Bromoperoxidase activity of vanadate-substituted acid phosphatases
from
Shigella flexneri
and
Salmonella enterica
ser.
typhimurium
Naoko Tanaka
1
, Vale
´
rie Dumay
1
, Qianning Liao
2
, Alex J. Lange
2
and Ron Wever
1
1
Institute for Molecular Chemistry, University of Amsterdam, The Netherlands;
2
Department of Biochemistry, Molecular Biology
and Biophysics, University of Minnesota Medical School and College of Biological Sciences, Minneapolis, Minnesota, USA
Vanadium haloperoxidases and the bacterial class A non-
specific acid phosphatases have a conserved active site. It is
shown that vanadate-substituted recombinant acid phos-
phatase from Shigella flexneri (PhoN-Sf) and Salmonella
enterica ser. typhimurium (PhoN-Se) in the presence of H
2
O

Keywords: acid phosphatase; brominating activity; enantio-
selective sulfoxidation; vanadium bromoperoxidase; vana-
dium chloroperoxidase.
Vanadium haloperoxidases are enzymes that catalyse the
oxidation of a halide by hydrogen peroxide to the corres-
ponding hypohalous acids according to:
H
2
O
2
þ H
þ
þ X
À
! H
2
O þ HOX
The enzymes are named after the most electronegative
halide ion they are able to oxidize, therefore chloroperoxi-
dase oxidizes Cl
–
,Br
–
,I
–
and bromoperoxidase oxidizes Br
–
and I
–
. This class of enzymes binds vanadate (HVO

from Escherichia blattae was determined [15]. Figure 1B
shows the active-site structure of this acid phosphatase. The
similarity of the residues involved in binding oxyanions is
remarkable. Sulfate cocrystallises with the acid phosphatase,
and its binding site (Fig. 1B) is comparable to that of
vanadate in the chloroperoxidase (Fig. 1A), confirming that
these families are indeed evolutionary related and share the
same ancestor [8]. Hemrika et al. [8] showed that apo-
chloroperoxidase has some phosphatase activity, although
the turnover with p-nitrophenyl phosphate as a substrate is
only 1.7 min
)1
, which is about 10 000 times slower than
that of various acid phosphatases. However, the K
m
for the
substrate is less than 50 l
M
[8,16], which is of the same order
of magnitude as various acid phosphatases. These data
show that the active site of chloroperoxidase has a good
affinity for the substrate but is not optimized for phospha-
tase activity. On the basis of the similarity of the active sites
and the fact that the phosphatase activity of phosphatases is
inhibited by vanadate [17,18], we expect that vanadate-
substituted phosphatase has haloperoxidase activity. Indeed,
Correspondence to R. Wever, Institute for Molecular Chemistry,
University of Amsterdam, Nieuwe Achtergracht 129, 1018 WS
Amsterdam, the Netherlands.
Fax: + 31 20 5255670, Tel.: + 31 20 5255110,

holds the gene III signal sequence for secretion of the
recombinant protein into the periplasmic space.
Expression and purification of recombinant PhoN-Se
S. enterica ser. typhimurium phoN gene was cloned in the
pBAD/gIIIA expression plasmid as follows. The mature
sequence (i.e. phoN gene without the 5¢ end coding for the
secretion signal) was PCR amplified from S. enterica
chromosomal DNA using the forward primer 5¢-A
CCA
TGGAATATACATCAGCAGAA-3¢ and the reverse pri-
mer 5¢-CGC
AAGCTTTCACCTTTCAGTAATT-3¢ (the
NcoIandHindIII sites, respectively, are underlined). The
PCR was performed using the Expand
TM
High fidelity PCR
System (Roche) with the following conditions: 1 lg chro-
mosomal DNA, 1 l
M
each primer, 200 l
M
each dNTP,
1.5 m
M
MgCl
2
, 2.6 U high-fidelity polymerase mix in a final
volume of 100 lL. A Ôhot startÕ of 2 min at 94 °Cwas
followed by 30 cycles of denaturation (15 s at 94 °C),
annealing (30 s at 55 °C) and extension (1 min at 72 °C)

Tris/HCl,pH8,2.5m
M
EDTA) to A
600
¼ 5 and incubated on ice for 10 min. The
secreted PhoN-Se was obtained in the supernatant (osmotic
shock fluid) after centrifuging for 10 min at 4 °C. The
osmotic shock fluid was dialysed overnight at 4 °C against
20 m
M
sodium acetate buffer (pH 6.0). The solution was
passed through a 0.45-l
M
filter (Millipore) and then
applied to an SP Sepharose Fast Flow ion-exchange
column (Pharmacia Biotech). The recombinant protein
was eluted with a linear gradient of NaCl (0–0.3
M
)in
20 m
M
sodium acetate buffer (pH 6.0).
Expression and purification of recombinant PhoN-Sf
Sh. flexneri phoN was cloned under control of the T7
promoter in pET3a as described below. It was generated
by PCR using pKU102 as a template and suitable primers
that allowed cloning of phoN between NdeIandHindIII
sites of pET3a. The construct was transformed into the
T7 polymerase-expressing strain BL21(DE3). PhoN-Sf
Fig. 1. Structure of the active site of (A) vanadium chloroperoxidase from C. inaequalis (PDB ID: 1 IDQ) and (B) the acid phosphatase from E. blattae

phosphatases were eventually dialysed against 100 m
M
Tris/
HCl (pH 7.5) and 1 m
M
EDTA which has no effect on the
phosphatase activity.
The protein concentration was determined by using a
protein assay kit (Bio-Rad) with BSA as the standard.
Enzymatic assay of phosphatase activity
The phosphatase activity was measured by hydrolysis of
10 m
M
p-nitrophenyl phosphate as a substrate in 100 m
M
Mes (pH 6.0). The reaction mixtures were quenched with
0.5
M
NaOH to change the pH to 12 and the production of
p-nitrophenol was measured at 410 nm (absorption coeffi-
cient 16.6 m
M
)1
Æcm
)1
).
Enzymatic assay of bromoperoxidase activity
Assay of PhoN-Sf brominating activity. The brominating
activity of the recombinant phosphatases was measured
qualitatively by the bromination of 40 l

)was
quantitatively measured by monitoring the bromination of
50 l
M
monochlorodimedon (MCD) at 290 nm (absorption
coefficient 20.2 m
M
)1
Æcm
)1
) in 100 m
M
sodium acetate
buffer (pH 4.6) containing 200 m
M
Br
–
and 2 m
M
H
2
O
2
on a Cary 50 [23]. The kinetic parameters were determined
using the
ENZYMEKINETICS
program from Trinity Software.
Assay of PhoN-Se brominating activity. The brominating
activity of PhoN-Se was measured by the phenol red assay
as mentioned above but using sodium acetate (pH 4.6)

phosphatases was demonstrated using thioanisole as a
substrate [20]. Thioanisole at a concentration of 2 m
M
was
incubated with 2 m
M
H
2
O
2
, 100 l
M
vanadate and 100 n
M
enzyme in 100 m
M
acetate buffer (pH 5.0) at 25 °Cin
1.7-mL sealed glass vials to prevent evaporation of the
substrate. After overnight incubation, H
2
O
2
remaining in
the reaction mixture was quenched with Na
2
SO
3
.The
enantiomeric products were extracted with dichloroethyl-
ene, evaporated to 20 lL, and dissolved in 1 mL hexane/

alignment (not shown) of vanadium chloroperoxidase with
these enzymes points to conservation of three separate
domains. Domain 1 contains Lys353 and Arg360; domain
2, Ser402, Gly403, His404, and domain 3, Arg490 and
His496. This shows clearly that the binding pocket for
vanadate in the peroxidases is very similar to the phosphate-
binding site in phosphatases. However, the overall similarity
between vanadium chloroperoxidase and these phosphatases
is very low (see also [8]), and the domains are connected by
regions that are highly variable. Both phosphatases were
expressed as recombinant proteins in E. coli, as described in
Materials and methods. No acid phosphatase activity was
detected in E. coli host strains TOP10 or BL21(DE3). In the
absence of inducer, neither TOP10, which harbours the
expression vector for PhoN-Se, nor BL21(DE3), which
harbours the expression vector for PhoN-Sf, showed
2164 N. Tanaka et al.(Eur. J. Biochem. 269) Ó FEBS 2002
relevant levels of acid phosphatase activity. On induction,
the specific activity of acid phosphatase in both strains was
about 40 UÆmg
)1
.
During purification, the acid phosphatase activity always
cochromatographed with a protein of about 30 kDa, in
agreement with the molecular mass of each phosphatase.
The final preparations with a yield of 1–2 mg Pho-Sf per L
of culture medium were judged to be at least 90% pure by
SDS/PAGE. There is a minor band present with a slightly
lower molecular mass. However, this band originates from
proteolytic degradation of the native phosphatase according

not shown). Many other phosphatases are inhibited by
vanadate [17,18], which is homologous in structure to
phosphate. Although it has no sequence similarity to the
bacterial acid phosphatases, the crystal structure of the
vanadate-substituted rat acid phosphatase shows clearly
that vanadate binding was strikingly similar to that in the
vanadium chloroperoxidase from C. inaequalis [10]. There-
fore, it is likely that vanadate binds to the active site of
PhoN and causes the peroxidase-like activity.
Further quantitative kinetic studies were carried out using
the MCD assay. Figure 2A shows that % 10 l
M
vanadate is
necessary to obtain full activity of 500 n
M
PhoN-Sf. From a
Hill plot (not shown) it was possible to obtain a K
d
of
% 1 l
M
at pH 4.6. In the presence of 100 l
M
vanadate, it
takes % 20 min to fully induce the brominating activity of
PhoN-Sf (result not shown). Therefore, at least 30 min of
preincubation with 100 l
M
vanadate was carried out with
PhoN-Sf as described in Materials and methods. Figure 2B

value of the group involved in the
bromination activity of these phosphatases. As only a
limited amount of enzyme was available, the determination
of the optimum pH of PhoN-Sf was based on a single
substrate concentration (200 m
M
KBr and 2 m
M
H
2
O
2
).
For PhoN-Se it was possible to measure K
m
and V at each
pH value. Figure 3B shows the pH-dependence of V.The
data suggest that a group with a pK
a
of % 4.3 is involved in
the bromination reaction. The K
m
forbromidewasalso
pH-dependent and increases with increasing pH (not shown).
A steady-state kinetic study of the brominating activity of
vanadate-substituted PhoN-Sf and PhoN-Se was carried
out. For PhoN-Sf, a K
m
of % 350 m
M

M
PhoN-Sf was preincubated for
1.5 h with 100 l
M
vanadate in 100 m
M
Tris/HCl (pH 7.5), and the
activity was measured by the MCD assay. (B) PhoN-Se was preincu-
bated for 1.5 h with various concentrations of vanadate, and the
activity was measured by MCD assay. K
m
and V for KBr at each pH
were recorded. The activity measurements were carried out in tripli-
cate.
Ó FEBS 2002 Haloperoxidase activity of phosphatases (Eur. J. Biochem. 269) 2165
of 120–180 UÆmg
)1
observed for vanadium haloperoxidases
[26,27]. However, the turnover for the brominating activity
of the acid phosphatases is of the same order of magnitude
as the phosphatase activity of apo-chloroperoxidase
(1.7 min
)1
)[8].TheK
m
for H
2
O
2
was also determined,

Æs
)1
and 2
M
)1
Æs
)1
for bromide oxidation by PhoN-Sf and
PhoN-Se, respectively. If one compares these values with the
specificity constant for bromide oxidation [28] by the
bromoperoxidase from A. nodosum (1.8 · 10
5
M
)1
Æs
)1
), it
is clear that the vanadate-substituted acid phosphatases are
poor catalysts in bromide oxidation.
As several vanadium haloperoxidases are able to catalyse
the enantioselective sulfoxidation of thioanisole [19,20], we
investigated whether the PhoN-Sf and PhoN-Se catalysed
this reaction. Indeed, when 0.5 l
M
PhoN-Sf was incubated
overnight with 2 m
M
thioanisole and 2 m
M
H

2
,a
minor amount of a racemic mixture resulted. It is clear that
vanadate is essential for the enantioselective sulfoxidation
activity of PhoN-Sf. PhoN-Se also catalyzes the sulfoxida-
tion of thioanisole, but in this case the S enantiomer was
produced with a selectivity of 36%. Surprisingly, in the
absence of vanadate an enantioselective conversion was also
observed (e.e. 24%). However, the conversion was much
slower than when vanadate was present. As further
incubation of PhoN-Sf, the sulfide and H
2
O
2
with 1 m
M
EDTA resulted in a lower e.e., the sulfoxidation observed in
the absence of vanadate may be due to metal contamination
in the preparation that was not completely removed by
dialysis against 1 m
M
EDTA. Recently, it has been reported
that vanadate-incorporated phytase [29], an unrelated
phosphatase that mediates the hydrolysis of phosphate
esters, also catalyses the enantioselective sulfoxidation of
prochiral sulfides with H
2
O
2
to the S-sulfoxides. However,

the basis of its active site is going to be more difficult than
expected.
ACKNOWLEDGEMENTS
This work was supported by the Council of Chemical Sciences of the
Netherlands organization for Scientific Research, the E.U. Research
Training Network on Peroxidases in Agriculture, the Environment and
Fig. 4. Bromoperoxidase activity of vanadate-substituted PhoN-Sf
(0.2 l
M
) at pH 4.6 and PhoN-Se (1 l
M
)atpH4.2asafunctionofthe
substrate concentration. PhoN-Sf was preincubated for 1 h in 100 m
M
Tris/HCl (pH 7.5) with 100 l
M
vanadate and the activity measured in
the MCD assay. (A) PhoN-Sf in 2 m
M
H
2
O
2
and variable concen-
trations of Br
–
.(B)PhoN-Sfin300m
M
Br
–

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