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FEMS MicrobiologyL,etters130 (19%) 211-214
PhoN-type acid phosphatases of a heavy metal-accumulating
Citrobacter sp.: resistance to heavy metals and affinity towards
phosphomonoester substrates
B.C. Jeong, L.E. Macaskie
*
School of Biological Sciences, The University of Birmingham, Edgbaston, Birmingham BIS 2TT, UK
Received 11 May 1995; revised 30 May 1995; accepted30 May 1995
Abstract
An atypical Citrobucter
sp. isolated from a heavy metal-polluted site has a PhoN-type acid phosphatase that is
responsible for the precipitation of heavy metals as cell-bound metal phosphates via liberated inorganic phosphate, supplied
via enzymic cleavage of a phosphomonoester substrate. Phosphatase activity, comprising two isoenzymes designated CPI
and CPII, was resistant to cadmium (II), zinc (II) and lead (II), but sensitive to uranyl and vanadyl oxycations and cupric ion.
‘Ihe monovalent cations of mercury (I) and silver (I), and trivalent yttrium (III) were inhibitory, particularly to CPII. The
anionic counterion did not influence metal toxicity. The K, of CPII towards some phosphomonoester substrates was higher
than than that of CPI, suggesting that although the two isoenzymes were shown previously to be closely related, subtle
differences exist between them that justify their classification as separate isoenzymes, for which the physiological function is
still obscure.
Keywords:
Phosphatase;Isoenzyme; Citrobacter; Metal toxicity; Phosphomonoester;Heavy metal
1. Introduction
Correspondingauthor. Tel: (0121) 414 5889; Fax: (0121) 414
6557.
molecule (phosphomonester substrate) with co-deposition of liberated phosphate with incoming metal
ions at nucleation sites at the cell surface [4]. A
comprehensive study of the purified acid phosphatase of this atypical strain 5) revealed two isoenzymes that were designated as CPI and CPII on the
basis of their stability, and fractionation behaviour
on ion exchange resins. The enzymes were immunologically cross-reactive, with similar physical properties and N-terminal sequences [5].
In view of the potential of this strain for industrial
waste decontamination, and the resistance of the
enzyme to cadmium [6] the effect of other heavy
metals was investigated. Some differences in metal
sensitivity reinforced the concept of two distinct, but
0378-1097/95/$09.50 8 1995 Federation of European Microbiological
Societies. All rights reserved
An atypical
Citrobacter
sp. has been extensively
studied with respect to its potential for the decontamination of aqueous solutions laden with heavy metals
[l]. Metals are desolubilized onto the cell surface as
crystalline deposits identified as metal phosphates,
e.g. CdHPO, and HUO,PO, for cadmium and uranyl
ion respectively [2]. Metal deposition is mediated via
the activity of a PhoN-type [3] acid phosphatase that
liberates IWO:- from an organic phosphate ‘donor’
l
SSDI 0378-1097(95)00208-l
B.C. Jeong, L.E. Macaskie/ FEMS Microbiology Letters 130 (1995) 211-214
212
related, enzymes. The different sensitivities towards
some cationic metal species might suggest converse
differences in the affinity of the enzymes for nega-
tively charged substrate molecules. Accordingly, the
affinity (K,) of some phosphomonoester
substrates
for the isoenzymes was determined.
IB
E
6 do4
D
~166
01
Od
96
Pb*’
0’ rrl6D
0
Zn
260
660
4W
I26 166
240
666
4
a
VW60
Concentration
126
166
240
61
(mM, pM)
Fig. 1. The effect of heavy metals on the activity of phosphatase &enzymes CPI (open symbols) and CPII (filled symbols). The enzymes
(0.2 pg) were incubated in 40 mM MOPS buffer, pH 7, in the presence of metals (concentrations as shown) and assayed for phosphatase
activity after 1 h as described. Activities are expressed as a percentage of the control (metal unsupplemented) activity. A: cadmium nitrate;
B: Zinc nitrate; C: Cupric nitrate; D: Lead nitrate; E: Uranyl nitrate; F: Vanadyl sulphate; G: Yttrium nitrate; II: Silver nitrate; and I:
Mercurous nitrate. Data are means f standard errors from 3 enzyme preparations.
B.C. Jeong, L.E. Macaskie/FEMS
Microbiology Letters 130 (1995) 211-214
2. Materials and methods
Table 1
The Ki values of metals for CPI and CPII
2.1. Organism and growth conditions
Metal salt
Ki (PM1 ’
sp. and growth conditions were
as described previously [l-5]. The cells were harvested from glycerol-glycerol 2-phosphate-based
minimal medium after overnight aerobic incubation,
and the phosphatases were purified to homogeneity
from cell extracts as described previously [5].
Cadmium nitrate
Cadmium sulphate
Zinc nitrate
Zinc sulphate
Lead acetate
Lead nitrate
Cupric nitrate
Cupric acetate
Uranyl acetate
Uranyl nitrate
Vanadyl sulphate
Mercurous nitrate
Silver nitrate
Yttrium nitrate
CPI
2150
2000
700
750
670
600
4
5
35
30
130
25
80
50
The Citrobacter
2.2. Phosphatase purification and assay
Briefly, purification was by ammonium sulphate
fractionation followed by chromatographic separation using anion and cation exchange resins, and
fractionation using hydroxyapatite and finally phenyl
sepharose to obtain pure samples of the phosphatases, designated CPI and CPI, which had identical migration behaviour on SDS-PAGE electrophoresis [5]. Routine phosphatase assay, in 200 mM MOPS
buffer, pH 7, was by the liberation of p-nitrophenol
from p-nitrophenyl phosphate (PNPP: 5), with phosphatase specific activity (unit> defined as nmol of
product/min/mg
bacterial protein. In some tests
other phosphatase substrates were used as described;
here the initial rate of phosphate release was determined [5].
2.3. Inhibition by heavy metals
Metal inhibition was determined by pre-incubation of enzyme (0.2 pg) in 40 mM MOPS buffer,
pH 7, with the metal salt under test for 1 h at 30°C.
The reaction was intiated by the addition of PNPP
(46.5 mM) with measurement of the liberated pnitrophenol as above. Where the reaction yielded an
insoluble metal precipitate this was allowed to settleout prior to estimation of the liberated p-nitrophenol
at &cl versus corresponding enzyme-free blanks.
3. Results and discussion
The effects of heavy metals on the phosphatases
are shown in Fig. 1, and summarized as apparent Ki
values in Table 1. In all cases the choice of counterion had little effect (sulphate in the case of Cd and
Zn; acetate in the case of Cu, Pb and U> and data are
213
CPII
2130
2000
730
750
870
750
6
8
25
20
50
8
45
35
The phosphatase isoenzymes were incubated in the presence of
the metal and assayed as described using PNPP. * The apparent
values for K, are calculated from the half-maximal inhibition of
the rate of p-nitrophenol release from the data of Fig. 1 and
corresponding data using other counterions (not shown).
generally shown for the nitrate salts. In accordance
with previous results using the whole-cell enzyme [6]
the enzymes were highly resistant to Cd (possibly
attributable to the low content of sulphydryl-containing amino acids: 51, less so to zinc and lead, and
sensitive to cupric ion (Fig. 1). The differential
toxicity may be explained in terms of the hydroxylation behaviour of the metal cations. The pK, values
for the formation of hydroxide complexes of Cd and
Zn (Cd(H,O),(OH)+ and Zn(H,O),(OH)+) are 9.0
and 9.9, respectively [7,8], i.e. at pH 7 the hydroxylated form of Zn would predominate, whereas here
much of the Cd is still in the form of Cd’+ [8]. In
the case of copper the formation of hydroxylated
metal species (pK, = pH 8.0: 7) begins at a lower
pH [7,8]; at pH 7 the metal is predominantly hydroxylated but at the low concentrations of Cu used
precipitation was not apparent. The high toxicity of
C&I) as compared to Zn(I1) may be attributable also
to its higher polarizing ability, and stronger ligand
complexes (higher formation constants) with amino
acids other than cysteine [7]. The oxycations of
uranium and vanadium (uranyl ion, UOi+ and
vanadyl ion, V02+ ) were also inhibitory; in each
case CPII was the more sensitive (Fig. 1). Extensive
hydroxylation of many1 ion in aqueous solution is
B.C. Jeong, L.E. Macaskie/FEMS
214
Table 2
The K, of CPI and CPII for phosphomonoesters
Substrate
K,
PNPP a
Glycerol 1-P b
MUFP ’
BCIP d
CPI
32+11
190 f 159
104+4
106*33
(PM)
CPII
40*10
354 * 45
160 f 39
80+23
The values for K, were calculated from double reciprocal plots
(l/S vs. l/V) using different concentrations of substrate (S) and
measurement
of the initial rate (V) of phosphate
release
(nmol/min/mg
protein: 5). The amounts of enzyme taken were:
PNPP: 0.2 pg; glycerol l-P, 0.375 pg; MUFP, 0.25 pg; BCIP,
0.5 pg. Data are means + standard errors from 4 experiments,
each using a separate enzyme preparation.
a:p-Nitrophenyl
phosphate; b: Glycerol l-phosphate; ‘: 4-methylumbelliferyl dihydrogen phosphate; d: 5-bromo-4-chloro-3-indolyl
phosphate.
well-documented [9]; little UO:’ is present in aqueous solution at pH 7, and the toxic form was possibly
UO,(OH)+ or one of several other hydrolysis products [9]. In contrast to the divalent cations (except
Cu), Hg+, Ag+ and Y3’ were inhibitory, with CPII
showing greater sensitivity (Fig. 1).
The above shows subtle differences in the sensitivity of CPI and CPII to metal cations, even though
the isoenzymes are very similar [5]. The affinity of
the isoenzymes for anionic substrate species was also
tested. The K, values for some phosphomonoester
substrates are shown in Table 2. Accordingly, where
a significant difference was seen (for MUFP, and
Glycerol 1-P; the significance was confirmed by
analysis of variance and a r-test) the K, was higher
in the case of CPII.
Although these differences were significant in
vitro, their biochemical significance in vivo cannot
be assumed. The K, for a substrate molecule may
be pH-dependent, while the pH-dependent speciation
of metals is described above. The localised pH of the
periplasmic [5] enzyme in vivo is not known and
would be, to some extent, dependent upon the external pH, since the cellular pH gradient is maintained
across the cytoplasmic, and not the outer membrane.
Further, it is not known whether the enzymes exist in
vivo as soluble or membrane-bound complexes. In
common with the PhoN phosphatase of Salmonella
typhimurium [lo] poor extraction by standard techniques of osmotic shock, aberrent chromatographic
behaviour in gel filtration columns and poor migra-
Microbiology Letters 130 (1995) 211-214
tion on non-denaturing polyacrylamide gel electrophoresis suggest that the enzymes may exist as
high molecular weight or membrane-bound complexes in vivo [&lo]. Hence, studies on the interaction of the purified enzymes with charged species in
aqueous solution may be of limited value for elucidation of their physiological roles, but do provide
useful tools to demonstrate that the CPI and CPII
have different properties that justify their classification as isoenzymes. It is not known whether these
represent two gene products, or are a single product
of phoN, modified post-translationally.
Acknowledgements
B.C.J. acknowledges, with thanks, the receipt of
financial support from the Government of South
Korea.
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