Metal-dependent inhibition of glyoxalase II: A possible mechanism to regulate the enzyme activity

Article abstract of DOI:10.1016/j.jinorgbio.2010.03.005

Glyoxalase II (GLX2, EC 3.1.2.6., hydroxyacylglutathione hydrolase) is a metalloenzyme involved in crucial detoxification pathways. Different studies have failed in identifying the native metal ion of this enzyme, which is expressed with iron, zinc and/or manganese. Here we report that GloB, the GLX2 from Salmonella typhimurium, is differentially inhibited by glutathione (a reaction product) depending on the bound metal ion, and we provide a structural model for this inhibition mode. This metal-dependent inhibition was shown to occur in metal-enriched forms of the enzyme, complementing the spectroscopic data. Based on the high levels of free glutathione in the cell, we suggest that the expression of the different metal forms of GLX2 during Salmonella infection could be exploited as a mechanism to regulate the enzyme activity.

Full text of DOI:10.1016/j.jinorgbio.2010.03.005

Journal of Inorganic Biochemistry 104 (2010) 726–731
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Metal-dependent inhibition of glyoxalase II: A possible mechanism to regulate the
enzyme activity
Valeria A. Campos-Bermudez a,1, Jorgelina Morán-Barrio a, Antonio J. Costa-Filho b, Alejandro J. Vila a,
⁎
a
IBR (Instituto de Biología Molecular y Celular de Rosario), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas,
Universidad Nacional de Rosario, Suipacha 531, S2002LRK, Rosario, Argentina
b
Biofísica Molecular Sérgio Mascarenhas, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Glyoxalase II (GLX2, EC 3.1.2.6., hydroxyacylglutathione hydrolase) is a metalloenzyme involved in crucial
detoxification pathways. Different studies have failed in identifying the native metal ion of this enzyme,
which is expressed with iron, zinc and/or manganese. Here we report that GloB, the GLX2 from Salmonella
typhimurium, is differentially inhibited by glutathione (a reaction product) depending on the bound metal
ion, and we provide a structural model for this inhibition mode. This metal-dependent inhibition was shown
to occur in metal-enriched forms of the enzyme, complementing the spectroscopic data. Based on the high
levels of free glutathione in the cell, we suggest that the expression of the different metal forms of GLX2
during Salmonella infection could be exploited as a mechanism to regulate the enzyme activity.
© 2010 Elsevier Inc. All rights reserved.
Received 18 August 2009
Received in revised form 15 February 2010
Accepted 9 March 2010
Available online 20 March 2010
Keywords:
Glyoxalase II
Iron
EPR
Product inhibition
Paramagnetic NMR
1. Introduction
as metallo-β-lactamases [7,8], N-acyl homoserine (AHL) lactonases
[9,10] and tRNAseZ [11], whereas the terminal oxidase rubredoxin:
The ubiquitous glyoxalase system is composed of two metalloen-
zymes (glyoxalase I and glyoxalase II), whose physiological role is to
catalyze the conversion of methylglyoxal to the corresponding 2-
hydroxycarboxylic acids [1]. Glyoxalase I (GLX1, EC 4.4.1.5., lactoylglu-
tathione methylglyoxal lyase) converts a thiohemiacetal (formed from
methylglyoxal and glutathione) to S-D-lactoylglutathione (SLG), while
glyoxalase II (GLX2) takes the latter as a substrate and catalyzes its
hydrolysis to yield D-lactic acid and glutathione (GSH) [2,3] (Fig. 1A).
Although GLX2 is able to hydrolyze many different glutathione
thioesters, S-D-lactoylglutathione (SLG) is the preferred substrate for
this enzyme from most sources including human, yeast, and plants.
The glyoxalase enzymatic system is essential for chemical detoxifica-
tion, and therefore has been identified as a target for the development
of anti-cancer and anti-protozoan drugs [4–6].
oxygen oxidoreductase (ROO) from D. gigas contains a binuclear iron
site [12–14]. GLX2, despite exhibiting the same coordination sphere
displayed by other hydrolases from this superfamily (Fig. 1B), has been
an outstanding exception regarding the metal ion dependence with
different metal cofactors [15–20].
In general, the wild type forms of the recombinant GLX2 isoen-
zymes characterized up to now were isolated with varying amounts of
iron, zinc and manganese bound to their active sites [15–20]. Sur-
prisingly, the different metal derivatives show comparable high cata-
lytic efficiencies [15–20]. An exception to this observation is human
GLX2, which has been reported to be inactive in its Mn-bound form
[20]. Despite the existence of three crystallographic structures (2QED,
1QH5, 1XM8) [18,19,21] which have been refined with different metal
ions in its active sites, this puzzling promiscuity has precluded the
identification of the native metal ion in GLX2.
GLX2 belongs to the metallo-β-lactamase superfamily, which is
characterized by an αβ/βα fold containing a conserved motif able to
bind up to two metal ions in their active sites. Most hydrolases from
this superfamily use ZnII, a redox inactive metal ion for their roles, such
Most mechanistic proposals so far have considered exclusively the
di-ZnII derivative, based on crystallographic, kinetic and spectroscopic
studies, and (more recently) theoretical calculations at the quantum
level [17,21,22] (Fig. 1B). However, one may argue whether this mech-
anism could be general to all metal derivatives.
⁎
Corresponding author. Fax: +54 341 4390465.
Here we report a spectroscopic and kinetic study on GloB, the
glyoxalase II from Salmonella typhimurium [19], upon addition of
substrate S-D-lactoylglutathione (SLG) or the product glutathione
(GSH). We have interrogated the enzyme purified from rich medium,
containing mixed metallic centers, as well as Fe-, Zn- and Mn-enriched
forms [19]. We have found that GloB is differentially inhibited by
E-mail addresses: campos@cefobi-conicet.gov.ar (V.A. Campos-Bermudez),
moran@ibr.gov.ar (J. Morán-Barrio), ajcosta@if.sc.usp.br (A.J. Costa-Filho),
vila@ibr.gov.ar (A.J. Vila).
1
Present address: CEFOBI (Centro de Estudios Fotosintéticos y Bioquímicos), Consejo
Nacional de Investigaciones Científicas
y Técnicas (CONICET), Facultad de Ciencias
Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, (S2002LRK),
Rosario, Argentina.
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.03.005

V.A. Campos-Bermudez et al. / Journal of Inorganic Biochemistry 104 (2010) 726–731
727
Fig. 1. (A) Reaction catalyzed by glyoxalase II (GLX2). (B) Proposed reaction mechanism for dimetallic-GLX2 based on crystallographic, kinetic and theoretical calculations. The metal
ion bound to 3 His residues (M1) is proposed to deliver the attacking nucleophile, while the M2 site favors S–C bond cleavage by stabilizing the negative charge in the sulfur atom of
the glutathione moiety.
glutathione depending on the bound metal ion, and we provide a
structural model for this inhibition mode.
forms, minimal media M9 was employed in culture growth supple-
mented with metal ions from stock solutions of Fe(NH4)SO4, MnCl2,
or ZnCl2, to reach a final concentration of 100 μM in the culture.
The minimal media contained 4 g/L D-(+)-glucose (Sigma), 12.8 g/L
Na2HPO4·7H2O, 3 g/L KH2PO4, 0.5 g/L NaCl, 1.0 g/L ammonium sul-
fate, 10 μM CaCl2 and 1 mM MgSO4. The metal ions were added in the
moment of induction, when the growth cultures reached an OD600 of
0.6–0.8. The purification protocol is described in Ref. [19]. Thus, we
obtained the iron-enriched GloB (FeGloB), manganese-enriched GloB
(MnGloB) and zinc-enriched GloB (ZnGloB).
2. Experimental procedure
2.1. General
S-D-lactoylglutathione (SLG) was purchased from Sigma-Aldrich.
All chromatographic steps were performed in an Amersham Bios-
ciences liquid chromatography system operating at 4 °C. Metal stan-
dards were purchased from Fisher Scientific and were diluted with
distilled water. All other chemicals used in this study were purchased
commercially and were of the highest quality available.
2.3. Protein concentration
Enzyme concentration was determined by measuring the A280 nm
and using the extinction coefficient ε280 nm =28,030 M−1 cm−1 [19].
This parameter was calculated from amino acid composition infor-
mation by applying a modified Edelhoch method [23].
2.2. Protein overexpression and purification
GLX2 (GloB) from S. typhimurium 14028s recombinantly produced
in E. coli, was used for this study. Protein overexpression and purifi-
cation were performed using the pET32-gloB vector, as previously
described [19]. Fractions with GLX2 activity were pooled and dialyzed
against 10 mM 4-morpholinepropanesulfonic acid (MOPS) 0.2 M NaCl
at pH 7.2. In order to selectively produce metal-enriched enzyme
2.4. Metal analysis
The metal content of the GloB samples was measured using atomic
absorption spectroscopy in a Metrolab 250 AA instrument. Purified

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V.A. Campos-Bermudez et al. / Journal of Inorganic Biochemistry 104 (2010) 726–731
enzymes were diluted with 10 mM MOPS pH 7.2, to a concentration
of 30 μM and analyzed for zinc, manganese and iron. The metal
content of GloB produced in minimal medium was the following:
FeGloB: 0.86 0.02 eq iron, 0.05 0.02 eq manganese and 0.04
0.01 eq zinc; MnGloB: 0.04 0.01 eq iron, 0.81 0.06 eq manganese
and 0.04 0.01 eq zinc; ZnGloB: 0.08 0.01 eq iron, 1.42 0.01 eq
zinc and manganese was not detected. The metal content data pre-
sented in this paper represent an average from at least three pre-
parations of each growth condition.
3. Results
GloB was obtained as previously described in Campos-Bermudez
et al. [19]. The typical metal content of enzyme samples expressed in
rich medium is 0.2 zinc; 0.6 iron and 0.3 manganese per protein.
Selectively metallated variants were produced by expressing the pro-
tein in minimal medium adequately supplemented with the required
metal ion. The behavior of these proteins upon the addition of sub-
strate or product was analyzed by different spectroscopic techniques.
2.5. 1H NMR spectroscopy
3.1. Electron paramagnetic resonance
NMR spectra were recorded in a Bruker Avance II 600 spectrom-
eter operating at 600.13 MHz. 1H NMR spectra were recorded under
conditions to optimize detection of the fast-relaxing paramagnetic
resonances, either using the superWEFT pulse sequence [24,25] or
water presaturation. Spectra were acquired over large spectral widths
with acquisition times ranging from 16 to 80 ms, and intermediate
delays from 2 to 35 ms. 1D experiments with solvent presaturation
were used to record isotropically shifted signals closer to the dia-
magnetic envelope. The samples were loaded into Wilmad 5-mm
NMR tubes. The substrate or product was prepared as 100 mM stock
solutions in 100 mM MOPS at pH 7.2 (pD 6.8). A final 1:10 enzyme
to reactant ratio was used to record the spectra in presence of the
adducts.
The EPR spectrum of resting-state GloB recorded at 4.7 K (Fig. 2A)
disclosed the coexistence of different paramagnetic species. As already
shown, the different observed signals can be assigned on the basis of
spectral simulations [19]. The main components in the EPR spectra are
due to two different iron centers. Resonances at geff ∼4.3 and 9.1 are
characteristics of magnetically isolated high-spin FeIII in a rhombic
environment [15,18,19,26–28]. The broad feature at gb2 corresponds
to an antiferromagnetically coupled dinuclear center which involves
a high-spin (S=5/2) FeIII ion and a high-spin (S=2) FeII ion [15,18,
19,28]. The six-line pattern centered around g=2.0 is due to MnII
bound to the active site (Fig. 2A).
S-D-lactoylglutathione (SLG, the enzyme substrate) was added to
GloB, and the EPR spectrum was recorded after manual mixing and
sample freezing. The spectrum (Fig. 2B) revealed changes in the reso-
nances stemming from the iron centers at geff ∼4.3, 9.1 and b2, while
the signals arising from the MnII site could be accounted for in the
simulation by the same set of parameters obtained for resting-state
GloB, suggesting that the MnII ions are not perturbed. Addition of
glutathione (GSH, a reaction product), gave rise to similar changes on
the EPR signals corresponding to the iron centers, while those from
the MnII sites also remained unperturbed (Fig. 2C).
2.6. Electron paramagnetic resonance spectroscopy
X-band (9.5 GHz) EPR spectra were measured on a Bruker ELEXSYS
E580 system (Bruker BioSpin, Germany) at 4.7 K. The temperature
was controlled with an Oxford ITC503 cryogenic system. EPR samples
containing a convenient amount of protein or the protein to which
the substrate or product were added (up to a 1:10 ratio) were frozen
by immersion in liquid nitrogen and then placed in the spectro-
meter rectangular cavity. All EPR data were corrected by subtracting
a baseline corresponding to the EPR signal of the buffer. Other acqui-
sition conditions: modulation amplitude, 1 mT; modulation frequen-
cy, 100 kHz; microwave power, 4 mW. A typical EPR sample was 1 mM
in 10 mM MOPS pH 7.2 buffer.
2.7. UV–visible spectroscopy
Spectra of GloB samples in the absence and presence of substrate
or product in different ratios, were recorded between 240 and 800 nm
at 25 °C in a Jasco 550 UV–vis spectrophotometer in quartz cuvette of
1 cm of path length. Differential spectra were obtained by subtracting
the spectrum of the enzyme in its unbound form. Typical samples of
GloB containing different metal ions were up to 1 mM in protein,
while the spectra of Fe-enriched, Mn-enriched and Zn-enriched were
recorded on samples typically ca. 30 μM.
2.8. End product inhibition studies of SLG hydrolysis
Inhibition studies were performed on selectively metal-enriched
GloB using glutathione, one of the products of SLG hydrolysis. Steady
state kinetic studies of purified GloB variants in the presence of vary-
ing concentrations of GSH (0.2 to 5 mM) were conducted by mea-
suring the rate of hydrolysis of a fixed concentration of SLG (400 μM)
at 240 nm (ε240 =3100 M−1 cm−1). A glutathione stock solution
was prepared in 100 mM MOPS, pH 7.2. The enzyme (400 nM) and
inhibitor were incubated, and then the substrate was added. IC50
values were determined by fitting the data of initial rates to the
following equation: (1−vi /vo)⁎100=Imax.[I]/(IC50 +[I]). Measure-
ments were performed at least by triplicate in a reaction volume
of 1 ml of 10 mM MOPS, 0.2 M NaCl pH 7.2 buffer, at 30 °C in a Jasco
550 UV–vis spectrophotometer.
Fig. 2. Experimental (upper traces) and calculated (lower traces) X-band EPR spectra of
1 mM GloB (A) as isolated; (B) after the addition of SLG and (C) after the addition of
GSH. The spectra were recorded at 4.7 K and simulated as described in the text. The
arrows indicate approximate g-values.

V.A. Campos-Bermudez et al. / Journal of Inorganic Biochemistry 104 (2010) 726–731
729
Analysis of the individual features revealed that in the presence of
SLG or GSH, the uncoupled signal (geff ∼4.3) presented higher zero-
field splittings (D=0.55 cm−1 and E/D=0.285) compared to resting-
state GloB (D=0.33 cm−1 and E/D=0.195). The increase in D-values
can be attributed to a change in the coordination sphere of the iron
ion, with more electron-donating atoms; while the higher E/D ratio
reveals a larger rhombicity in the FeIII center. Both parameters are
consistent with the binding of an additional ligand to the FeIII ion
[29,30].
Regarding the signal accounting for the coupled center (gb2), the
addition of either SLG or GSH led to lineshape and g-value differen-
ces (Fig. 2B–C), revealing that the environment of the binuclear center
is significantly perturbed in both cases. Spectra of coupled centers
are very sensitive to changes in the coordination environment of the
binuclear site which can be reflected in the magnitude of the super-
exchange coupling (J) between the metal centers, and the zero-field
splittings (D) of each ion, among other features [31–34]. Effective
g-values for these systems could be easily determined assuming an
S=1/2 spin system and yielded, in the presence of ligands, 1.95, 1.87,
and 1.71 (gav =1.84). Thus, the addition of exogenous ligands (SLG
or GSH) is reflected as a significant increase in gav value for GloB in
system. Resonances corresponding to an uncoupled FeIII center can-
not be detected, since considerably broader lines are expected [35].
The addition of excess substrate (SLG) to GloB, resulted in a new set
of resonances sharper than those from the resting-state enzyme
(Fig. 3B). Addition of GSH to GloB rendered a spectrum very similar to
that resulting upon substrate addition (Fig. 3C). Since NMR spectra are
recorded at room temperature, in contrast with EPR, and NMR acqui-
sition times are rather long (in relation with the GloB kcat =170 s−1),
the time stability of the monitored species suggests that all studied
spectra correspond to an enzyme–product adduct. We must recall
that the glutathione moiety is present both in the substrate and pro-
duct molecules (Fig. 1A).
3.3. UV–vis spectroscopy
To test the hypothesis that the formed species is an enzyme–
product complex, the interaction of GloB with SLG and GSH was fol-
lowed by UV–vis spectroscopy. We initially attempted these experi-
ments with a sample of GloB obtained from expression in rich media,
containing Fe, Zn and Mn. The addition of excess SLG resulted in
the appearance of a blue color, corresponding to an absorption band
centered at 590 nm, with an estimated extinction coefficient of
300 M−1 cm−1 (Fig. 4A). This spectral feature vanished after several
hours, confirming the formation of a stable adduct with the enzyme.
This feature is in good agreement with the presence of a ligand-to-
metal charge transfer (LMCT) band of a Cys ligand to a high-spin FeIII
in an octahedral coordination, reminiscent of those observed in super-
oxide reductase and model complexes [36–38]. An additional intense
band at 320 nm is observed in the differential spectrum (Fig. 4A),
which can be assigned to a (Cys)S–FeII LMCT [38]. Similar UV–vis
specta were obtained for Tflp, a ferredoxin-like protein from Thermo-
anaerobacter tengcongensis [39] belonging to the metallo-β-lactamase
superfamily, where a disulfide bond is near the di-Fe center in the
active site.
the presence of ligands (gav =1.84) as compared to pure GloB (gav
=
1.76), which suggests a stronger coupling regime upon ligand binding.
The EPR spectra of Fe-enriched GloB before and after addition of
substrate (SLG) or product (GSH) revealed the same spectral features
that for the heterogeneous sample of GloB (not shown). The spectra
disclosed mononuclear and binuclear Fe centers, and a weak six-line
pattern. Double-integration of the spectra showed that this six-line
pattern is responsible for less than 5% of the total signal intensity,
indicating that this is the result of a very minor contribution from Mn
centers in the sample, which is in agreement with metal analyses. The
spectra showed that addition of substrate (SLG) or product (GSH), led
to the same spectral alterations that those observed in Fig. 2B–C.
3.2. 1H nuclear magnetic resonance
When GSH was added to GloB, the same spectral features were
observed (Fig. 4A). These results further confirm that the spectra
The 1H NMR spectrum of GloB was recorded under conditions
tailored to optimize detection of the fast-relaxing signals close to the
paramagnetic metal center [25]. The spectrum of GloB reveals a set
of hyperfine-shifted signals attributed to the His and Asp ligands of
the metal site (Fig. 3) of a weakly antiferromagnetic coupled FeIII/FeII
Fig. 4. Differential UV–visible spectrum of GloB produced in rich medium after the
addition of substrate or product (A) and from minimal medium supplemented either
with FeII (FeGloB), MnII (MnGloB) or ZnII (ZnGloB) after the addition of 10 equivalents
of substrate SLG (B). The differential spectra were obtained by subtracting the spectrum
of unbound GloB. The observed features are in good agreement with the presence of a
ligand-to-metal charge transfer (LMCT) band of a Cys ligand to iron ions.
Fig. 3. 1H NMR spectra of GloB (A) as isolated, (B) after the addition of SLG and (C) after
the addition of GSH. The spectra were recorded at 600 MHz, pH 7.2, and 298 K in 10 mM
MOPS buffer, 0.2 M NaCl.

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recorded after addition of SLG correspond to a stable enzyme–product
(E–P) adduct, formed as the result of the interaction of the metal site
with the Cys-containing glutathione moiety of the substrate (Fig. 1A).
These data are consistent with the crystal structure of the human
enzyme complexed with GSH (1QH5), in which the sulfur atom of this
Cys residue is at 2.86 Å from one of the metal ions [21]. The absorption
feature at 590 nm observed for the GloB–GSH complex displays a
relatively low extinction coefficient, which is consistent with the
relatively long S–FeIII bond reported in the crystal structure. The lack
of a spectral feature at 280 nm allows us to discard the presence of a
Cys–MnII LMCT, i.e., confirming that this adduct is exclusively formed
by the iron form of GLX2 [36].
respectively (Fig. 1B). A sequential mechanism for product release has
been proposed [17,21,22].
Here we report a metal-selective product inhibition, which allow
us to obtain some mechanistic insights. By using complementary spec-
troscopic techniques, we provide evidence supporting that: (1) addi-
tion of either substrate (SLG) or product (GSH) to GloB results in the
formation of a stable enzyme–product (GloB–GSH) complex; (2) this
adduct is formed by the iron variants of the enzyme, and not by the
manganese derivatives; (3) a thiol–Fe bond is formed between the
Cys moiety of GSH and the metal site; and (4) the dinuclear iron
sites show an enhanced antiferromagnetic coupling in the GloB–GSH
adduct.
When the same experiments were performed in the Fe-enriched
form, obtained by expression in minimal medium supplemented with
FeII, we observed the same LMCT bands upon addition of either SLG
or GSH. These experiments unequivocally confirm that the features
observed in the mixed GloB sample correspond to the iron form.
Instead, when similar titrations were conducted in the Mn-enriched
and Zn-enriched forms of GloB, no distinctive features could be de-
tected in the differential spectra even upon addition of a 10-fold
excess of the exogenous ligands up to 240 nm (Fig. 4B). Minor changes
in the spectra can be attributed to alterations in the absorption fea-
tures of the aromatic residues, as results from the fine structure of
the differential spectra. These data allow us to discard the formation
of a Cys–MnII CT band, which is expected to appear at 280 nm. This is
validated by comparison with the spectrum obtained with the Zn
form of the enzyme, which it is not expected to exhibit a LMCT in this
spectral range.
The finding of a stable thiol–Fe bonding interaction can be readily
attributed to product inhibition by glutathione, supporting a sequen-
tial, ordered mechanism for product release after hydrolysis [17,21,
22]. The reported lower KI for glutathione suggests that it is bound
more tightly to the active site and therefore is released after D-lactic
acid [17].
EPR spectroscopy reveals that the coupling between the two iron
ions is stronger in the enzyme–product complex. This coupling is ex-
pected to occur by involving Asp127 as a bridging ligand (Fig. 5). One
possible rationale for this observation is to assume an equilibrium
between the bridged and non-bridged forms in the free enzyme, which
is consistent with the variable geometries observed in different crystal
structures [15,18,19,21], that would be shifted towards the bridged
one upon formation of the EP adduct (Fig. 5A). Sharing the Asp127
ligand between the two metal ions upon thiolate binding may com-
pensate the increase in negative charge in the metal coordination
sphere. Another possibility is that the sulfur atom from glutathione
forms a strong hydrogen bond to the bridging hydroxide, thus impart-
ing more oxo character in the bridging unit, giving rise to a stronger
coupling.
Finally, we could also assume that the thiolate group of glutathi-
one is bound to the metal site bridging the two iron ions (Fig. 5B),
in a position equivalent to the one occupied by the nucleophile in
the resting-state enzyme. The GloB–GSH complex displays S–thiol–
FeIII and S–thiol–FeII: CT bands (Fig. 4). This observation could be ac-
counted for by assuming a bridging glutathione moiety, confirming
again that product release is ordered in a sequential fashion.
The most relevant observation regarding this enzyme–product
complex concerns its metal selectivity. MnII–thiolate bonds are less
covalent and weaker than iron–thiolate bonds, providing a rationale
for this differential behavior [36]. This is also consistent with the find-
ing that, in general, the MnII forms of different GLX2s are the most
efficient ones [15,16,19,41].
3.4. Product inhibition of the specific metallated forms
The differential behavior of the different metallated forms towards
the formation of enzyme–product complexes led us to analyze pro-
duct inhibition of each metal form of GloB. We obtained the Mn-
enriched, Zn-enriched and Fe-enriched forms of GloB, by expression
in minimal medium as already described [19], and we tested their
inhibition by glutathione. Fe- and Zn-GloB were inhibited at submil-
limolar concentrations of glutathione, while the IC50 value for Mn-
GloB is 1.5 mM. These results are in agreement with the spectrosco-
pic data, which show strong product binding to the iron-substituted
enzyme, and not to the manganese forms. These data also reveal that
the behavior of the Zn form resembles that of Fe-GloB.
4. Discussion
FeII and MnII are regarded as the most important transition metal
ions involved in host–bacterial pathogen interactions [43]. The acqui-
sitions of both metal ions, and particularly MnII, are required for in-
tracellular survival and replication of Salmonella enterica serovar
typhimurium in macrophages in vitro and for virulence in vivo [44,45].
Thus, fine regulation of metal ions availability in vivo could determine
the pathogen survival inside host cells. Since the levels of free gluta-
thione in vivo are high, this differential inhibition mode might be
taken as an indication that MnII is the native metal ion, in line with our
results. However, additional experimental work is needed to state
that.
The glyoxalase system in Salmonella strains is the main defense
mechanism to the accumulation of methylglyoxal generated in the
phagolysosome during infection [42]. In contrast to other genes
involved in methylglyoxal detoxification, that are induced in the
Salmonella-containing vacuole during infection (such as those coding
GLX1), the expression levels of gloB are constant through the patho-
gen infection cycle [42]. Based on the evidence herein presented that
shows that inhibition by GSH is metal-dependent, the assembly of a
specific metal-enriched form of GLX2 could be exploited as a mech-
anism to regulate the enzyme activity in vivo.
Glyoxalase II belongs to the metallo-β-lactamase superfamily, which
includes several zinc-dependent hydrolases, such as metallo-β-lacta-
mases, endonuclease tRNase Z, AHL lactonase and phosphorylcholine
esterase [40]. GLX2 is an exception in that different metal derivatives
show sizably high catalytic performances for most isoforms [15–20].
This is valid for the plant and bacterial isozymes characterized so far,
since, despite high sequence homology, human GLX2 is inactive in
its MnII form [20]. This fact suggests that metal discrimination could
play a role in glyoxalase activity.
The proposed mechanism for the di-Zn enzyme assumes binding
of S-D-lactoylglutathione substrate to the enzyme, mainly by recog-
nition of the glutathione moiety by active site residues, excluding
a direct interaction with the metal ion (Fig. 1B) [22]. The metal ion
bound to 3 His residues (M1) is proposed to deliver the attacking
nucleophile, at the same time stabilizing the development of negative
charge in an oxygen atom during formation of a tetrahedral inter-
mediate. After the nucleophilic attack, the M2 site favors S–C bond
cleavage by stabilizing the negative charge in the sulfur atom of
the glutathione moiety. After protonation and bond cleavage, a lac-
tate and a glutathione moiety remains bound to the M1 and M2 ions,

V.A. Campos-Bermudez et al. / Journal of Inorganic Biochemistry 104 (2010) 726–731
731
Fig. 5. Cartoon of the active site of GloB during catalysis. The equilibrium between a bridging Asp (left, top) and non-bridging Asp (left, bottom) in the free enzyme is shifted towards
the bridged one in the GSH-bound form (right) (A). The thiolate group of glutathione bound to the metal site bridging the two iron ions upon formation of the EP adduct (B).
[15] O. Schilling, N. Wenzel, M. Naylor, A. Vogel, M. Crowder, C. Makaroff, W. Meyer-
Abbreviations
Klaucke, Biochemistry 42 (2003) 11777–11786.
GLX
EPR
RMN
SLG
GSH
E–P
glyoxalase
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This work was supported by grants from ANPCyT and HHMI to AJV
and from PRONEX/FAPESP/CNPq (Grant no 03/09859-2) and CNPq
(307102/2006-8) to AJCF. VCB and JMB are recipients of fellowships
from CONICET. AJV is a Staff member from CONICET and an Inter-
national Research Scholar of the Howard Hughes Medical Institute. The
600 MHz NMR spectrometer was purchased with funds from ANPCyT
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