Experimental Parasitology 125 (2010) 152155

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Experimental Parasitology
journal homepage: www.elsevier.com/locate/yexpr

Activation of Leishmania (Leishmania) amazonensis arginase at low temperature

by binuclear Mn2+ center formation of the immobilized enzyme on a Ni2+ resin
Edson R. Silva a,*, Lucile M. Floeter-Winter b
a
b

Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de So Paulo, So Paulo, SP, Brazil

Departamento de Fisiologia, Instituto de Biocincias, Universidade de So Paulo, So Paulo, SP, Brazil

a r t i c l e

i n f o

Article history:
Received 17 August 2009
Received in revised form 15 January 2010
Accepted 18 January 2010
Available online 28 January 2010
Keywords:
Arginase (EC 3.5.3.1)
Metalloenzyme
Leishmania
Chemotherapy

a b s t r a c t
In Leishmania, arginase is responsible for the production of ornithine, a precursor of polyamines required
for proliferation of the parasite. In this work, the activation kinetics of immobilized arginase enzyme from
L. (L.) amazonensis were studied by varying the concentration of Mn2+ applied to the nickel column at
23 C. The intensity of the binding of the enzyme to the Ni2+ resin was directly proportional to the concentration of Mn2+. Conformational changes of the enzyme may occur when the enzyme interacts with
immobilized Ni2+, allowing the following to occur: (1) entrance of Mn2+ and formation of the metal
bridge; (2) stabilization and activation of the enzyme at 23 C; and (3) an increase in the afnity of the
enzyme to Ni2+ after the Mn2+ activation step. The conformational alterations can be summarized as follows: the interaction with the Ni2+ simulates thermal heating in the articial activation by opening a
channel for Mn2+ to enter.
2010 Elsevier Inc. All rights reserved.

1. Introduction
Arginase is a trimeric metalloenzyme containing a binuclear
manganese cluster in the active site of each subunit (Reczkowski
and Ash, 1992). In the mammalian liver, it is a cytosolic enzyme involved in the urea cycle, which converts arginine into ornithine
and urea (Krebs, 1942). In non-hepatic tissues, arginase is involved
in regulating the cellular concentration of L-arginine for nitric
oxide production (Forstermann et al., 1995) and ornithine for proline and polyamine biosynthesis (Wu and Morris, 1998; Chang
et al., 1998). In humans, an imbalance in the activity of arginase
in extra-hepatic tissue is related to a number of physiological disorders, such as hypertension and sexual impotence (Bagnost et al.,
2008; Cox et al., 1999). In the bacteria Helicobacter pylori, arginase
is responsible for the mechanism of escape from the immunologic
system (Gobert et al., 2001). In the parasitic protozoa Leishmania,
arginase plays a pivotal role in macrophage infection (Roberts
et al., 2004; Iniesta et al., 2001).
Establishing a metal bridge in the binuclear manganese center
is required for maximal activation of the native enzyme. Generally,
metal bridges are obtained in vitro by incubating the enzyme with
MnCl2 (50 mM) at 30, 40 or 55 C. Dissociation of one of the manganese atoms generates an enzyme with half of its catalytic activity (Hirsch-Kolb et al., 1971). In humans, H. pylori and Leishmania
arginase can be considered as a target for drug development, but

* Corresponding author.
E-mail address: edsilva@usp.br (E.R. Silva).
0014-4894/$ - see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.exppara.2010.01.013

the required presence of 1 or 2 Mn2+ in each monomer must be taken into account. This requirement may be crucial for interpretation of the inhibitory results because the failure to prevent
arginase action could be associated with variation in its activation
state.
In an attempt to purify the enzyme through immobilized metal
afnity chromatography (IMAC), we discovered that the enzyme
could be activated without the need for heating in the presence
of Mn2+, which restores enzyme activity (da Silva et al., 2008). As
described in the molecular model that we proposed for parasite
arginase, it is possible that the major differences between parasite
arginase and rat hepatic arginase occur in the amino- and carboxyl-end groups, mainly at the S-shaped motif of the carboxylterminus. This work presents the kinetics of activation of the enzyme at low temperature, which point to particular features of
the Leishmania enzyme structure that allow its activation under
different conditions than those described for any other arginase
characterized so far.
2. Material and methods
2.1. Expression of recombinant arginase of L. (L.) amazonensis
Arginase was expressed without a His-tag on the BL21(DE3)pLysS strain of Escherichia coli containing a pRSET-Arg (plasmid
with the gene of arginase) and cultivated at 37 C in Super Optimal
Broth (SOB) medium containing 100 lg/mL ampicillin and 35 lg/
mL chloramphenicol to a culture optical density at 600 nm

E.R. Silva, L.M. Floeter-Winter / Experimental Parasitology 125 (2010) 152155

(OD600) of 0.6 (da Silva et al., 2008). Expression of pRSET-Arg was

induced by adding isopropyl b-D-1-thiogalactopyranoside (IPTG)
until a nal concentration of 1 mM was reached. After a 3 h induction period, the cells were harvested by centrifugation, and the pellets were frozen at 80 C. The pellets from 1-L cultures were
resuspended in 50 mL of lysis buffer (100 mM 3-(N-morpholino)propanesulphonic acid (MOPS)NaOH, 5 mM imidazole,
1 mM of phenylmethylsulphonyl uoride (PMSF), and 200 U of
deoxyribonuclease (DNAse), pH 7.2) and lysed by three cycles of
freezethaw in liquid nitrogen and a 42 C water bath. The broken
cells were centrifuged at 12,000g for 15 min at 4 C (da Silva et al.,
2008). The supernatant was ltered through a Millipore lter
(0.22 lm) and was used in the kinetic assay of arginase activation
by IMAC.
2.2. Activation of recombinant arginase by IMAC
Activation of the enzyme was performed by immobilized metal
afnity chromatography (IMAC). IMAC was performed using a
5 mL HiTrap Chelating Sepharose column charged with Ni2+. The
column was previously equilibrated with 10 mL of buffer A
(100 mM MOPSNaOH, and 5 mM imidazole buffer, pH 7.2). The
column was washed with 20 mL of buffer A, and the activation
was performed by applying 20 mL of buffer A containing 50 mM
MnSO4. After activation, the enzyme was eluted with 20 mL of buffer A containing 50 mM imidazole. These procedures were performed at temperatures of 6 and 23 C. To study the kinetics of
the activation of the enzyme by IMAC, we performed six independent experiments using 5 mL of the protein extract in each one.
The manganese concentration utilized in the activation step was
variable (0, 1, 5, 10, 20 and 50 mM). The elution was performed
by application of a 5250 mM imidazole (pH 7.2) linear gradient,
and the fractions containing arginase activity were pooled and
used to determine the specic activity. This step was used to observe the interaction of arginase with the immobilized Ni2+ in the
column, but it was not needed for the purication itself, as described by da Silva et al. (2008). The chromatography experiments
were performed in an KTA Explorer HPLC system.
2.3. Arginase assay
The enzyme was incubated at 37 C in a 1.0 mL mixture of 0.1 M
TrisHCl buffer (pH 8.3), 50 lg of L-glutamate dehydrogenase,
0.2 mg of urease, 0.5 lM nicotinamide adenine dinucleotide phosphate (NADPH) and 50 mM L-arginine. The course of the reaction
was monitored by the consumption of NADPH observed at
340 nm. A unit of specic activity of arginase was dened as the
amount of enzyme that produced 1 lmol of urea in 1 min/mg of
protein.
2.4. Models
The gures containing models of arginase were created with
RasMol 2.7.4.2 (http://www.rasmol.org).
3. Results and discussion
The recombinant arginase from L. (L.) amazonensis was immobilized on a resin charged with Ni2+ and subjected to activation by an
inux of 100 mM MOPS buffer (pH 7.2) containing 50 mM MnSO4
at 23 C (sample A) or 6 C (sample B). The enzyme activity was
determined to be 677 U and 406 U for samples A and B, respectively. After that, samples A and B were subjected to thermal activation (Hirsch-Kolb et al., 1971), resulting in a 17% increase in the
activity of sample A (677792 U) and a decrease of 7% for sample B

153

(406378 U). We also observed that without the application of

manganese to the column while the enzyme was immobilized,
thermal activation of arginase after elution was not possible.
Because the maximum activity of the enzyme is obtained when
the binuclear center is formed by the binding of two Mn2+ ions to
each monomer, we conclude that most of the subunits of the enzyme activated at 23 C already have the binuclear center formed
at the time the enzyme is still attached to the nickel column. This
behavior is different from mature human arginase II, which demonstrates a 3-fold increase in its activity after heat activation (Colleluori et al., 2001).
In a previous work, we observed that heat activation of L. (L.)
amazonensis arginase after nickel column purication increased
its activity by 2.25-fold (da Silva et al., 2008). Those data indicated
that there is a minimum mandatory presence of two Mn2+ linked to
the native enzyme (trimer) to support heating and then increase its
activity. Thus, the thermal insertion of four more Mn2+ ions into
the quaternary structure of the enzyme can promote a 3-fold increase in the activity. As such, we can assume that the decrease
in activity observed in sample B may be due to a failure to reach
the minimum amount of Mn2+ that confers thermal stability to
the protein.
Subunitsubunit interactions in trimeric arginase are dependent on the S-shaped motif at the carboxyl-terminus (Lavulo
et al., 2001). Previously, we proposed that the amino acid His324 in the S-shaped motif of Leishmania arginase and His-3 could
be interacting simultaneously with Ni2+ from the chromatographic
resin. This binding causes a decrease in the S-shaped interaction of
the trimer. The interaction of His-324 and His-3 with the nickel
column may promote the formation of an open channel, favoring
the entrance and binding of Mn2+ to the active site in a monomer
subunit (Fig. 1). The kinetics of Mn2+ binding to immobilized arginase showed that enzyme activation is proportional to the concentration of Mn2+. The minimal concentration of Mn2+ needed to
produce maximal specic activity at 23 C was approximately
20 mM. This was determined by varying the concentration of
Mn2+ applied to the Ni2+ column to activate arginase (Fig. 2). To obtain half activation, the concentration of Mn2+ was <1 mM.
Because the Mn2+ binding increases the amount of positive
charge of the enzyme, we expected that the elution of the column
of Ni2+ would be facilitated. The experiments showed the opposite,
however: an increase in the amount of Mn2+ in the enzyme increased the demand for imidazole needed to elute the enzyme
from the column (Fig. 3). Therefore, the binding of His-324 and
His-3 to the Ni2+ is enhanced by the formation of the metal bridge
at the binuclear Mn2+ centers. Once attached, the Mn2+ cations act
as a lock that stabilizes the enzyme and the binding of His-3 and
His-324 to Ni2+.
The thermostability of rat hepatic arginase diminishes when the
binuclear manganese cluster is altered, but the observed temperature for stability of the unfolded fraction of the wild type enzyme
is >65 C with a Tm = 75 C (Scolnick et al., 1997). These observations
raised a question concerning the meaning of the thermostability obtained by the presence of two Mn2+ in organisms whose body temperatures are around 37 C. The same characteristic that provides
thermostability in thermophilic bacterial arginase (Bewley et al.,
1996) could be conserved in complex organisms as an alternative
that regulates catalytic activity instead of conferring thermostability. Arginase activation of a rat hepatic cytosol preparation is possible via incubation of the enzyme for 1 h with 1, 10 or 100 mM of
MnCl2 at 4 C. The same result can be achieved by incubation of
the preparation with 50 mM MnCl2 at 50 C for 10 min (Bond et al.,
1983). Manganese concentrations of 10 mM or 50 mM, used to activate arginase in vitro, represent, respectively, 50 and 250 times the
limiting concentration of 0.2 mM that is supported by cultured
broblasts (Parat et al., 1995). At a concentration of 1 mM Mn2+ in

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E.R. Silva, L.M. Floeter-Winter / Experimental Parasitology 125 (2010) 152155

Fig. 1. Proposed model for activation of arginase immobilized on Ni2+ resin by an inux of Mn2+ at low temperatures. S-shaped motif (red) in the carboxyl-terminus interacts
with nickel, disrupting the trimeric arrange and allowing the entrance of Mn2+ to the active site. After elution, active arginase is formed. (For interpretation of color mentioned
in this gure the reader is referred to the web version of the article.)

high Mn2+ concentration and the high temperature activation of

arginase in vitro are not physiological. However, the treatments induce structural changes in the protein, leading to the manganese
binding and correct trimer formation. The activation of immobilized
arginase in a nickel column described here is another way to induce
the structural changes needed to activate the enzyme at a low concentration of Mn2+ and a moderate temperature. Based on this information and the results shown here, we propose the existence of
molecular mechanisms in Leishmania that could regulate arginase
activity by altering its conformation for the binding of one or two
Mn2+ ions to the active site for a low concentration of Mn2+ and at
a physiologic temperature. Finally, the curve of arginase elution,
with loading of 1 or 2 Mn2+, shows that movement of the structural
organization of the trimer is necessary to activate the enzyme.
Fig. 2. Activity of arginase eluted by IMAC after activation with various Mn2+
concentrations.

Acknowledgments
The authors thank Otvio H. Thiemann, Glaucius Oliva and
Richard Garrat for their incentives to develop this work. The research was supported by Conselho Nacional de Desenvolvimento
Cientco e Tecnolgico (CNPq) and Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP). E.R.S. received fellowships
from FAPESP.

References

Fig. 3. Elution of arginase immobilized on Ni2+ resin after activation by different

concentrations of Mn2+.

the cell culture, a cytotoxicity of 20% was observed, while at 2.5 mM,
the toxicity reached 100%. The toxic dose where 50% of the cells are
affected is 1.3 mM Mn2+ (Parat et al., 1995). It is clear that both the

Bagnost, T., Berthelot, A., Bouhaddi, M., et al., 2008. Treatment with the arginase
inhibitor Nx-hydroxy-nor-L-arginine improves vascular function and lowers
blood pressure in adult spontaneously hypertensive rat. Journal of
Hypertension 26, 11101118.
Bewley, M.C., Lott, J.S., Baker, E.N., Patchett, M.L., 1996. The cloning,
expression and crystallisation of a thermostable arginase. FEBS Letters
386, 215218.
Bond, J.S., Failla, M.L., Unger, D.F., 1983. Elevated manganese concentration and
arginase activity in livers of streptozotocin-induced diabetic rats. The Journal of
Biological Chemistry 258, 80048009.
Chang, C.I., Liao, J.C., Kuo, L., 1998. Arginase modulates nitric oxide production in
activated macrophages. American Journal of PhysiologyHeart and Circulatory
Physiology 274, H342H348.
Colleluori, D.M., Morris Jr., S.M., Ash, D.E., 2001. Expression, purication, and
characterization of human type II arginase. Archives of Biochemistry and
Biophysics 389, 135143.
Cox, J.D., Kim, N.N., Traish, A.M., Christianson, D.W., 1999. Arginase-boronic acid
complex highlights a physiological role in erectile function. Nature Structural
Biology 6, 10431047.
da Silva, E.R., da Silva, M.F., Fischer, H., et al., 2008. Biochemical and biophysical
properties of a highly active recombinant arginase from Leishmania

E.R. Silva, L.M. Floeter-Winter / Experimental Parasitology 125 (2010) 152155

(Leishmania) amazonensis and subcellular localization of native enzyme.
Molecular & Biochemical Parasitology 159, 104111.
Forstermann, U., Kleinert, H., Gath, I., Schwarz, P., Closs, E.I., Dun, N.J., 1995.
Expression and expressional control of nitric oxide synthases in various cell
types. Advances in Pharmacology 34, 171186.
Gobert, A.P., McGee, D.J., Akhtar, M., et al., 2001. Helicobacter pylori arginase inhibits
nitric oxide production by eukaryotic cells: a strategy for bacterial survival.
Proceedings of the National Academy of Sciences of the United States of
America 98, 1384413849.
Hirsch-Kolb, H., Kolb, H.J., Greenberg, D.M., 1971. Nuclear magnetic resonance
studies of manganese binding of rat liver arginase. The Journal of Biological
Chemistry 246, 395401.
Iniesta, V., Gomez-Nieto, L.C., Corraliza, I., 2001. The inhibition of arginase by
N(omega)-hydroxy-l-arginine controls the growth of Leishmania inside
macrophages. The Journal of Experimental Medicine 193, 777784.
Krebs, H.A., 1942. Urea formation in mammalian liver. Biochemical Journal 36, 758
767.

155

Lavulo, L.T., Sossong Jr., T.M., Brigham-Burke, M.R., et al., 2001. Subunitsubunit
interactions in trimeric arginase. Generation of active monomers by mutation of
a single amino acid. The Journal of Biological Chemistry 276, 1424214248.
Parat, M.O., Richard, M.J., Leccia, M.T., et al., 1995. Does manganese protect cultured
human skin broblasts against oxidative injury by uva, dithranol and hydrogen
peroxide? Free Radical Research 23, 339351.
Reczkowski, R.S., Ash, D.E., 1992. EPR evidence for binuclear manganese(II) centers
in rat liver arginase. Journal of the American Chemical Society 114, 10992
10994.
Roberts, S.C., Tancer, M.J., Polinsky, M.R., et al., 2004. Arginase plays a pivotal role in
polyamine precursor metabolism in Leishmania. Characterization of gene
deletion mutants. The Journal of Biological Chemistry 279, 2366823678.
Scolnick, L.R., Kanyo, Z.F., Cavalli, R.C., et al., 1997. Altering the binuclear manganese
cluster of arginase diminishes thermostability and catalytic function.
Biochemistry 36, 1055810565.
Wu, G., Morris Jr., S.M., 1998. Arginine metabolism: nitric oxide and beyond.
Biochemical Journal 336, 117.