain. Plasmid,
pEAP320 containing the AP/PTase gene phoC of
Escherichia blattae,10) was used as a template in PCR
mutagenesis. Plasmid, pUC18 (Takara Shuzo, Japan),
was used as a cloning vector. E. coli transformants were
cultured aerobically at 37 ꢀC on Luria–Bertani medium
containing ampicillin (50 ꢀg/ml).
Synthesis of 50-IMP by E. coli overproducing mutant
PhoC. Cells of each E. coli JM109 transformant were
cultured in LB medium and harvested from culture broth
by centrifugation at 10;000 ꢁ g for 10 m. The harvested
cells were washed with 10 mM potassium phosphate
buffer (pH 7.0) and then suspended in the same buffer.
Cell growth was estimated turbidimetrically by means of
a dry-cell calibration curve for absorbance at 610 nm:
0.24 of dry cell weight per ml was equivalent to 1.0 of
optical density at 610 nm. The standard reaction mixture
for 50-IMP synthesis contained various concentrations of
inosine, 150 g/l (200 mM) disodium hydrogen pyrophos-
phate, 1 mM sodium acetate buffer (pH 4.0), and 10 g/l
(dry weight) of cells in 10 ml. The reaction was carried
out at 30 ꢀC with moderate shaking and stopped by
adding 1 ml of 2 N HCl. Synthesized 50-IMP was
DNA mutagenesis and purification of mutant AP/
PTases. All basic recombinant DNA procedures, such as
isolation and purification of DNA and transformation of
Escherichia coli, were performed as described by
Sambrook et al.13) Site-directed mutagenesis was per-
formed on the E. blattae acid phosphatase gene, cloned
in pUC1812) using the Quick Change mutagenesis kit
(Stratagene) according to its protocol. Mutations were
confirmed by sequencing by the dideoxynucleotide
chain termination method with a Dye Terminator Cycle
sequencing kit (Perkin-Elmer) and a DNA sequencer
(model 373A, Perkin-Elmer). Synthesized universal and
fragment-specific oligonucleotides for each clone were
used as primers.
calculated as IMP 2Na 7.5H O (MW 527).
2
ꢂ ꢂ

1048
Y. MIHARA et al.
Fig. 1. Amino Acid Alignment of the Deduced Amino Acid Sequences of AP/PTases around Target Residues.
Numbering of the amino acids starts at the amino-terminus of the mature proteins. Asterisks show identical amino acid residues. The positions
of target residues are boxed in gray. The Gly74 and Ile153 residues, of which substitutions to Asp and Thr respectively were reported to be
effective for the improvement of 50-IMP productivity in M. morganii AP/PTase,8) are boxed in gray. The sequences shown are: Mmo,
Morganella morganii; Pst, Providencia stuartii; Eae, Enterobacter aerogenes; Ebl, Escherichia blattae; Kpl, Klebsiella planticola. The
conserved residues are marked with an asterisk.
Table 1. Plasmids in Which AP/PTase Genes Were Cloned in This Study
Nucleic acid
substitutions
Amino acid
substitutions
Plasmid
Parent
Mutated enzyme
Reference
pEAP320
pEAP330
pEAP340
pEAP350
—
Wild type
I153T
G74D/I153T
10
11
pEAP320
pEAP330
pEAP340
T458C
I153T
G74D
L63Q, A65Q, E66A
G221A, G222T
T188A, G193C,
C194A, A197C
C404A, C405A,
G408C
11
This study
L63Q/A65Q/E66A/G74D/I153T
pEAP360
pEAP370
pEAP350
pEAP360
T135K, E136D
L63Q/A65Q/E66A/G74D/T135K/E136D
/I153T
This study
This study
A205G, A211G,
G212C, A214G,
G215C
N69D, S71A, S72A
L63Q/A65Q/E66A/N69D/S71A/S72A/
G74D/T135K/E136D/I153T
pEAP380
pEAP370
T348A
D116E
L63Q/A65Q/E66A/N69D/S71A/S72A/
G74D/D116E/T135K/E136D/I153T
This study
Results and Discussion
Site-directed mutagenesis for an improved PPi-ino-
sine phosphotransferase activity mutant
We have isolated genes encoding an acid phosphatase
with regioselective phosphotransferase activity (AP/
PTase) from Enterobacter aerogenes, Escherichia blat-
tae, Klebsiella planticola, Morganella morganii, and
Providencia stuartii, and compared the primary struc-
tures and enzymatic characteristics of these enzymes.
Although they were highly homologous in primary
structure, the 50-IMP productivities of the E. aerogenes,
E. blattae, and K. planticola enzymes were inferior to
those of the M. morganii and P. stuartii enzymes.9) This
suggested that local sequence differences are responsible
for the differences in the phosphotransferase reaction.
Based on the alignment of the sequences of five AP/
PTases, candidate residues for amino acid substitution of
EB-AP/PTase were selected as shown Fig. 1, and each
mutation was introduced additively on the EB-AP/
PTase gene cloned on pUC18. The target residues that
located adjacently were mutated at one time. Each
mutant plasmid was used as the PCR template of the
next generation. Finally, the mutant plasmid coding the
mutant enzyme of which 11 amino acid residues were
substituted was constructed as shown Table 1. All
mutant enzymes were overexpressed in E. coli JM109.
No changes in the levels of production of mutant
enzymes in each E. coli transformant were observed
under the culture conditions used.
Fig. 2. 50-IMP Synthesis by E. coli Overproducing Each AP/PTase.
The time course of 50-IMP synthesis by resting cells of E. coli
JM109 (pEAP340);
, E. coli JM109 (pEAP350);
, E. coli
JM109 (pEAP360); , E. coli JM109 (pEAP370); , and, E. coli
JM 109 (pEAP380); was measured. The reaction was carried out
at pH 4.0 and 30 ꢀC in a reaction mixture consisting of 0.1 M sodium
acetate buffer (pH 4.0) containing 40 g/l (148 mM) inosine, 150 g/l
(676 mM) disodium hydrogen pyrophosphate and 1 g/l (dry weight)
of each type of cell.
Synthesis of 50-IMP by E. coli overproducing the
mutant PhoC acid phosphatase
The time course of 50-IMP production using E. coli
JM109 overproducing each mutant enzyme is shown in
Fig. 2. As we described previously, substitution of
G74D/I153T increases the 50-IMP productivity of EB-
NSAP.10) In spite of the fact that its productivity was
inferior to that of the M. morganii G72D/I151T mutant

Improved E. blattae Acid Phosphatase/Phosphotransferase
1049
AP/PTase, 104 g/l 50-IMP was produced with a molar
yield of 52% from inosine using E. coli overproducing
the G74D/I153T mutant of the EB-AP/PTase. As the
number of substituted amino acid residues increased, the
productivity of the mutant enzyme increased. Using
E. coli overproducing the 10 amino acid substituted
mutant, the L63Q/A65Q/E66A/N69D/S71A/S72A/
G74D/T135K/E136D/I153T mutant EB-AP/PTase,
145 g/l of 50-IMP was obtained with a molar yield of
74% from inosine. By the mutation of D116E, the
phosphotransferase reaction rate increased almost two
times, and using E. coli overproducing the 11 amino
acid substituted mutant, L63Q/A65Q/E66A/N69D/
S71A/S72A/G74D/D116E/T135K/E136D/I153T mu-
tant EB-AP/PTase, 156 g/l of 50-IMP was obtained with
a molar yield of 79% in 24 h. This was almost the same
productivity as that of the E. coli overproducing
M. morganii G72D/I151T mutant AP/PTase.
I153T mutant AP/PTase (43 mM).8) This result under-
lines the importance of lower Km values for efficient
nucleotide production. The D116E mutation appeared
not to contribute to the decrease in the Km value for
inosine, but the Vmax value of the 11 amino acids
substituted mutant EB-AP/PTase in the phosphotrans-
ferase reaction increased approximately two times. EB-
AP/PAase also catalysed the dephosphorylation reac-
tion, and a decrease in the 50-nucleotidase activity was
another key distinction of the improved mutant. The
Vmax value of the 5 amino acids substituted mutant in the
dephosphorylation reaction decreased to almost half of
that of the G74D/I153T mutant. There were only small
changes in the kinetic parameters by the 5 subsequential
mutations. The Vmax value in the dephosphorylation
reaction was also increased by the D116E mutation.
Although the target residues were selected only for
the comparison of primary structures of homologous
proteins, all 11 residues were placed on one side of the
EB-AP/PTase, where the active site was located. We
have demonstrated that Ser72, Gly74, and Ile153,
situated near an inosine binding site and that S72A,
G74D and I153T contribute to the decrease in the Km
value for inosine.11) Contrary to our expectations, 7 of
11 amino acid residues, Leu63, Ala65, Glu66, Asn69,
Ser71, Thr135, and Glu136, were located far from the
substrate binding pocket. It is likely that a local
structural change due to the substitutions of these
residues had an effect on the structure of the substrate
binding site. Since Asn69 and Ser71 are situated
comparatively close to the substrate binding pocket, it
is possible that the N69D and S71D mutations gave a
positive charge and strengthened the interaction with
inosine. Thr135 and Glu136 are located comparatively
close to the putative phosphate binding site. The
mutation of Glu136 to Asp, a shorter side chain than
Glu, might reduce repulsion against the phosphate
residue. As D116 is situated near an N-terminal residue,
Characterization of mutant acid phosphatases
Each mutant enzyme was purified from crude extracts
of E. coli JM109 transformant and its enzymatic
characteristics were investigated. The kinetic constants
of these enzymes in the transphosphorylation and
dephosphorylation reactions are summarized in Table 2.
One key distinction of the improved mutant was a
reduction in the Km value for inosine in the phospho-
transferase reaction to approximately one-third of that in
the wild type. In spite of the decrease due to the effect of
the two mutations, the Km value for inosine in the
phosphotransferase reaction of the G74D/I153T mutant
was 109 mM, which was extremely high, and above the
achievable inosine concentration of 80 mM. As the
number of substituted amino acid residues increased,
the Km value for inosine decreased without affecting the
Vmax value very much, and the 10 amino acid substituted
mutant EB-AP/PTase exhibited almost the same Km
value for inosine as that of the M. morganii G74D/
Table 2. Kinetic Constants for Transphosphorylation Reactions
The enzyme activities were assayed as described in the Material and Method section. Initial velocities were determined, and the steady-state kinetic
constants were calculated by using a Hanes–Woolf plot.
Km
Vmax
Enzyme
Activity
Substrate
(ꢀM)
(units/mg)
Wild type
G74D/I153T
202,000
109,000
85,000
1.83
1.39
1.03
L63Q/A65Q/E66A/G74D/I153T
L63Q/A65Q/E66A/N69D/S71A/S72A/
G74D/T135K/E136D/I153T
Phosphotrans-
ferase
Inosine
42,000
43,000
1.15
2.60
L63Q/A65Q/E66A/N69D/S71A/S72A/
G74D/D116E/T135K/E136D/I153T
Wild type
G74D/I153T
180
220
340
49.4
6.00
3.11
L63Q/A65Q/E66A/G74D/I153T
L63Q/A65Q/E66A/N69D/S71A/S72A/
G74D/T135K/E136D/I153T
L63Q/A65Q/E66A/N69D/S71A/S72A/
G74D/D116E/T135K/E136D/I153T
50-Nucleotidase
50-IMP
150
4.39
6.96
57.0

1050
Y. MIHARA et al.
3) Yoshikawa, M., Kato, T., and Takenishi, T., Studies of
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it was speculated that a mutation of Asp136 to Glu, a
longer side chain than Asp, caused the structural change
and reduced possible interruption of the enzymatic
reaction by the N-terminal residues.
As described above, the present study was undertaken
in order to increase the phosphotransferase activity of
EB-AP/PTase, which is a side reaction of the enzyme.
By comparing the primary structures of higher produc-
tivity and lower productivity enzymes, candidate resi-
dues of substitution were selected. Then a total of 11
amino acid substitutions were conducted with sequential
substitutions, and we succeeded in enhancing the
phosphotransferase activity of EB-AP/PTase to the
same level as that of MM-AP/PTase. With this in hand,
a number of positions, which might be important for
phosphotransferase activity but whose relevance was not
established by structural analysis, were elucidated. The
result shows that Leu63, Ala65, Glu66, Asn69, Ser71,
Asp116, Thr135, and Glu136 also play an important role
in the phosphotransferase activity of EB-AP/PTase. We
have already found that a substitution of S72F signifi-
cantly reduced the Km value for inosine and increased
the productivity of 50-IMP in the EB-AP/PTase.10)
Combining this knowledge, further improvement of the
EB-AP/PTase to increase the productivity of 50-GMP is
in progress.
Acknowledgments
The authors thank T. Utagawa and H. Kawasaki for
their advice on this work.
11) Ishikawa, K., Mihara, Y., Shimba, N., Ohtsu, N.,
Kawasaki, H., Suzuki, E., and Asano, Y., Enhancement
of nucleoside phosphorylation activity in an acid
phosphatase. Protein Engineering, 15, 539–543 (2002).
12) Yanisch-Perron, C., Vieira, J., and Messing, J., Improved
M13 phage cloning vectors and host strains: nucleotide
sequence of the M13mp18 and pUC19 vectors. Gene, 33,
103–119 (1985).
13) Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular
cloning: a laboratory manual, 2nd edn., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New
York (1989).
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