Cloning, expression, and characterization of a thermostable PAP2L2, a new member of the type-2 phosphatidic acid phosphatase family from Geobacillus toebii T-85

Article abstract of DOI:10.1271/bbb.80305

Most members of the type-2 phosphatidic acid phosphatase (PAP2) superfamily are integral membrane phophatases involved in lipid-related signal transduction and metabolism. Here we describe the cloning of a novel gene from Geobacillus toebii T-85, encoding

Full text of DOI:10.1271/bbb.80305

Biosci. Biotechnol. Biochem., 72 (12), 3134–3141, 2008
Cloning, Expression, and Characterization of a Thermostable PAP2L2,
a New Member of the Type-2 Phosphatidic Acid Phosphatase Family
from Geobacillus toebii T-85
Yong ZHANG, Zhenxing YANG, Xiaodong HUANG, Jing PENG, Xiangwei FEI,
y
Shaohua GU, Yi XIE, Chaoneng JI,y and Yumin MAO
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University,
Shanghai 200433, P.R. China
Received May 2, 2008; Accepted September 8, 2008; Online Publication, December 7, 2008
[doi:10.1271/bbb.80305]
Most members of the type-2 phosphatidic acid
phosphatase (PAP2) superfamily are integral membrane
phophatases involved in lipid-related signal transduc-
tion and metabolism. Here we describe the cloning of a
novel gene from Geobacillus toebii T-85, encoding a
PAP2-like protein, Gtb PAP2L2, which contains 212
amino acids and shows a limited homology to other
known PAP2s, especially at conserved phosphatase
motifs, and a similar six-transmembrane topology
structure. This enzyme was expressed, and purified in
Escherichia coli. Recombinant Gtb PAP2L2s from the
membrane fractions were solublized with 0.3% (v/v)
Triton X-100 and purified by Ni2þ affinity chromatog-
raphy. The purified enzyme showed broad substrate
specificity to phosphatidic acid, diacylglycerol pyro-
phosphate, and lysophosphatidic, but preferred phos-
phatidic acid and diacylglycerol pyrophosphate in vitro.
Gtb PAP2L2 is a thermal stable enzyme with a half-life
of 30 min at 60 ꢀC. The enzyme was strongly inhibited
by 1% SDS, 10 mM veranda, and Zn2þ, whereas it was
independent of the Mg2þ ion, and insensitive to N-
ethylmaleimide. The purified recombinant Gtb PAP2L2
was catalytically active and highly stable, making it
ideal as a candidate on which to base further PAP2
structure/function studies.
lipids, such as lysophosphatidic acid (LPA), phosphatidic
acid (PA), ceramide-1-phosphate (C1P), diacylglycerol
pyrophosphate (DGPP), and sphingosine-1-phosphate
(S1P), in a Mg2þ independent, N-ethylmaleimide
(NEM) insensitive manner.2,3) All of these phospholipid
molecules and the corresponding dephosporylated prod-
ucts (e.g., diacylglycerol, ceramide, and sphingosine)
have been found to be potent signaling molecules. On
the basis of this and their cellular membrane local-
izations, these enzymes in the PAP2 family are
postulated to play vital roles in lipid signal transduction
and metabolism.4–6) This class of enzymes has been
shown to be involved in the regulation of cell migration
and differentiation, e.g., Drosophila Wunen7) and rat
Dri428) as homologs of PAP2. At least four different
PAP2 isoforms have been described in humans, hPAP2a,
2b, 2c, 2d, and all of these enzymes have broad substrate
specificity.9,10) In addition to these identified eukaryotic
PAP2s, a large number of membrane-associated PAP2-
like proteins have been found in bacteria. These
bacterial membrane proteins, as eukaryotic PAP2 close
homologs, have also been found to be lipid phosphate
phosphatases.11,12)
Despite its apparent importance, relatively little is
known as to structural and functional information on this
class of integral membrane phosphatases in the PAP2
family. Previously, a critical insight was that PAP2
phosphatase motifs are also found in structure-known
soluble fungal vanadium-dependent choroperoxidase13)
and nonspecial bacterial acid phosphatase.14) Subse-
quently, amino acids sequence comparison, mutational
analysis, and extrapolations on the physical organization
of the PAP2 family proteins, suggested that three
conserved motifs constitute the active sites of the
enzymes.15,16) But none of the actual structure of
Key words: type 2 phosphatidic acid phosphatase; ther-
mostability; integral membrane phospha-
tase; cloning; purification
The type-2 phosphatidic acid phosphatase (PAP2)
superfamily is defined by three conserved phosphatase
motifs.1) Differently from other phosphatases, most
members of this family are integral membrane phospha-
tases catalyzing dephosphorylation of various phospho-
y
To whom correspondence should be addressed. Chaoneng JI, Fax: +86-21-65642502; E-mail: chnji@fudan.edu.cn; Yumin MAO, Fax: +86-21-
65642502; E-mail: ymmao@fudan.edu.cn
Abbreviations: PAP2, type-2 phosphatidic acid phosphatase; PA, phosphatidic acid; DGPP, diacylglycerol pyrophosphate; LPA, lysophosphatidic
acid; pNPP, p-nitrophenyl phosphate; NEM, N-ethylmaleimide

A Thermostable PAP2L2 Phosphatase from Geobacillus spp.
3135
membrane-associated PAP2 is available, partly due to
tight association with membranes and the low abun-
dance of PAP2 family enzymes, which hampered large-
scale purification attempts and related study. In fact,
thermostable proteins from thermophilic organisms have
been found to be good models for understanding the
function and structure of their mesophilic counterparts,
and their thermostability would facilitate protein puri-
fication and further study.17) Furthermore, comparative
studies of mesophilic and thermophilic protein homo-
logues might provide insight into the mechanisms of
protein thermal adaptation as well as protein evolution.
In this study, we cloned a gene coding for a
thermostable PAP2 homolog, Gtb PAP2L2, from ther-
mophilic Geobacillus toebii T-85, and successfully
overexpressed, purified, and characterized this novel
member of the PAP2 family.
Construction of expression vector and expression of
recombinant protein. The Gtb PAP2L2 coding sequence
without start coden ATG was amplified by polymerase
chain reaction (PCR) from plasmid pTP, using specific
primers, Pf 5-CTAGCTAGCAACAAATGTCTTCTAT-
CTG-3 and Pr 5-CCGCTCGAGAAATCCATGGCTTT-
CGTTG-3, containing NheI and XhoI restriction sites
respectively (underlined). The resulting PCR product
was digested with corresponding restriction enzymes
and cloned into the NheI and XhoI sites of pET-28b
(Novagen, Madison, WI). The fidelity of the insertion
in pET vectors was confirmed by sequencing. In this
case, Gtb PAP2L2 was expressed with an additional
27 residues, including the N-terminal hexa-his tag
sequence. E. coli Rosetta (DE3) pLyS harboring the
constructed expression plasmid was grown in 1 liter LB
medium containing kanamycin (100 mg/l) and chlor-
amphenicol (34 mg/l), and was induced by the addition
of 0.5 mM IPTG at OD600 0.6–0.8. After induction for
10 h at 30 ꢀC, the cells were harvested by centrifugation.
Materials and Methods
Materials. The enzymes used in vector construction
were from New England Biolabs (Beijing, China). All
chemicals were purchased from Sigma-Aldrich (St.
Louis, MO) unless otherwise specified.
Preparation of membrane fraction and purification of
Gtb PAP2L2 fusion protein. The harvested cell pellet
was suspended in a standard buffer (50 mM NaH2PO4,
300 mM NaCl, 10 mM imidazole, pH 8.0) and ultra-
sonicated on ice. The lysate was centrifuged at
100;000 ꢁ g for 50 min at 4 ꢀC. The supernatant was
used as the soluble fraction and the pellet as the
membrane fraction after resuspension in an equal
volume of the standard buffer. Triton X-100 was added
to the membrane fraction to a final concentration of
0.3% (v/v). After stirring for 2 h, the mixture was then
stored overnight at 4 ꢀC. Crude extract was obtained by
centrifugation at 100;000 ꢁ g for 50 min to remove
insoluble materials. Then, the crude extract was incu-
bated at 60 ꢀC for 30 min and centrifuged at 14;000 ꢁ g
for 30 min. The supernatant obtained was batch-bound
to 2 ml of pre-equilibrated Ni-NTA Superflow resin
(Qiagen, Hilden, Germeny). The material was loaded
into a 0:8 ꢁ 8 cm chromatography column and washed
extensively with washing buffer (0.2% Triton X-100,
50 mM NaH2PO4, 300 mM NaCl, pH 8.0) with an
increasing imidazole concentration (650 mM) to remove
unspecifically bound proteins. Finally, the bound en-
zyme was completely eluted with an elution buffer
(0.2% Triton X-100, 250 mM imidazole, NaH2PO4,
300 mM NaCl, pH 8.0). Active fractions were pooled
and concentrated by ultrafiltration (Amicon-Ultra-15
column, Millipore, MA).
Construction of genomic libraries and screening for
thermostable PA phosphatase positive clones. Genomic
DNA from thermophilic bacterial was prepared by the
phenol-chloroform extraction method18) and partially
digested with Sau3AI. The DNA fragments, >2 kb, were
recovered from agarose DNA gel and ligated into
BamHI-digested dephosphorylated plasmid pUC19.
E. coli DH 5ꢀ was transformed with the ligation mixture
and plated on Luria-Bertani (LB) agar plates containing
100 mg/ml ampicilline and 0.1 mM isopropyl-D-thioga-
lactopyranoside (IPTG) to form about 103 colonies/
10-cm dish. Then all the colonies were lifted onto
nitrocellulose filter papers, and the filters were placed,
colony-side up, on a 10-cm plate and incubated with 3 ml
of buffer (0.1 M Tris–HCl, pH 8.5, and 1% Triton X-100)
at 70 ꢀC for 5 min. Ten ml of 0.6 M p-nitrophenyl
phosphate (pNPP) (Merck, Darmstadt, Germany) was
supplemented and incubated an additional 5 min at
70 ꢀC. Colonies producing phosphatase hydrolyze pNPP,
releasing pNP yellow in color. Corresponding to the
original plates, putative positive colonies were found and
chosen. And plasmids (termed pTP) from these colonies
were prepared and sequenced by submission to the DNA
sequencing facility of Unit Gene (Shanghai, China).
Sequence analysis. Homology searches were per-
formed by BLAST at the National Center for Biotech-
nology Information (NCBI) web server (http://www.
Protein assay. Analytical 12% sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS–PAGE) and
native polyacrylamide gel electrophoresis (native–
PAGE) were performed, gels were stained, and the
proteins were visualized using Coomassie brilliant blue
R-250.18) Protein purity was determined by densitometry
using a gel documentation system, Gel–Doc EQ (Bio–
Rad, Hercules, CA). Recovered protein from the native
ncbi.nlm.nih.gov/BLAST).
A hydropathy plot was
obtained using the hydropathy analysis program (http:
//www.tcdb.org/progs/hydro.php). A phylogenetic tree
was inferred by the maximum parsimony method using
MEGA program version 4.0.

3136
Y. ZHANG et al.
gel electrophoresis was also subjected to western blot
assay with an anti-hexa-His antibody (Qiagen, Hilden,
Germeny). Detection was done using an ECL detection
kit (Pierce, Rockford, IL). The protein concentration
was determined by BCA protein assay (Pierce) with
bovine serum albumin (BSA) as the standard protein.
and pH. Strain T85 was a rod-shaped, not motile Gram-
positive bacillus that grew optimally at about 65 ꢀC at
pH 7.5. On the basis of morphological, physiological,
and 16S rRNA gene characteristics, strain T85 was
assigned to Geobacillus toebii T85.
Cloning and sequence analysis of Gtb PAP2L2
Through activity-based screening of the thermophilic
strain T85 genomic library, two clones with apparent
phosphatase activity were detected in approximately
10,000 transformed colonies. According to the sequenc-
ing results, one of these two clones encoded a putative
alkaline phosphatase, but the other one demonstrated an
open reading frame (ORF) of 639 bp encoding 212
amino acids, smaller than previously known and
predicted PAP2s, varying form 216 to 357 amino acids
in length.1) A search of conserved domain databases of
NCBI by RPS-BLAST analysis suggested that the
deduced protein contained a single-domain PAP2like2.
The following three highly conserved phosphatase
motifs were found in the deduced protein sequence:
motif 1, KXXXXXXRP; motif 2, PSGH; and motif 3,
SRXXXXXHXXXD (Fig. 1A, C). The deduced protein
sequence displayed a relatively limited sequence iden-
tity with other known eukaryotic PAP2 homologs
(<22% identity), whereas a hydropathy analysis of the
deduced protein revealed six remarkable hydrophobic
regions of sufficient length to span the membrane,
indicating the presence of six trans-membrane segments
(Fig. 1B). Similar sequence features were also noted in
human and other known membrane-bound PAP2 homo-
logs, which suggest that this ORF product is a new
member of the PAP2 superfamily. Hence, the ORF was
designated the Gtb PAP2L2 gene (GenBank accession
no. EF535727). Accordingly, the evolutionary relation-
ships among the various PAP2 homologs of human and
bacterial origin were analyzed by phylogenetic tree
(Fig. 1D). The results confirmed that the Gtb PAP2L2
protein is conserved as a novel PAP2 homolog.
Enzyme characterization. The substrate specificity of
the enzyme was examined using DGPP (8), PA (8), and
LPA (18) (Avanti Polar Lipids, Alabaster, AL) by
malachite green colorimetric assay.19) The assay was
performed in 94-well surface assay plates (Nunc,
Roskilde, Denmark) in 50 ml of assay buffer (0.1 M
Tris–HCl, pH 7.5, 0.2% Triton X-100, 10 mM EDTA)
containing DGPP, PA, or LPA at 50 mM in the absence
and the presence of Gtb PAP2L2 at 3.0 mg. The assay
plates were placed into a thermal incubation chamber
(Yuejin, Shanghai, China), and the reaction was carried
out at 65 ꢀC for 5 min. It was stopped with 100 ml of
malachite green reagent (Biomol Green, Plymouth
Meeting, PA), and the absorbance was read at 620 nm
according to the manufacturer’s instructions. Kinetic
data were analyzed with a surface dilution kinetics
model-fitting program according to the Michaelis-
Menten equation.20) A standard assay for phosphatase
activity was carried out in 0.5 ml of reaction mixture
containing 0.1 M Tris–HCl buffer (pH 7.5), 2.4 mM
pNPP, and 3.0 mg of the enzyme. After incubation at
65 ꢀC for 5 min, the reaction was terminated by adding
an equal volume of 2.0 M NaOH, and the released pNP
(18,380 Mꢂ1 cmꢂ1) was measured at 405 nm using a
spectrophotometer (Hitachi, Tokyo). The optimum pH
for enzyme activity was determined in 0.1 M acetate
buffer (pH 2.5–6.5), 0.1 M Tris–HCl (pH 6.5–9.0), and
0.5 mM diethanolamine (pH 9.0–13.0) buffer at 65 ꢀC
with pNPP as the substrate. Temperature effects on
activity and thermostability were investigated by stand-
ard assay at the indicated temperatures. To determine
the effects of metal ions and inhibitors, the enzyme was
pre-incubated with various divalent ions and inhibitors
for 1 h, and then the activity was assayed by standard
procedure.
Furthermore, we noted that possible orthologs of Gtb
PAP2L2 are also present in some human pathogens, e.g.,
several putative membrane-associated phospholipid
phosphatases from Bacillus cereus (Genbank accession
no. NP977723) and Bacillus anthracis (Genbank acces-
sion no. ZP00391615). Membrane-associated members
of the PAP2 family are found across kingdoms,
suggesting important roles of these enzymes in cell
physiology.
Results and Discussion
Characterization of thermophilic strain T85
Thermophilic bacteria were isolated from a hot spring
in China with LB agar media at 65 ꢀC. The bacterial 16S
rRNA gene was amplified as described by Marchesi
et al.21) The strain T85 rRNA gene partial sequence
(GenBank accession no. EF535794) exhibited a high
level of homology with those of Geobacillus toebii
strains, e.g., 99.6% homology with G. toebii strain
BGSC 99A1, 99.2% homology with G. toebii strain
DPE7, and 99.0% homology with G. toebii strain
E26323. Additionally, we analyzed the phenotype of
strain T85 by routine methods, including microscopic
inspection, Gram staining, optimal growth temperature
Expression and purification of the recombinant Gtb
PAP2L2 protein
The recombinant Gtb PAP2L2 protein was expressed
in E. coli. By optimizing the expression conditions, we
found significant elevation of thermostable phosphatase
activity in the induced sample. Enzymatic activity was
detected mainly in prepared membrane fractions, as
compared with extremely low activity in the soluble
fractions (data not shown). This result is consistent with

A Thermostable PAP2L2 Phosphatase from Geobacillus spp.
3137
A
B
C
D
Fig. 1. Sequence Analysis of Gtb PAP2L2 Phosphatase.
A, The sequence of Gtb PAP2L2. The three conserved phosphatase motifs are in shaded boxes. Predicted transmembrane regions are indicated
by the bars under the sequence; B, Hydropathy profile of Gtb PAP2L2 amino acid sequence. The hydropathy of Gtb PAP2L2 was analyzed by
the method of Kyte and Doolittle22) using a window size of 19 residues; C, Alignments of the three highly conserved type-2 phosphohydrolase
motifs of Gtb PAP2L2 and several human PAP2s; D, Phylogenetic tree of Gtb PAP2L2 and PAP2 homologs. The sequences of several PAP2
family members were aligned by ClustalW. Numbers above the horizontal lines indicate the evolutionary distance between one protein and
another. The GenBank accession numbers are NP003702 (2a: hPAP2a), NP003704 (2b: hPAP2b), NP003703 (2c: hPAP2c), NP001032394 (2d:
hPAP2d), NP287919 (eco: E. coli pgpB), and ABQ12440 (gtb: Gtb PAP2L2).
the above membrane protein prediction, and suggests
that Gtb PAP2L2 was correctly folded and inserted into
the plasma membrane of E. coli.
PAP2L2 from membrane fragments was effectively
solubilized with 0.3% (v/v) Triton X-100. After heat
treatment, the majority of thermolabile E. coli proteins
were removed by centrifugation. Then a single step of
Ni2þ-chelated affinity chromatography was successful in
removing the residual contaminating proteins. The
recombinant Gtb PAP2L2 was purified to approximately
93% pure, as shown in Fig. 2A. As is often the case with
strongly hydrophobic membrane proteins,23) boiling of
protein samples in SDS loading buffer prior on SDS–
PAGE often results in significant aggregation of Gtb
PAP2L2s so that they do not enter the resolving gel.
Protein samples were therefore incubated in SDS
loading buffer at room temperature before gel loading.
The observed protein band migrated to 23.5 kDa
In preliminary experiments, we tested several deter-
gents, such as Triton X-100, Na cholate, and CHAPS, to
solubilize the recombinant Gtb PAP2L2 from the
membrane fraction at concentrations from 0 to 2%
(data not shown). Finally, most membrane-associated
recombinant Gtb PAP2L2s were liberated into the
supernatant when the membrane fragments were incu-
bated with Triton X-100. Higher concentrations of
Triton X-100 released more membrane-associated pro-
tein, but resulted in an inhibition of Gtb PAP2L2
phosphatase activity and disturbed the resolution of
SDS–PAGE. In this experiment, the recombinant Gtb

3138
Y. ZHANG et al.
Table 1. Purification of Recombinant Gtb PAP2L2 from E. coli
Cells
A
B
Total
proteins activity activity
Total
Special
Yield Purification
fold
(%)
(mg)a
(U)b
(U/mg)
Lysate
224.9
172.2
109.8
2.5
407.4
164.2
123.2
81.5
1.8
1.0
100
1
Crude extract
Heat treatmentc
Ni-chelation
40.3
30.2
20.0
0.5
0.6
18.4
1.1
33.3
aTotal protein was determined by BCA assay with BSA as standard.
bActivity unit was expressed in mmol/min with pNPP as substrate.
cData for heat treatment were taken directly after the heating process, not
reflecting that denatured proteins were removed by centrifugation.
Fig. 2. Electrophoresis and Western Blot Analysis of Purified
Recombinant Gtb PAP2L2.
Table 2. Kinetic Constants for Recombinant Gtb PAP2L2a
A, Analysis of recombinant Gtb PAP2L2 expression and
purification on 12% SDS–PAGE. Lane M, molecular weight
marker; lane 1, total protein of induced bacteria; lane 2, Supernatant
of the induced bacteria; lane 3, crude extract after heat treatment at
60 ꢀC for 30 min; lane 4, purified recombinant Gtb PAP2L2 protein;
lane 5, purified recombinant Gtb PAP2L2 after concentration; B,
Western blot analysis of purified recombinant protein. The recov-
ered recombinant Gtb PAP2L2 protein from native PAGE was
further confirmed by Western blotting using the anti-hexa-his tag
antibody.
Km(app)
Vmax(app)
(unitsb/mg)
Vmax=Km
(units/mg/mol %)
Substrate
(mol %)
PA
LPA
DGPP
1.5
2.6
1.1
0.44
0.10
0.36
0.29
0.04
0.32
aThe concentrations of PA, DGPP, and LPA in the mixed micelles are
expressed in terms of surface concentration (in mol %).
bUnit of activity defined as mmol/min.
according to a molecular weight marker (Qiagen). This
value is smaller than the theoretical value of 26.8 kDa as
calculated from the deduced amino acid sequence of
recombinant Gtb PAP2L2 with the ExPASY proteomics
tool.24) Migration shift is a common feature of mem-
brane proteins.23) It might be due to the absence of a
boiling step unfolding the highly hydrophobic mem-
brane protein in this experiment.
In order further to confirm the identity of the purified
recombinant enzyme, the purified samples were sub-
jected to native PAGE at 4 ꢀC. Following electropho-
resis, one lane from the gel was stained with Coomassie
blue. It showed a single major band. The corresponding
gel band on a second lane from the native gel was
excised, minced in assay buffer and homogenized, and
divided into two fragments. One fragment was detected
by Western blot, and a 23.5 kDa band was verified using
anti-His6-tag antibodies (Fig. 2B). In addition, the other
fragment was assayed for enzymatic activity, and
phosphatase activity was found in the reaction mixture.
These results confirmed purified recombinant Gtb PAP2
phosphatase. The efficiency of the purification process is
shown in Table 1. The typical yield of the purified
recombinant Gtb PAP2L2 was approximately 2.0 mg per
1 liter culture.
performed using well-defined phospholipid/Triton
X-100 mixed micelles.20) Acting on detergent solubi-
lized substrates, Gtb PAP2L2 exhibited typical surface
dilution kinetics, in which the rate of substrate hydrol-
ysis depends on the molar fraction of the substrate in the
mixed detergent and lipid micelle (data not shown).
Surface dilution kinetics is a model system that mimics
the physiological surface of the membrane, where two-
dimensional surface interactions occur.20) The kinetic
constants for the enzyme were obtained using the
Michaelis-Menten equation, and are summarized in
Table 2. Gtb PAP2L2 phosphatase catalyzed dephos-
phorylation of PA, LPA, and DGPP, showing broad
substrate specificity, but preferred DGPP and PA
(Vmax=Km ¼ 0:29, 0.04, and 0.32 units/mg/mol % re-
spectively). The Km values indicate that the affinity of
the enzymes for each substrate. The Gtb PAP2L2
phosphatase exhibited relatively high affinity for DGPP
and PA (Km ¼ 1:1 and 1.5 mol %). Similar Km values for
the PAP2 enzyme hydrolyzing PA was found in human
PAP2s (Km ¼ 0:04–3.4 mol %).25) As a lipid phosphate
phosphatase, Gtb PAP2L2 differs from the DGPP
phosphatases from E. coli26) and from S. cerevisiae27)
with respect to substrate specificity. Those DGPP
phosphatases displayed about 9-fold greater phosphohy-
drolase specificity for DGPP than for PA. However,
recombinant Gtb PAP2L2 phosphatase degraded PA and
DGPP at similar rates.
Characterization of recombinant Gtb PAP2L2
Since the physiologically relevant substrates for Gtb
PAP2L2 are still unclear, the enzyme was tested for
activity against a variety of phosphorylated lipid
substrates. The substrate specificities of the Gtb PAP2L2
enzyme for PA, DGPP, and LPA were examined under
optimal assay conditions. Kinetic experiments were
Considering the stability of the tested phospholipids
substrates at high temperatures and extreme pH, the
activity against p-NPP, a general phosphatase substrate,
was determined to examine the biochemical properties of
Gtb PAP2L2 phosphatase. Several membrane-associated

A Thermostable PAP2L2 Phosphatase from Geobacillus spp.
3139
members of the PAP2 family, such as E. coli diacylgly-
A
cerol pyrophosphate phosphatase (DGPPase),26) yeast
DGPPase,27) and rat PAP2,28) have been described to be
optimally active at pH 6–7. Here, a similar pH-activity
profile was found in recombinant Gtb PAP2L2, which
displayed higher activity at pH 6 to 8, with an optimal
pH of 7.5 (Fig. 3A). Negligible phosphatase activity for
recombinant Gtb PAP2L2 was detected at acidic pH,
below pH 5.0, and this clearly distinguished this enzyme
from bacterial acid phosphates.14) The optimal catalytic
reaction temperature for the recombinant Gtb PAP2L2
was at about 70 ꢀC (Fig. 3B). The purified recombinant
Gtb PAP2L2, which showed no loss of phosphatase
activity at 55 ꢀC after incubation for 1 h, was a thermo-
stable enzyme, as compared to a measured half-life of
55 s at 50 ꢀC for E. coli DGPPase.26) Further analysis
indicated that the half-life of recombinant Gtb PAP2L2
for thermal inactivation at 60 ꢀC was up to 30 min
(Fig. 3C).
B
The activities of PAP2s from yeast and mammalian
cells have been characterized as being Mg2þ independent
and NEM insensitive.1,26) The effects of various divalent
cations on bacterial Gtb PAP2L2 were examined. Mg2þ
and Ca2þ at a concentration of 10 mM had no effect on
Gtb PAP2L2 phosphatase activity. Cd2þ, Co2þ, Mn2þ
exhibited a partly inhibitory effect on activity, whereas
the Gtb PAP2L2 enzyme lost nearly 100% of its original
activity in presence of 10 mM Zn2þ (Table 3). These
observations corroborate previous findings with other
membrane-associated members of the PAP2 family.27)
However, further analysis revealed that the inhibition of
Zn2þ on Gtb PAP2L2 activity was reversible, since the
phosphatase activity was completely restored by treat-
ment with EDTA. This reversible inhibitory effect
probably reflects a specific interaction of Gtb PAP2L2
with Zn2þ. It calls for additional study to verify it.
Inhibition studies revealed that the enzyme was com-
pletely inactivated by ionic detergent 1% SDS, but a
non-ionic detergent, 1% Trion X-100, suppressed only
about 45.6% of the enzyme activity. Vanadate at 10 mM
suppressed of nearly 100% enzyme activity. Molybdate,
an analog of phosphate, is recognized as a potent
inhibitor of various phosphatases. The activity of Gtb
PAP2L2 was strongly inhibited by 2 mM molybdate (the
residual activity was 10.31%). However, the addition of
molybdate at 10 mM to the assay system resulted in a
decreased inhibition effect on enzyme activity (46% of
activity retained). The reason for this remains unknown.
Both inorganic phosphate and pyrophosphate exhibited
marked dose-dependence. Slight inhibition of activity
was observed with 2 M urea. The measured phosphatase
activity of Gtb PAP2 before and after EDTA treatment
demonstrates that there are no significant differences.
The enzyme also exhibited insensitivity to inhibition by
thioreactive compounds: NEM and dithiothreitol (DTT).
Thus purified recombinant Gtb PAP2 phosphatase
displayed typical biochemical characteristics of PAP2
homologs.
C
Fig. 3. Activity Profiles of Recombinant Gtb PAP2L2.
A, effects of pH on activity. Enzyme activity was determined in
the following pH buffers: 0.1 M acetate buffer (pH 2.5–6.5), 0.1 M
Tris–HCl (pH 6.5–9.0), and 0.5 mM diethanolamine (pH 9.0–13.0);
B, Temperature effects on activity. Gtb PAP2L2 phosphatase
activity was determined by standard assay from 25 to 100 ꢀC; C,
Thermostability. Residual activity was measured by standard assay
after incubation of the enzyme at 55 ꢀC ( ), 60 ꢀC ( ), and 65 ꢀC
( ) for the indicated times; Each data point in above figures
represents an average of three determinations with pNPP as the
substrate.
Conclusion
This study was undertaken to provide a source of
large quantities of purified integral membrane phospha-
tase of the PAP2 family. Facile production and
purification of protein is a prelude to in-depth analysis

3140
Y. ZHANG et al.
Table 3. Effect of Various Chemicals on Recombinant Gtb PAP2L2
Activitya
membrane lipid phosphatases/phosphotransferases:
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Relative activity
(%)
Metal ions and inhibitors
Concentration
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None
Mg2þ
100 ꢃ 6:5
103:5 ꢃ 4:3
101:2 ꢃ 2:4
2:0 ꢃ 0:6
10 mM
10 mM
10 mM
10 mM
10 mM
10 mM
1%
Ca2þ
Zn2þ
Cd2þ
42:6 ꢃ 1:7
76:6 ꢃ 2:7
80:0 ꢃ 1:1
0:1 ꢃ 0:5
Co2þ
Mn2þ
SDS
Triton X-100
Inorganic phosphate
1%
45:6 ꢃ 1:3
82:4 ꢃ 1:3
55:8 ꢃ 2:1
72:8 ꢃ 6:7
65:5 ꢃ 3:9
10:3 ꢃ 4:3
43:6 ꢃ 2:0
6:1 ꢃ 0:5
1 mM
10 mM
1 mM
10 mM
1 mM
10 mM
1 mM
10 mM
3 M
Pyrophosphate
Molybdate
Vanadate
0:2 ꢃ 0:2
Urea
34:0 ꢃ 0:8
103:1 ꢃ 2:1
96:5 ꢃ 1:7
104:1 ꢃ 2:1
NEM
DTT
EDTA
5 mM
2 mM
10 mM
aThe enzyme was pre-incubated in absence and the presence of various
divalent metal ions and inhibitors for 1 h, and then the effects of the ions and
inhibitors on activity were examined by standard procedure. All of the
examined metal ions were in the chloride form. Data represent mean ꢃ
standard deviation.
of structure and function. In this study, we cloned,
expressed, and purified a PAP2-like phosphatase, Gtb
PAP2L2, from thermophilic Geobacillus toebii T-85. As
a novel member of the PAP2 superfamily, Gtb PAP2L2
was a thermostable protein, exhibiting a half-life of
30 min at 60 ꢀC. To our knowledge, this is the first
highly thermostable member of the PAP2 superfamily to
be described. Purified recombinant Gtb PAP2L2 was
catalytically active and highly stable, making it ideal for
further structure/function studies.
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Chippaux, M., and Denizot, F., BcrC from Bacillus
subtilis acts as an undecaprenyl pyrophosphate phos-
phatase in bacitracin resistance. J. Biol. Chem., 280,
28852–28857 (2005).
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Lecreulx, D., Identification of multiple genes encoding
membrane proteins with undecaprenyl pyrophosphate
phosphatase (UppP) activity in Escherichia coli. J. Biol.
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between integral membrane phosphatases and soluble
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2423 (2000).
15) Toke, D. A., McClintick, M. L., and Carman, G. M.,
Mutagenesis of the phosphatase sequence motif in
diacylglycerol pyrophosphate phosphatase from Saccha-
romyces cerevisiae. Biochemistry, 38, 14606–14613
(1999).
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Acknowledgments
This study was supported by the National Basic
Research Program of China (973 Program,
2007CB914304), the New Century Excellent Talents
in Universities Program (NCET-06-0356), and the
National Talent Training Fund in Basic Research of
China (J0630643).
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