Cloning of cold-active alkaline phosphatase gene of a psychrophile, Shewanella sp., and expression of the recombinant enzyme

Article abstract of DOI:10.1271/bbb.66.754

A psychrophilic alkaline phosphatase (EC 3.1.3.1) from Shewanella sp. is a cold-active enzyme that has high catalytic activity at low temperature [Ishida et al. (1998) Biosci. Biotechnol. Biochem., 62, 2246-2250]. Here, we identified the nucleotide sequen

Full text of DOI:10.1271/bbb.66.754

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Cloning of Cold-active Alkaline Phosphatase Gene of
a Psychrophile, Shewanella sp., and Expression of
the Recombinant Enzyme
a
a
ab
a
Takeshi MURAKAWA , Hiroshi YAMAGATA , Hiroki TSURUTA & Yasuo AIZONO
a
Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of
Agriculture, Kobe UniversityNada-ku, Kobe, Hyogo 657-8501, Japan
b
Research Fellow of the Japan Society for the Promotion of Science (JSPS)
Published online: 22 May 2014.
To cite this article: Takeshi MURAKAWA, Hiroshi YAMAGATA, Hiroki TSURUTA & Yasuo AIZONO (2002) Cloning of Cold-active
Alkaline Phosphatase Gene of a Psychrophile, Shewanella sp., and Expression of the Recombinant Enzyme, Bioscience,
Biotechnology, and Biochemistry, 66:4, 754-761, DOI: 10.1271/bbb.66.754
To link to this article: http://dx.doi.org/10.1271/bbb.66.754
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Biosci. Biotechnol. Biochem., 66 (4), 754–761, 2002
Cloning of Cold-active Alkaline Phosphatase Gene of a Psychrophile,
Shewanella sp., and Expression of the Recombinant Enzyme†
Takeshi MURAKAWA, Hiroshi YAMAGATA, Hiroki TSURUTA, †† and Yasuo AIZONO †† †
Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture,
Kobe University, Nada-ku, Kobe, Hyogo 657-8501, Japan
Received September 14, 2001; Accepted December 5, 2001
A psychrophilic alkaline phosphatase (EC 3.1.3.1)
from Shewanella sp. is a cold-active enzyme that has
tween the ‰exibility and the heat-lability of cold-ac-
tive enzymes remain to be discovered experimentally.
To investigate the relationship between structure
and function of cold-active enzymes, we previously
high catalytic activity at low temperature [Ishida et al
.
(
1998) Biosci. Biotechnol. Biochem., 62, 2246–2250].
7
)
Here, we identiˆed the nucleotide sequence of a gene en-
coding the enzyme after cloning with the polymerase
chain reaction (PCR) and inverted PCR techniques. The
deduced amino acid sequence of the enzyme contained
conserved amino acids found among mesophilic alka-
line phosphatases and showed some structural charac-
teristics including a high content of hydrophobic amino
isolated SCAPase and characterized it. This phos-
2+
phatase functioned in the presence of Mg ions as a
monomer with the molecular mass of 41 kDa. The
enzyme at 0
temperature, 40
ly lost even at 20
Many species of APase were found in various
9
C showed 39
C, although its activity was gradual-
C.
z of activity at the optimal
9
9
8
)
acid residues and the lack of single
a-helix compared
organisms including Escherichia coli and Homo
9)
with the alkaline phosphatase of Escherichia coli, which
were possibly e‹cient for catalytic reaction at low tem-
peratures. The recombinant enzyme expressed in E. coli
was puriˆed to homogeneity with the molecular mass of
sapiens
.
APases are physiologically important
8)
enzymes; for example, the APases of E. coli and
10)
Bacillus subtilis were induced when the organisms
were starved for phosphate, and secreted into the ex-
tracellular space. Then phosphate could be supplied
to the organisms by the function of the enzyme.
Particularly, there have been numerous investiga-
tions on APase of E. coli. This enzyme functions as a
4
1 kDa. The recombinant enzyme had a speciˆc activity
of 1,500 units mg and had high catalytic activity at low
W
temperatures.
2
+
2+
Key words: alkaline phosphatase; cold-active enzyme;
dimer with two Zn ions and one Mg ion in the
11)
‰
exible structure; psychrophile
active site. Furthermore, physicochemical proper-
ties including the catalytic mechanism with three
1
2)
13)
Cold-active enzymes are produced by psychrophil-
ic and ectothermic organisms living in low tempera-
ture environments and deˆned by high catalytic activ-
ity at low temperature with lower activation energy
metal atoms and the crystal structure of the en-
zyme protein are well known. Together with infor-
mation about other APases, we judged that SCAPase
was suitable for investigation of the structure-func-
tion relationship of cold-active enzyme. However,
the primary-structure of SCAPase was not yet
known.
1
)
than those of mesophilic counterparts. Some of the
2
–4)
enzymes have been puriˆed and characterized.
It
was suggested that this highly e‹cient catalysis in the
low temperature range could result from the ‰exible
structure of the enzymes, which causes the thermal
In this paper, we describe cloning of the gene en-
coding SCAPase and some enzymatical characteris-
tics of the recombinant SCAPase expressed in E.
coli, and discuss the structural properties leading to
high activity at low temperature in comparison with
5
)
6)
lability. The recent thermodynamic studies on
several cold-active enzymes speculated that the limit-
ing structure neighboring the active centers might be
‰
exible, but the structures unrelated to the catalytic
APase of E. coli.
reaction have been rigid. However, the common
structural factors for the ‰exibility and the link be-
†
The nucleotide sequence data reported here appears in the DDBJ, EMBL, and GenBank data banks under the accession number
AB073982
††
Research Fellow of the Japan Society for the Promotion of Science (JSPS)
†††
To whom all correspondence should be addressed. Fax: +81-78-803-5874, E-mail: aizono
＠
kobe-u.ac.jp
„
Abbreviations: APase, alkaline phosphatase; GST, glutathione-S-transferase; IPCR, inverted PCR; pBSK, pBluescript II SK ; SCAPase,
cold-active alkaline phosphatase of Shewanella sp.

Cloning of Cold-active Alkaline Phosphatase Gene
755
Materials and Methods
IARYTGIACYTCIGCRAA-3
?
,
corresponding to
partial amino acid sequences in the peptide L-1 and
L-4, respectively (Y, H, R, and I indicated C or T, A
or C or T, A or G, and inosine, respectively). For 1st
Materials. Acromobacter Protease I (lysyl en-
dopeptidase, 2.8 AU mg) was purchased from Wako
W
Pure Chemicals.
, size 2.1
m
Bondasphere C18 column (300 Å
inverted PCR, F1: 5
GGCTCTTCG-3 and R1: 5
GCGTTTATTGATGTCG-3 , corresponding to the
?-ggggaattccTAAAGTACTCG-
5
m
q
×
150 mm) was from Waters. A
?
?
-ggggaattccAAAGG-
Hybond-N nylon ˆlter, Megaprime random prime
DNA labeling kit, proteinase K, and ribonuclease A
were from Amersham. Restriction enzymes were
from New England Biolabs. New RX50 X-ray ˆlm
was from Fuji Photo Film. T
and competent cells of E. coli DH5
?
partial sequence in nPII fragment ampliˆed by PCR
with the genomic DNA as a template and the de-
generate sense and antisense primers (FL-1 and RL-4,
4
DNA ligase, pBSK,
were from
respectively). For 2nd inverted PCR, F2: 5
aattccGTGTTGACGTCCAAGTCTTT-3 and R2:
-ggggaattccGAGTTGCATGGTTCACTTGA-3
?-gggg-
a
?
Toyobo. AmpliTaq GOLD DNA polymerase was
from Perkin-Elmer. SequiTherm Excell Long-Read
DNA Sequencing kit, and IRD41 infrared dye
labeled primers (M13 forward- and reverse-primers)
were from Aloka. pGEX-6p-2 plasmid, glutathione-
5
?
?,
corresponding to the partial sequence of DNA frag-
ment ampliˆed by 1st inverted PCR. For construc-
tion of expression-plasmid, pGSCAP, NT: 5
aattccAATGATTATCATGGTCGGCG-3 and CT:
-ggggctcgagATCAGTTAACTTTCGGCAAT-3
?-gggg-
?
Sepharose 4B, Mono-Q PC1.6 5 and PreScission
5
?
?,
W
3
2
Protease were from Pharmacia Biotech. [
a
- P]
corresponding to the deduced N-terminal and C-ter-
minal region of SCAPase, respectively. Small letters
represent the designed cleavage sites of restriction
enzymes.
dCTP (3,000 Ci mol) was from ICN Biomedicals.
W
Other chemicals used were of analytical grade.
Puriˆcation of SCAPase. SCAPase was puriˆed to
homogeneity from a psychrophile (Shewanella sp.) as
Southern hybridization for restriction mapping.
7
)
described. The enzyme had a speciˆc activity of
The psychrophile genomic DNA (6 mg) was digested
1
,394 units mg. The molecular mass of the puriˆed
with 20 units of each restriction enzyme, denatured,
and transferred onto a Hybond-N nylon ˆlter as de-
W
phosphatase was 41 kDa by SDS-PAGE.
1
4)
scribed. Hybridization was done at 55
9
C for 16 h
10 cpm) in a solution containing
SSC contains 3 NaCl, 0.3 tri-sodi-
um citrate dihydrate), 5 Denhart's solution, 0.1
(w v) SDS, and 0.1 mg ml denatured salmon sperm
7
Analysis of amino acid sequence. For analysis of
the partial amino acid sequences of SCAPase, the
with a probe (1.0
SSC (20
×
6
×
×
M
M
puriˆed enzyme (35.3
with 0.3
00 l of 50 m
m
g, 0.82 nmol) was incubated
C for 16 h in
Tris-HCl (pH 9.0) containing 4
×
z
m
g of lysyl endopeptidase at 309
W
W
1
m
M
M
DNA. The probe for hybridization was prepared as
follows: the DNA fragment (nPII) was labeled with
urea. The mixture was put through reverse phase
3
2
high pressure liquid chromatography using
Bondasphere C18 column equilibrated with 0.1
a
z
[a
- P] dCTP using a Megaprime random prime
DNA labeling kit. The ˆlters were washed twice for
5 min with 2 SSC-0.1 (w v) SDS and 0.1
SSC-0.1 (w v) SDS at 55
ˆlm with an intensifying screen at „80
m
(
w v) tri‰uoroacetic acid. Peptide fragments were
×
z
9
×
W
W
separated with a linear-gradient of acetonitrile,
from 0 to 60 (v v). The amino acid sequences were
analyzed by automated Edman degradation using a
Shimadzu PPSQ-10 protein sequencer.
z
C, then exposed to X-ray
W
z
9C for 1 day.
W
IPCR. IPCR was done by the method of Triglia
1
5)
et al
units of BamHI and Bgl II, and with Sal I, in 200
of 10 m Tris-HCl (pH 7.9) containing 50 m NaCl,
10 m MgCl and 1 m dithiothreitol at 37 C for
.
The genomic DNA (1 mg) was digested with 20
Preparation of genomic DNA from Shewanella sp.
The genomic DNA was prepared from Shewanella
sp. as follows. Bacterial cells (0.2 g) were suspended
ml
M
M
M
2
M
9
in 5 ml of TEG-buŠer [25 m
M
Tris-HCl (pH 8.0) con-
EDTA and 50 m glucose]. After ad-
g of proteinase K, the suspension was
C for 1 h. The DNA was extracted
three times with phenol–chloroform–isoamylalchol
25:24:1 by volume) and treated with 50 g of
ribonuclease A at 37 C for 16 h. Finally, the DNA
was collected by ethanol-precipitation.
16 h. The digested DNA fragments were extracted
with phenol-chloroform-isoamylalchol and collected
by ethanol precipitation. Each fragment was self-
taining 10 m
dition of 50
M
M
m
incubated at 65
9
ligated with 10 units of T
4
DNA ligase in 500
Tris-HCl (pH 8.0) containing 10 m MgCl
dithiothreitol, 1 m ATP, and 25 g of BSA
ml of
50 m
10 m
at 15
M
M
2
,
(
m
M
M
m
9
9C for 16 h. PCR was done with 100 pmol of the
synthetic oligonucleotides as sense and antisense
primers and the ligated DNAs as a template in 100
of 10 m Tris-HCl (pH 8.3) containing 50 m KCl,
2.5 m
m
l
Oligonucleotides used as primers for PCR.
Synthetic oligonucleotides for PCR were designed; as
M
M
M
MgCl
2
, four deoxynucleotides at 0.2 m
M
degenerate primers, FL-1: 5
?
-ggggaattccTAYAAYG-
each, and 5 units of AmpliTaq GOLD DNA poly-
merase. The ampliˆcation conditions were 30 cycles
GIGCIATHGCIGT-3
?
and RL-4: 5 -ggggaattccGG-
?

7
56
T. MURAKAWA et al.
of 1 min at 95
9
C, 1 min at 55
9
C, and 2 min at 72
C, fol-
C. The PCR
9
C
homogenized with aluminium oxide by using a mor-
tar and pestle. The homogenate was centrifuged at
after the initial denaturation for 10 min at 95
9
lowed by a ˆnal 10-min incubation at 72
9
35,000
×
g
for 1 h, and the supernatant was put on a
2.0 2.0 cm)
products were subcloned into the EcoRI site of
glutathione-Sepharose 4B column (
q
×
pBSK.
equilibrated with buŠer A. After the column was
washed with buŠer A, adsorbed material was devel-
Analysis of nucleotide-sequence. Nucleotide se-
quencing was done by the cycle sequencing method
using a SequiTherm Excell Long-Read DNA Se-
quencing kit-LC and IRD41 infrared dye labeled
primers (M13 forward- and reverse-primers) with a
oped with buŠer A containing 5 m glutathione. The
M
phosphatase activity appeared in this eluate (data not
shown). After dialysis against buŠer A, the eluate
material (4.0 mg) containing GST-SCAPase was in-
cubated with 40 units of PreScission protease at 4
for 16 h. The mixture was put onto a Mono-Q
PC1.6 5 column ( 0.16 5 cm) equilibrated with
buŠer A, and the adsorbed proteins were eluted with
a linear gradient of 0 to 0.3 NaCl in buŠer A. The
phosphatase activity appeared at 0.22 to 0.26 NaCl
9C
4
100L DNA sequencer, LI-COR. Analysis and trans-
lation of the obtained sequence were done using the
Genetyx Mac 7.3 software package (Software De-
velopment).
q
×
W
M
M
Expression of fusion protein, GST-SCAPase. For
construction of the expression plasmid ( pGSCAP
of GST-SCAPase fusion protein, the coding region
of the SCAPase gene was ampliˆed by PCR with
in the gradient. The active fractions were pooled,
diluted three times with buŠer A, and put on a
)
Mono-Q PC1.6 5 column again. The adsorbed
W
proteins were eluted with a linear gradient of 0.05 to
1
00 pmol of NT- and CT-oligonucleotide primers
and 10 ng of the genomic DNA as a template in
00 l of 10 m Tris-HCl (pH 8.3) containing 50 m
KCl, 2.5 m MgCl , four deoxynucleotides at
.2 m each, and 2.5 units of AmpliTaq GOLD
DNA polymerase. The ampliˆcation conditions were
0 cycles of 1 min at 94 C, 1 min at 55 C, and 2 min
at 72 C after the initial denaturation for 10 min at
0.25
M
NaCl in buŠer A. The activity appeared as sin-
gle peak at 0.2 to 0.25
M
NaCl in the gradient. Active
1
m
M
M
fractions were pooled and used as the ˆnally puriˆed
enzyme preparation.
M
2
0
M
Measurement of phosphatase activity. Phos-
phatase activity of the GST-SCAPase fusion protein
and the SCAPase excised from GST-SCAPase was
3
9
9
9
9
4
9C, followed by a ˆnal 10-min incubation at 72
9C .
measured in a reaction mixture (100
25 m sodium carbonate, 2.5 m MgCl
bitol, and 1 m -nitrophenyl phosphate (pNPP),
pH 10.6. After incubation at 70 C for 2 min, 1.0 ml
m
l) comprising
2
The PCR product (about 1.2 kbp) was subcloned into
M
M
, 0.5 sor-
M
an EcoRI site and an XhoI site at each end of pBSK
M
p
(
pBSCAP).
9
The insert DNA was conˆrmed as the SCAPase
of 0.1 N NaOH was added to stop the reaction, then
the absorbance at 410 nm was measured. When the
dependence of the activity of the recombinant en-
zyme on pH was examined, the buŠer used in the
gene by sequencing. After digestion of the plasmid
with EcoRI and XhoI, the DNA fragment was insert-
ed into pGEX-6p-2 and the expression plasmid was
introduced into E. coli DH5
a
.
reaction mixture was 25 m
8.4–11.5). The product of the enzyme reaction was
measured by a calibration curve obtained with
M
sodium carbonate (pH
For expression of GST-SCAPase fusion protein,
the transformed cells were inoculated in 10 liters of
p-
LB medium [Luria-Bertanis' broth: 1
z
(w v) tryp-
W
nitrophenol. One unit of phosphatase activity was
deˆned as the amount of enzyme that hydrolyzed 1
W
tone, 0.5
pH 7.5] containing 1 mg of ampicillin. After incuba-
tion at 37 C for 3 h, expression of the fusion protein
was induced by addition of 0.2 m (ˆnal concentra-
tion) IPTG and incubation at 25 C for 48 h.
z z
(w v) yeast extract and 1 (w v) NaCl,
W
m
mol of pNPP for 1 min at 709C and pH 10.6.
9
M
Measurement of protein concentration. The
amounts of native and recombinant SCAPase were
measured spectrophotometrically on the basis of the
9
Puriˆcation of recombinant SCAPase. An expres-
sion plasmid ( pGSCAP) of GST-SCAPase was con-
structed by ligation of the inserted DNA (EcoRI-
XhoI fragment) in pBSCAP into the pGEX-6p-2
plasmid. The pGSCAP was introduced into E. coli
molecular extinction coe‹cient at 280 nm; 8.03
×
4
„1
„1
„1 7)
10 cm
・
mol
・
liter
.
SDS-PAGE. SDS-PAGE was done using 15
z
gel
1
6)
as described. Proteins in a gel were stained with
17)
DH5
Puriˆcation of the recombinant SCAPase was
done at 4 C. Transformed cells (30 g) from a 10-liter
culture were suspended in 100 ml of buŠer A [25 m
Tris-HCl (pH 7.8) containing 5 m MgCl , 1 sor-
bitol, and 0.5 (w v) leupeptin]. After incubation
with 5 mg of lysozyme on ice for 1 h, cells were
a
, and GST-SCAPase was expressed with IPTG.
silver.
9
Results
M
M
2
M
Partial amino acid sequences of SCAPase
Four peptides obtained by digestion of the enzyme
with lysyl endopeptidase with subsequent high
z
W

Cloning of Cold-active Alkaline Phosphatase Gene
757
pressure liquid chromatography showed the amino
acid sequences as follows; L-1: (K)SYNGAIAV,
to 20 9C were 380–700 mmol min mg of the enzyme
W
W
protein.
L-2: (K)M ARGMSTGVAVTAQVNHATPA, L-3:
W
(
K)GYQHITELAQ and L-4: (K)VLGLFAEVQLP.
Discussion
The amino acid sequence of the N-terminal region of
the enzyme protein could not be analyzed because the
amino group of the N-terminal amino acid was prob-
ably modiˆed.
Identiˆcation of the primary structure of SCAPase
is one of important subjects for understanding the
reaction mechanism leading high catalytic e‹ciency
at low temperature of cold-active enzymes.
Restriction map of the SCAPase gene
A DNA fragment (1.7 kbp) containing the SCA-
Pase gene was cloned using PCR and IPCR tech-
niques and sequenced. The deduced amino acid se-
quence of the SCAPase included all of four peptide
sequences, L-1, L-2, L-3, and L-4 that were found in
the puriˆed native enzyme.
As a probe for Southern hybridization, a DNA
fragment (nPII ) of 370 nucleotides in length was
used, which was ampliˆed by PCR with the degener-
ate primers (FL-1 and RL-4) and the genomic DNA
as a template. The amino acid sequence deduced
from the nucleotide sequence of this DNA fragment
contained that of peptide L-2 and 3. A restriction
map based on the results of Southern hybridization
showed that cleavage sites with restriction enzymes
BamHI, HindIII, AccI, Sal I, PstI, Bgl II, SacI, and
XbaI were located around the SCAPase gene (data
not shown).
As shown in Fig. 3B, the recombinant enzyme
showed higher activity (380–700
0–20
APase. The higher catalytic activity at low tempera-
mmol min mg) at
W
W
9
C than the mesophilic counterpart, E. coli
7
)
ture was similar to that of the native SCAPase, in-
dicating that the cloned gene encoded SCAPase and
that the recombinant enzyme was classiˆed as a cold-
active enzyme.
Cloning of SCAPase gene with IPCR
The recombinant SCAPase expressed in E. coli
On the basis of the restriction map around the
SCAPase gene, the ˆrst IPCR was done by using F1
and R1 oligonucleotide primers and the genomic
DNA self-ligated at BamHI-BglII sites. The second
IPCR was done using F2 and R2 primers and the
genomic DNA self-ligated at Sal I sites. The nucleo-
tide sequences of the ampliˆed DNAs were then ana-
lyzed. The same manipulations were done over three
times, and all obtained sequences of clones were
identical.
showed the speciˆc activity of 1,500 units mg under
W
the optimal conditions (70
9
C and pH 10.6), which is
comparable with the value (1,394 units mg) for the
W
7
)
native enzyme. But the optimal conditions for the
recombinant SCAPase were distinctively diŠerent
7
)
9
from that (40 C and pH 9.8) of the native enzyme.
Although a reason for the shift of the optimal
conditions toward high temperature and pH
remains unknown, a similar shift was also found in
the recombinant cold-active protein-tyrosine-
phosphatase from Shewanella sp. that was expressed
Based on the sequences obtained by PCR, ˆrst
IPCR, and second IPCR, the nucleotide sequence of
the DNA fragment (1.7 kbp) containing the SCA-
Pase gene was analyzed (Accession number
AB073982).
The four peptide sequences of L-1, L-2, L-3, and
L-4, which were determined by Edman degradation,
appeared in the deduced amino acid sequence
2
1)
in E. coli
.
Accordingly, the mechanism of protein-
folding in E. coli might in‰uence on the thermal-sta-
bility of these recombinant enzymes.
To date, it has been thought that cold-active en-
zymes were heat-labile in exchange for acquirement
5
)
of the ‰exible conformation. The ‰exible conforma-
tion could provide the high e‹cient catalysis at low
temperature for cold-active enzymes. However,
recently, Thermus thermophilus 3-isopropylmalate
dehydrogenases (IPMDH) adapted to the low tem-
perature range were produced by evolutionary
(
Fig. 1), indicating that this gene certainly encoded
SCAPase. This result showed the SCAPase consisted
of 398 amino acids, and its molecular weight was
4
2,919.
2
2)
molecular-engineering methods. Two of the pro-
duced enzymes, which had respective single-amino
acid residues replaced, Gly12Ser or Lys21Thr, could
Properties of recombinant SCAPase
As shown in Fig. 2, the homogeneity of the puri-
ˆed enzyme preparation was judged by SDS-PAGE.
Its molecular mass was 41 kDa. The recombinant en-
decrease the activation energy at 309C, in spite of
their similar thermal stability in that of wild type
IPMDH. This ˆnding implies that thermal stability
and e‹cient catalytic activity at low temperatures are
not physical trade-oŠ requirements.
zyme (1.7
mg) puriˆed from 30 g of transformed cells
showed a speciˆc activity of 1,500 units mg (70
9
C
W
and pH 10.6) when pNPP was used as the substrate.
The optimal pH of the activity was observed near
pH 10.6 (Fig. 3A). As shown in Fig. 3B, the recom-
binant SCAPase had the maximal activity at 70
and the activities in the low temperature range from 0
Likely, the high catalytic activity around 70 9C of
the recombinant SCAPase indicated that the recom-
binant enzyme was more stable in the high tempera-
9C,
7)
ture range than the native SCAPase in addition to

7
58
T. MURAKAWA et al.
Fig. 1. Comparison of the Amino Acid Sequence of SCAPase with Those of Other APases.
Asterisks indicate identical amino acids among four APases, SCAPase, Shewanella sp. cold-active APase; Bac, Bacillus subtilis
18)
19)
20)
APase III; Ent, Enterococcus faecalis APase; Esc, Escherichia coli APase. The wavy-lined regions represent sequences that match
the partial amino acid sequences found in the puriˆed native SCAPase. Other symbols represent as follows; Boxes under Mg Zn1, and
Zn2, metal-binding sites; sharp, the catalytic residue; double lines on roman numerals, -helix; single lines -sheet; large box, the loca-
tion of PQR-minidomain. Information was obtained from the structure of E. coli APase.
,
a
, b
20)

Cloning of Cold-active Alkaline Phosphatase Gene
759
Fig. 2. Homogeneity of Puriˆed Recombinant SCAPase.
The enzyme preparation was put through SDS-PAGE on a
gel. Lane 1: molecular mass markers, consisting of rabbit
15
z
phosphorylase a (97,400), bovine serum albumin (66,267), rab-
bit aldolase (42,400), bovine carbonic anhydrase (30,000), and
soybean trypsin inhibitor (20,100). Lane 2: puriˆed recombinant
SCAPase.
high catalytic activity at low temperature. It was sug-
gested that the high activity at low temperature and
thermal-stability of recombinant SCAPase might be
elicited independently.
The structural features of the SCAPase for elicit-
ing the high activity at low temperature must be
found out even in the primary structure. Recently,
the crystal structure of a cold-active a-amylase of Al-
teromonas haloplanctis provided the evidence that
the hydrophobic residues in the cold-active enzyme
2
3)
Fig. 3. Dependence of the Activity of Recombinant SCAPase on
pH ( panel A) and Temperature ( panel B).
The activity of recombinant SCAPase (1.2 ng) toward pNPP
was measured as described in ``Materials and Methods''.
molecule were exposed to solvent. And, in the case
of a cold-active alkaline protease, subtilisin S41, it
was suggested that the decrease in numbers of elec-
trostatic interactions mediated by Asp, Glu, Arg, and
Lys residues and Pro residues which severely restrict-
ed the mobility of the loop regions contributed to the
2
+
APase of E. coli, two Zn ions bound in its catalytic
1
)
188
‰
exible structure of the protease. With regard to the
cleft and Arg
residue recognized a phosphate
amino acid composition of the recombinant SCA-
Pase, higher contents of hydrophobic residues (Ile,
Val and Met) and lower contents of hydrophilic
residues (Asp, Glu, Arg and Lys) and Pro residues
group of the substrate before hydrolysis catalyzed
124
mainly with the Ser residue in the active center. By
contrast, the phosphatase activity of the native SCA-
7)
2+
Pase was almost lost in the presence of Zn ions,
20)
2+
than those of APase of E. coli
were observed
though it has the conserved Zn -binding site. There-
fore, it was speculated that its conformation could be
slightly diŠerent from that of APase of E. coli and
this diŠerent structure might contribute to the ‰ex-
ibility of SCAPase.
despite these diŠerences between two enzymes being
less than 3 mole percentages.
The increased hydrophobic amino acids and the
decreased hydrophilic amino acids might contribute
to the ‰exible conformation of SCAPase, although
the role of the increased hydrophobic residues for
stabilization at high temperature remains obscure.
The SCAPase contained the amino acid sequences
conserved among APases of many organisms includ-
As a distinctive feature in the primary structure of
2
14
221
SCAPase, the amino acid-segment (Thr -Arg
)
constituting
lacking in the SCAPase. This
neighboring the catalytic site of the APase of E.
a
-helix VI in the APase of E. coli was
a
-helix VI was located
2
0)
18)
19)
12)
ing E. coli
,
B. subtilis
,
and E. faecalis (Fig. 1),
coli
.
Accordingly, the lack of a single helix in SCA-
which constitute the catalytic site and metal-binding
sites for two Zn ions and a Mg ion. In the case of
Pase might cause the ‰exible conformation leading to
a diŠerent reaction mechanism with metal ions from
2
+
2+

7
60
T. MURAKAWA et al.
APase of E. coli, although the conformation around
the catalytic site of recombinant SCAPase was unde-
ˆned as yet.
5) Feller, G. and Gerday, C., Psychrophilic enzymes:
molecular basis of cold-adaptation. Cell Mol. Life
Sci., 53, 830–841 (1997).
6
)
Lonhienne, T., Gerday, C., and Feller, G.,
Psychrophilic enzymes: revising the thermodynamic
parameters of activation may explain local ‰exibility.
Biochim. Biophys. Acta, 1543, 1–10 (2000).
Ishida, Y., Tsuruta, H., Tsuneta, S. T., Uno, T.,
Watanabe, K., and Aizono, Y., Characteristics of
Moreover, the following diŠerences were found
out between SCAPase and APase of E. coli. Since
SCAPase had no Cys residues, no intramolecular dis-
ulˆde bond, which is a factor to stabilize the confor-
7
)
2
4)
mation in protein, could be formed in this enzyme
molecule, unlike APase of E. coli, where two disul-
psychrophilic
alkaline
phosphatase.
Biosci.
1
8)
ˆde bonds were in one subunit. And the APase of
Biotechnol. Biochem., 62, 2246–2230 (1998).
3
4
37
E. coli could require two
b
-sheets (Ala to Asp and
8) Torriani, A., In‰uence of inorganic phosphate in the
formation of phosphatases by Escherichia coli.
Biochim. Biophys. Acta, 38, 460–479 (1960).
4
4
47
52
61
Ala to Leu ),
a-helix I (Thr to Asp ) at the N-
395
terminal region, and ``PQR minidomain'' (Ala to
4
22
21)
9
)
Gum, J. R., Hicks, J. W., Sack, T. L., and Kim, Y.
S., Molecular cloning of complementary DNAs en-
coding alkaline phosphatase in human colon cancer
cells. Cancer Res., 50, 1085–1091 (1990).
Met ) for formation of homodimer that were not
conserved in the primary structure of recombinant
SCAPase. These Cys residues and the factors for
dimerization were also lacking in APases of B. subti-
1
1
0) Hulett, F. M. and Jensen, K., Critical roles of spo0A
and spo0H in vegetative alkaline phosphatase produc-
tion in Bacillus subtilis. J. Bacteriol., 170, 3765–3768
2
2)
lis
,
which are mesophilic enzymes, suggesting that
these characteristics might not contribute to the ‰ex-
ibility of recombinant SCAPase directly.
(
1988).
In conclusion, SCAPase have some structural
characteristics including the higher content of
hydrophobic residues and the smaller contents of
hydrophilic residues than APase of E. coli, and the
1) Plocke, D. J., Levinthal, C., and Vallee, B. L., Alka-
line phosphatase of Escherichia coli: a zinc metal-
loenzyme. Biochemistry, 1, 373–378 (1962).
12) Stec, B., Holtz, K. M., and Kantrowitz, E. R., A
revised mechanism for the alkaline phosphatase reac-
tion involving in three metal ions. J. Mol. Biol., 299,
lack of single a-helix VI, which were important for
e‹cient catalytic reaction in the low temperature
range. The establishment of an expression system for
abundant recombinant SCAPase and the solution of
the crystal structure are essential for further investi-
gations of the relationship between structure and
function of cold-active enzymes.
1
303–1311 (2000).
1
1
3) Kim, E. E. and WyckoŠ, H. W., Structure of alkaline
phosphatases. Clin. Chim. Acta, 186, 175–187 (1990).
4) Sambrook, J., Fritsch, E. F., and Maniatis, T.,
Molecular cloning: A laboratory manual, 2nd eds.,
Cold Spring Habor Laboratory, Cold Spring Harbor
(
1989).
Acknowledgment
15) Trigria, T., Peterson, M. G., and Kemp, D. J., A
procedure for in vitro ampliˆcation of DNA segments
that lie outside the boundaries of known sequences.
Nucleic Acid Res., 16, 8186 (1988).
This work was supported in part by a Grant for a
JSPS research fellow from the Ministry of Educa-
tion, Science, Sports, and Culture of Japan, and a
Grant from the Japan Foundation for Applied
Enzymology.
1
6) Laemmli, U. K., Cleavage of structural proteins
4
during the assembly of the head of bacteriophage T .
Nature, 227, 680–685 (1970).
1
7) Morrisey, J. H. Silver stain for proteins in poly-
acrylamide gel:
enhanced uniform sensitivity. Anal. Biochem., 117,
07–310 (1981).
A modiˆcation procedure with
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