Biochimie 104 (2014) 50e60

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Biochimie
journal homepage: www.elsevier.com/locate/biochi

Research paper

The cold-adapted g-glutamyl-cysteine ligase from the psychrophile

Pseudoalteromonas haloplanktis
Antonella Albino a, Amalia De Angelis b, Salvatore Marco a, Valeria Severino c,
Angela Chambery c, Antimo Di Maro c, Doriana Desiderio b, Gennaro Raimo b,
Mariorosario Masullo d, Emmanuele De Vendittis a, *
a
Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita
di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italy
b
del Molise, Contrada Fonte Lappone, 86090 Pesche (IS), Italy
Dipartimento di Bioscienze e Territorio, Universita
c
di Napoli, Via Vivaldi 43, 81100 Caserta, Italy
Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, II Universita
d
Dipartimento di Scienze Motorie e del Benessere, Universita
di Napoli Parthenope, Via Medina 40, 80133 Napoli, Italy

a r t i c l e i n f o a b s t r a c t

Article history: A recombinant g-glutamyl-cysteine ligase from the psychrophile Pseudoalteromonas haloplanktis
Received 18 February 2014 (rPhGshA II) was produced and characterised. This enzyme catalyses the rst step of glutathione
Accepted 9 May 2014 biosynthesis by forming g-glutamyl-cysteine from glutamate and cysteine in an ATP-dependent reaction.
Available online 24 May 2014
The other ATP-dependent enzyme, glutathione synthetase (rPhGshB), involved in the second step of the
biosynthesis, was already characterised. rPhGshA II is a monomer of 58 kDa and its activity was char-
Keywords:
acterised through a direct radioisotopic method, measuring the rate of ATP hydrolysis. The enzyme was
Glutamyl-cysteine ligase
active even at cold temperatures in a moderately alkaline buffer containing a high concentration of
Glutathione biosynthesis
Psychrophile
Mg; 2-aminobutyrate could replace cysteine, although a lower activity was detected. The reaction rate
Cold adaptation of rPhGshA II at 15  C was higher than that reported for rPhGshB, thus suggesting that formation of g-
Pseudoalteromonas haloplanktis glutamyl-cysteine was not the rate limiting step of glutathione biosynthesis in P. haloplanktis. rPhGshA II
had different afnities for its substrates, as evaluated on the basis of the KM values for ATP (0.093 mM),
glutamate (2.8 mM) and cysteine (0.050 mM). Reduced glutathione acted as an inhibitor of rPhGshA II,
probably through the binding to an enzyme pocket different from the active site. Also the oxidised form
of glutathione inhibited the enzyme with a more complex inhibition prole, due to the complete mono-
glutathionylation of rPhGshA II on Cys 386, as proved by mass spectrometry data. When compared to
rPhGshB, rPhGshA II possessed more typical features of a psychrophilic enzyme, as it was endowed with
lower thermodependence and higher heat sensitivity. In conclusion, this work extends the knowledge on
glutathione biosynthesis in the rst cold-adapted source; however, another possible redundant g-glu-
tamyl-cysteine ligase (PhGshA I), not yet characterised, could participate in the biosynthesis of this
cellular thiol in P. haloplanktis.
2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

The tripeptide glutathione (g-glutamyl-cysteinyl-glycine, GSH)

Abbreviations: GshA, g-glutamyl cysteine ligase; GshB, glutathione synthetase;
gshA, gene encoding GshA; gshB, gene encoding GshB; Ph, Pseudoalteromonas hal-
oplanktis; Ec, Escherichia coli; GSH and GSSG, reduced and oxidised form of gluta-
thione; g-Glu-Cys, L-g-glutamyl cysteine; IC50, concentration of inhibitor leading to
50% reduction of the activity; IPTG, isopropyl-b-thiogalactopiranoside; Ea, energy of
activation; kin, rate constant of inactivation; ROS, reactive oxygen species; TCEP,
Tris-(2-carboxyethyl)-phosphine hydrochloride.
* Corresponding author. Tel.: 39 081 7463118; fax: 39 081 7463653.
E-mail address: devendit@unina.it (E. De Vendittis).
is a regulator of the physiological redox environment acting in both
eukaryotes and prokaryotes [1e10]. The main antioxidant action
displayed by GSH includes a defence against damages caused by
reactive oxygen species (ROS) on cellular components, detoxica-
tion of foreign compounds and toxic metals, preservation of protein
sulfhydryls against irreversible oxidation. Besides these protective
roles, this abundant biological thiol is involved in several cellular
functions, such as amino acid transport, metabolism of therapeutic
drugs, cell cycle regulation, carcinogenesis and apoptosis [11e14].

http://dx.doi.org/10.1016/j.biochi.2014.05.003
0300-9084/ 2014 Elsevier Masson SAS. All rights reserved.
 A. Albino et al. / Biochimie 104 (2014) 50e60
The mechanism of redox homoeostasis sustained by GSH is based
on the intracellular balance with its oxidised form (GSSG), obtained
through a disulde bridge between two molecules of GSH. More-
over, GSH, GSSG and S-nitrosoglutathione, another modied form
of glutathione originating from reactive nitrogen species, are
responsible for the S-glutathionylation on specic cysteine residues
of the target protein, and eventually affecting its functioning
[11,12,15e17].
The biosynthesis of GSH occurs with a mechanism conserved
throughout prokaryotes and eukaryotes. In particular, the enzy-
matic assemblage of L-Glu, L-Cys and Gly into GSH occurs in two
sequential steps, both coupled to the hydrolysis of one molecule of
ATP [1]. As described in the following scheme, the rst step is
catalysed by the enzyme g-glutamyl-cysteine ligase and leads to
the formation of L-g-glutamyl cysteine (g-Glu-Cys); the second step
produces GSH and includes the involvement of another enzyme,
called glutathione synthetase.
L-Glu L-Cys ATP / g-Glu-Cys ADP Pi (1)
g-Glu-Cys Gly ATP / GSH ADP Pi (2)
The enzymes involved in the GSH biosynthesis isolated from
prokaryotic and eukaryotic sources are somehow different in their
structural and functional organisation. In most prokaryotes, two
distinct genes, gshA and gshB, encode g-glutamyl-cysteine ligase
(GshA) and glutathione synthetase (GshB), respectively; the rst
enzyme is a monomer and the second one is organised as a
homotetramer [9]. On the other hand, few prokaryotes possess a
single bifunctional enzyme organised as a homodimer, supporting
both activities required for GSH biosynthesis [18e20]. In eukary-
otes, the activities of g-glutamyl-cysteine ligase and glutathione
synthetase are respectively sustained by a heterodimeric enzyme
[21,22] and a homodimeric glycosylated enzyme [23e25]. The
studies on GSH biosynthesis pointed to some features possessed by
g-glutamyl-cysteine ligase in different organisms. Indeed, the re-
action catalysed by this enzyme seems to be the rate limiting step of
the overall process [26]; the activity is feedback inhibited by GSH
[21,27]; the Mg2 ions, a specic requirement for ATP-dependent
enzymes, can be replaced by Mn2 ions, although this substitu-
tion reduces the activity of the enzyme and modies its afnity
towards different substrates [28e30].
GSH has a more crucial role in microorganisms characterised by
an oxidative metabolism [4]. Pseudoalteromonas haloplanktis, a
psychrophile isolated from the Antarctic sea, able to grow in the
4e20  C interval [31], is a model of aerobic cold-adapted bacteria.
The presence and the functional role of GSH in this microorganism
was inferred by the nding of glutathionylated adducts either in the
endogenous proteins from a growing culture of this bacterium [17]
or in some puried recombinant enzymes involved in the control of
the oxidative stress of the psychrophile [17,32]. In the presence of
sufcient nutrients and high aeration, P. haloplanktis grows to very
high density due to its efcient respiration, a behaviour implying a
good functioning of the defence mechanisms against the oxidant
environment [31]. Indeed, P. haloplanktis is exposed to high levels of
ROS for both the increased oxygen solubility in the cold sea water
and the enhanced stability of these toxic compounds at cold tem-
peratures [31,33]. Previous reports on the enzyme systems involved
in the defence against ROS damages in P. haloplanktis pointed to the
high efciency of superoxide dismutase [17,34,35] and thioredoxin
system [32,36]. Furthermore, the studies on GSH biosynthesis in
this microorganism already led to the characterisation of its cold-
adapted glutathione synthetase (rPhGshB) [37,38].
In order to continue the characterisation of the enzyme system
for GSH biosynthesis in P. haloplanktis, the present work regards the
51
enzyme g-glutamyl-cysteine ligase from this psychrophile
(PhGshA). The genome of P. haloplanktis, strain TAC125 contains
two chromosomes and a gene encoding a putative PhGshA was
annotated in chromosome I (Ph-gshA I) and II (Ph-gshA II) [31]. For
this reason, the cloning of both genes in suitable expression vectors
was considered for the heterologous production of recombinant
forms of PhGshA; however, the current study was mainly focused
on the detailed biochemical characterisation of the enzyme enco-
ded by the gene Ph-gshA II. Indeed, a recombinant enzyme, named
rPhGshA II, possessing the typical activity of g-glutamyl-cysteine
ligases, was obtained. In particular, rPhGshA II was able to catalyse
the rst step of GSH biosynthesis, involving the ATP-dependent
formation of g-Glu-Cys from L-Glu and L-Cys. The investigation
included the evaluation of the substrate specicity of rPhGshA II,
the determination of its kinetic and inhibition parameters and an
assessment on the effect of temperature, a relevant issue for a cold-
adapted enzyme. Therefore, this study, together with the previous
reports on glutathione synthetase [37,38], gives further information
on GSH biosynthesis to P. haloplanktis, a typical cold-adapted or-
ganism which seems to be highly responsive to glutathione meta-
bolism and homoeostasis.
2. Materials and methods
2.1. Materials
Restriction and modifying enzymes were from GE Healthcare or
Promega. Taq DNA polymerase from Takara was used in PCR ex-
periments. Plasmid pGEM-T Easy was from Promega, whereas
vector pET-22b() and the Escherichia coli BL21(DE3) strain were
from Novagen. Purication of plasmids and DNA fragments was
realised with Qiagen kits from M-Medical. Oligonucleotide syn-
thesis, nucleotide sequencing and synthesis of vectors were carried
out at Primm (Italy). Ampicillin, isopropyl-b-thiogalactopiranoside
(IPTG), b-mercaptoethanol, Tris-(2-carboxyethyl)-phosphine hy-
drochloride (TCEP), L-Cys, L-Glu, L-2-aminobutyric acid, L-2-
hydroxy-glutaric acid, N-methyl-L-glutamic acid, 2,3-
naphthalenedicarboxyaldehyde, GSH and GSSG were from Sigma-
eAldrich. [g-32P]ATP (2 mCi mL1; 10 Ci mmol1) was purchased
from Perkin Elmer. The chromatographic medium Ni-NTA agarose
was from Qiagen. HPLC-grade solvents were obtained from Carlo
Erba. All other chemicals were of analytical grade.
The following buffers were used: buffer A, 20 mM Tris$HCl, pH
7.8, 5 mM MgCl2, 150 mM KCl, 200 mM TCEP; buffer B, as buffer A
supplemented with 50% (v/v) glycerol; buffer C, 100 mM Tris$HCl,
pH 8.0, 20 mM MgCl2, 150 mM KCl.
2.2. Heterologous expression of the gshA gene from P. haloplanktis
The genomic DNA from P. haloplanktis, strain TAC125, was pre-
pared as previously described [39]. Transformation of bacterial
strains, preparation of plasmids and other details of DNA recom-
binant technology were carried out according to standard pro-
cedures [40]. The two genes encoding putative GshA in
chromosome I and II from P. haloplanktis, strain TAC125, have been
annotated as Ph-gshA I (locus tag: PSHAa0937) and Ph-gshA II (locus
tag: PSHAb0107), respectively [31]. For the production of a re-
combinant form of a psychrophilic GshA, the Ph-gshA II gene was
considered and its heterologous expression was performed essen-
tially as recently reported [41]. Its complete coding sequence was
amplied by PCR, using the genomic DNA from this psychrophile as
a template, and couples of oligonucleotide primers annealing to 50 -
or 30 -untranslated region of the gene. In particular, forward and
reverse primer for the amplication of Ph-gshA II were 50 d-
Ae15TTACAGAGGTTCATATG$ACG$TTA$T10-30 and 50 d-
52 A. Albino et al. / Biochimie 104 (2014) 50e60
T1528GCTCTTTTGCTC$GAG$CTT$ACC$TTC$T1503-30 . Numbering in
primers begins from starting codon (italics), whereas underlined
letters indicate mismatches introduced to create the NdeI and XhoI
restriction sites. The amplied segment was digested with NdeI and
XhoI and cloned in pET-22b() previously digested with the same
endonucleases. The resulting construct was controlled by nucleo-
tide sequencing to exclude any undesired mutation occurring
during the amplication/cloning procedure. The vector for the
expression of Ph-gshA II, named vPh-gshA II, allowed the produc-
tion of a recombinant form of a putative GshA from P. haloplanktis,
called rPhGshA II, in which the extrapeptide LE(H)6 replaced the C-
terminal V 505 of PhGshA II. After transformation of the E. coli
BL21(DE3) strain with vPh-gshA II, a culture of the resulting
transformant was grown at 30  C up to 0.5 A600 and the heterolo-
gous expression was continued for 3 h without or upon the addition
of 0.1 mg mL1 IPTG. Bacterial cells were collected by centrifuga-
tion, resuspended in buffer A (15 mL for 1-L culture) and then lysed
by a French Press (Aminco, USA) to obtain a cell homogenate. This
sample was then centrifuged at 100,000 g and the supernatant
was used as starting material for the purication of the recombi-
nant product by afnity chromatography on Ni-NTA agarose. To this
aim, the supernatant was added in batch to the Ni-NTA Agarose
resin, equilibrated in buffer A. After incubation overnight at 4  C,
the slurry was poured in a column, which was extensively washed
with the same buffer supplemented with 10 mM imidazole$HCl.
The bound proteins were then eluted with buffer A supplemented
with 200 mM imidazole$HCl and pure protein fractions, as analysed
by SDS-polyacrylamide gel electrophoresis, were pooled together
and extensively dialysed against buffer A. Some preparations of
rPhGshA II required a further purication step on Ni-NTA agarose
resin, in order to eliminate traces of triphosphatase contaminating
activities. The pure protein samples were nally stored at 20  C in
buffer B. Nearly 4e5 mg of protein were obtained from 1-L culture
of the transformant.
2.3. Biochemical assays
The activity of rPhGshA II was evaluated through the ATP hy-
drolysis promoted by this enzyme, coupled to the synthesis g-Glu-
Cys starting from L-Glu plus L-Cys (reaction (1)). The method is
almost identical to that successfully used for measuring the activity
of rPhGshB [38]. In particular, the ATPase activity was evaluated
through the release of radioactive inorganic phosphate (32Pi) from
[g-32P]ATP, using the phosphomolybdate method [42]. On the other
hand, the detection of the nal product g-Glu-Cys was carried out
through a uorimetric method, essentially as previously reported
[43]. Unless otherwise indicated, the reaction was carried out
kinetically at 15  C in buffer C, and the reaction rate was obtained
from the slope of linear kinetics. The kinetic parameters of the
ATPase catalysed by the psychrophilic enzyme, including the KM
values for the three substrates L-Cys, L-Glu and ATP, and the Vmax of
the reaction, were derived from LineweavereBurk plots of the ac-
tivity data. To this aim, the reaction mixture contained a xed and
essentially saturating concentration of two out of three substrates,
and various concentrations of the third substrate distributed
around the KM of the reaction. The inhibition prole on the rPhGshA
II ATPase by GSH or GSSG was obtained through activity mea-
surements carried out in the presence of various concentrations of
these glutathione forms. The kinetic parameters of the rPhGshA II
ATPase were determined also in the presence of a xed concen-
tration of GSH or GSSG. The energy of activation (Ea) of the ATPase
activity of rPhGshA II and of the heat inactivation process of this
psychrophilic enzyme was derived from Arrhenius plots; the other
thermodynamic parameters of activation (DH*, DS* and DG*) were
calculated as reported elsewhere [44]. The biochemical data
presented in each of Figs. 2e6, 9 and 10 reect one out of at least
two independent experiments realised with different protein
preparations and giving almost overlapping results. Furthermore,
the signicance of the linear t in kinetics and double reciprocal
plots was thoroughly evaluated through the squared correlation
coefcient r2. Where appropriate, the signicance level of the data
presented was evaluated by calculating the t-parameter and the
corresponding p-values; otherwise, error bars were included in the
graphical representations.
2.4. MALDI-TOF MS mapping
To identify the cysteinyl residue target of the S-gluta-
thionylation reaction, a tryptic peptide mapping was performed by
MALDI-TOF mass spectrometry on rPhGshA II incubated for 15 min
at 15  C in the absence or in the presence of 3.6 mM GSH or GSSG.
Treated and untreated protein samples were subjected to tryptic
digestion for 16 h at 37  C in 50 mM NH4HCO3 with trypsin
(1:1000). At the end of digestion, tryptic peptides were analysed on
a MALDI-TOF micro MX apparatus (Waters Co., Manchester, UK)
equipped with a pulsed nitrogen laser (l 337 nm). Prior to spectra
acquisition, 1 mL of each peptide solution was mixed with 1 mL of
saturated a-cyano-4-hydroxycinnamic acid matrix solution (10 mg/
mL in 1:1 acetonitrile:water, v/v, containing 0.1% triuoroacetic
acid) and 1 mL of the resulting mixture was placed on the mass
spectrometer sample target. The droplet was dried at room tem-
perature. Once the liquid was completely evaporated, the sample
was loaded into the mass spectrometer and analysed. The instru-
ment was externally calibrated using a tryptic alcohol dehydroge-
nase digest (Waters, Manchester, UK) in reectron mode. All
spectra were processed and analysed using the MassLynx 4.1 soft-
ware (Waters, Manchester, UK).
2.5. Other methods
Protein concentration was determined by the method of Brad-
ford, using bovine serum albumin as standard [45]. Purity of protein
samples was assessed by 12% SDS-PAGE according to standard
protocols [46]. The quaternary structure of the recombinant
enzyme was evaluated by gel-ltration on a Superdex 200 10/300
GL column connected to a FPLC apparatus (GE Healthcare). The
mass spectrometry analysis was performed on protein samples
desalted by RP-HPLC, as previously reported [34,47]. The number of
cysteine residues in protein samples was determined by the Ellman
assay [36,48]. Intracellular glutathione content was measured using
the EnzyChrome GSH/GSSG assay kit essentially as previously re-
ported [49]. Briey, 1 million cells from P. haloplanktis cultures were
sonicated and homogenised in 50 mM phosphate buffer, containing
1 mM EDTA. The suspension was centrifuged at 4  C for 15 min at
10,000  g and the supernatant was deproteinized with 5% meta-
phosphoric acid. After centrifugation at 14,000 rpm for 5 min,
GSH and GSSG contents were measured on supernatants following
the manufacturer's protocol. Multiple alignments were performed
by using the ClustalW2 on-line tool available at http://www.ebi.ac.
uk/Tools/msa/clustalw2 with the BLOSUM matrix and two open
gaps settings; all the other parameters were in the default setup.
The model of the rPhGshA II 3D structure was obtained by using the
automated I-TASSER service available at http://zhanglab.ccmb.med.
umich.edu/I-TASSER. The on line procedure yielded the 3D model
on the basis of multiple-threading alignments by LOMETS and
iterative TASSER simulations [50]. The molecular model of rPhGshA
II was constructed using as a template the crystallographically
derived coordinates of g-glutamyl-cysteine ligase from E. coli
(EcGshA, PDB entry code 2D33) rened to 2.6 resolution, sharing
42.4% amino acid sequence identity with rPhGshA II. The three
 A. Albino et al. / Biochimie 104 (2014) 50e60
dimensional model was visualised using PYMOL application soft-
ware (www.pymol.org).
3. Results
3.1. Molecular properties of a recombinant form of GshA from
P. haloplanktis
The P. haloplanktis genome contains two genes encoding puta-
tive g-glutamyl-cysteine ligases (Ph-gshA I in chromosome I and Ph-
gshA II in chromosome II). The amino acid sequence deduced from
these genes was pair wise-aligned either each other or with the
sequence of the corresponding single enzyme from E. coli, EcGshA,
whose structural and biochemical properties were extensively
investigated [21,27e29,51,52]. The percentage of amino acid iden-
tity between PhGshA I and PhGshA II was 43.2%; similar percent-
ages of amino acid identity were obtained from the alignment of
EcGshA with PhGshA I (44.6%) or PhGshA II (42.4%). Therefore, these
data could suggest a possible redundancy for g-glutamyl-cysteine
ligase activity in P. haloplanktis. The strategy followed for studying
this activity in the cold-adapted microorganism considered the
production of recombinant forms of both enzymes. However, the
rst one produced through the expression system described in the
Experimental section was rPhGshA II. For this reason, the present
work was focused on the characterisation of the molecular and
biochemical properties of this enzyme, obtained as a recombinant
protein fused to His-tag. Its production was achieved through the
E. coli BL21(DE3) strain transformed with the vPh-gshA II vector.
The inducible promoter of this heterologous expression system is
typically activated by IPTG; however, differently from what re-
ported for the production of rPhGshB [37,38], no IPTG induction was
apparently observed in this case. Indeed, the best experimental
conditions for obtaining rPhGshA II were reached when growth of
the BL21(DE3)/vPh-gshA II transformant continued for additional 3-
h at 30  C without IPTG induction, after the culture reached the
value of 0.5 OD600. The presence of a reducing agent, such as TCEP,
during purication procedure and storage of rPhGshA II ensured the
obtainment of an active form of this enzyme.
The purity of rPhGshA II was controlled by SDS/PAGE analysis,
which showed that the protein sample was homogenous and that
its electrophoretic mobility corresponded to a molecular mass of
58 kDa, a value coincident with that predicted for the recombinant
enzyme (Fig. 1). The molecular mass of rPhGshA II was then
Fig. 1. SDS-PAGE of rPhGshA II. Increasing amounts (1, 3 and 5 mg) of puried rPhGshA
II (lanes 1e3) were analysed on a 12% polyacrylamide gel. Migration of molecular mass
protein standards (lane M) is reported on the right.
53
determined by ESI/Q-TOF mass spectrometry after protein desalt-
ing by RP-HPLC. The accurate molecular mass was found to be
57662.50 Da (not shown), in good agreement with the theoretical
value of 57662.57 calculated for the recombinant enzyme, taking in
account the His-tail and the lack of the rst methionine residue.
This result excluded the presence of covalent adducts on the
enzyme, as previously observed on other recombinant antioxidant
enzymes from P. haloplanktis [17,32,34,38]. The molecular mass of
rPhGshA II was also determined by gel ltration chromatography on
a Superdex 75 10/300 GL column using non-denaturing condi-
tions. A single peak was obtained and its elution time, compared
with that of protein standards, corresponded to an extrapolated
molecular mass of 54.5 kDa (not shown). This value clearly in-
dicates that rPhGshA II behaved as a monomer under non-
denaturing conditions, similarly to what reported for EcGshA [51].
The molecular characterisation of rPhGshA II also included deter-
mination of the free cysteines in the puried protein sample, in
order to assess whether the purication procedure of rPhGshA II in
the presence of TCEP ensured maintenance of these residues in a
reduced state. Indeed, the Ellman assay, carried out on the puried
recombinant enzyme, allowed the determination of 3.0 mol
cysteine/mol enzyme, in perfect agreement with total three cys-
teines counted in the amino acid sequence; therefore, all cysteine
residues of the rPhGshA II sample were present as free thiols in the
recombinant protein sample.
3.2. Biochemical properties of rPhGshA II
As described in previous reports, the activity of GshA was usu-
ally evaluated through the ADP formed during the ATP-dependent
synthesis of g-Glu-Cys from L-Glu plus L-Cys; in particular, an in-
direct spectrophotometric method was employed, which deter-
mined the ADP formed through the NADH oxidation in the
presence of pyruvate kinase and lactate dehydrogenase [22]. In this
case, the direct effect of some parameters, such as temperature, pH
or ions, as well as of substrates or inhibitors, on the biochemical
properties of GshA was impaired by the presence of the other en-
zymes. To overcome this difculty, the activity of rPhGshA II was
determined through a direct radioisotopic assay, using the method
successfully employed for measuring the activity of rPhGshB [38].
In particular, the assay involved the determination of 32Pi released
from radiolabelled [g-32P]ATP in the presence of the specic sub-
strates of reaction (1). The effect of various substrates on the rate of
32
Pi release promoted by rPhGshA II is reported in Fig. 2. The data
show that the ATPase sustained by this enzyme required the
presence of both specic substrates L-Glu and L-Cys. Indeed, a linear
kinetics of ATP hydrolysis was evident in the presence of these
substrates, whereas no activity was measured in the presence of L-
Glu or L-Cys alone. It was concluded that the ATPase activity pro-
moted by rPhGshA II was likely coupled to the synthesis of g-Glu-
Cys, as expected from reaction (1). The results shown in Fig. 2 also
indicate that, in the presence of L-Glu, the substrate L-Cys could be
replaced by L-2-aminobutyric acid, although the rate of ATP hy-
drolysis measured in the presence of this substrate was signi-
cantly lower than that determined with L-Cys. On the other hand,
other substrates, such as L-2-hydroxy-glutaric acid or N-methyl-L-
glutamic acid, could not replace L-Cys. The substitution of L-2-
aminobutyric acid for L-Cys was already reported in other GshA
activities, although using a different assay [21,51]. When using
some preparations of the enzyme with a lower grade of purity, a
low and non linear rate of ATP hydrolysis was measured in the
presence of L-Glu alone (not shown). It cannot be excluded that this
nding was due to traces of triphosphatase contaminating activ-
ities present in these protein samples. The activity of rPhGshA II
was conrmed through the detection of the nal product of the
54 A. Albino et al. / Biochimie 104 (2014) 50e60
Fig. 2. Effect of various substrates on the ATPase activity catalysed by rPhGshA II. The
reaction mixture, containing 0.16 mM rPhGshA II in 350 mL of buffer C, was supple-
mented with the following substrates: no addition (B); 10 mM L-Glu (); 2.5 mM L-
Cys (); 10 mM L-Glu 2.5 mM L-Cys (C); 10 mM L-Glu 2.5 mM L-2-aminobutyric
acid (:); 10 mM L-Glu 2.5 mM L-2-hydroxy-glutaric acid (); 10 mM L-
Glu 2.5 mM N-methyl-L-glutamic acid (7). The reaction was carried out at 15  C and
started with the addition of 2 mM [g-32P]ATP (specic radioactivity 3.3 cpm pmol1).
At the times indicated, aliquots were withdrawn and analysed for the 32Pi released, as
described in Materials and Methods. The squared correlation coefcient r2 in (C) and
(:) was 0.99 and 0.92, respectively, whereas the p-values were lower than 0.0001 and
0.005, respectively.
reaction g-Glu-Cys using the reagent 2,3-
naphthalenedicarboxyaldehyde. Indeed, the rate of g-Glu-Cys for-
mation by rPhGshA II was of the same order of that measured
through the release of 32Pi from [g-32P]ATP. In particular, under the
same experimental conditions the calculated rates were 40.3 mol
g-Glu-Cys formed mol rPhGshA II1 min1 and 101.2 mol ATP
hydrolysed mol rPhGshA II1 min1. However, for the enzyme
characterisation the radioisotopic method was preferred to the
uorimetric one because of its higher sensitivity.
The pH and ionic dependence of the ATPase activity sustained by
rPhGshA II at 15  C was investigated (Fig. 3). The effect of pH was
studied in the 6.0e8.6 pH interval, using imidazole$HCl or Tris$HCl
buffers supplemented with 20 mM MgCl2 and 150 mM KCl. The
data reported in Fig. 3A indicate that the rPhGshA II-dependent
ATPase activity was affected by the pH and that maximum activ-
ity was reached in the 7.8e8.6 pH range, using the Tris$HCl buffer. A
reaction mixture containing 100 mM Tris HCl, pH 8.0 was consid-
ered as representative of optimum pH conditions and was used for
the evaluation of the ionic dependence on the ATPase activity
promoted by rPhGshA II. In order to study the requirement for a
specic divalent cation in the assay, this buffer was supplemented
with 150 mM KCl and increasing concentration of MgCl2 or MnCl2.
As shown in Fig. 3B, the presence of a divalent cation in the reaction
mixture was required for triggering the ATPase, because no activity
was essentially measured in the absence of Mg or Mn. Among
these two cations, Mg was by far the most effective one, as it
caused a strong dose-dependent enhancement of the activity up to
20 mM MgCl2. Compared to that exerted by Mg, the stimulation
provoked by Mn, if any, was hardly detectable (Fig. 3B); for
instance, the highest level of the Mn-dependent activity, measured
in the presence of 0.5 mM MnCl2, was 34-fold lower compared to
that measured with 20 mM MgCl2. The effect of monovalent cations
on the ATPase activity sustained by rPhGshA II was investigated by
supplementing the 100 mM Tris$HCl buffer at pH 8.0 with 20 mM
MgCl2 and increasing concentration of NaCl or KCl. As shown in
Fig. 3C, full activity was already evident in the absence of NaCl or
Fig. 3. pH and ionic requirement for triggering the ATPase activity of rPhGshA II. (A)
Effect of pH. The reaction mixture contained 0.16 mM rPhGshA II, 10 mM L-Glu and
2.5 mM L-Cys in 250 mL nal volume of 100 mM Tris$HCl (C) or 50 mM imidazole$HCl
buffer (:), at the pH indicated, supplemented with 20 mM MgCl2 and 150 mM KCl. (B)
Effect of divalent cations. The reaction mixture contained the same concentration of
rPhGshA II, L-Glu and L-Cys as in A, in 250 mL nal volume of 100 mM Tris$HCl, pH 8.0
buffer, supplemented with 150 mM KCl and the indicated concentration of MgCl2 (C)
or MnCl2 (:). (C) Effect of monovalent cations. The reaction mixture contained the
same concentration of rPhGshA II, L-Glu and L-Cys as in A, in 250 mL nal volume of
100 mM Tris$HCl, pH 8.0 buffer supplemented with 20 mM MgCl2 and the indicated
concentration of KCl (C) or NaCl (:). All the reactions were carried out at 15  C and
started with the addition of 2 mM [g-32P]ATP (specic radioactivity 3.5 cpm pmol1).
At selected times, 50 mL-aliquots were withdrawn from the reaction mixture and
analysed for the 32Pi released. The data were expressed as the rate of 32Pi release,
evaluated from the slope of linear kinetics (r2 values ranging between 0.97 and 0.99).
KCl and therefore, the presence of a monovalent cation was
apparently dispensable for triggering the ATPase of rPhGshA II.
However, the presence of Na or K up to 320 mM did not cause any
variation of the activity. In conclusion, a buffer containing 100 mM
Tris$HCl, pH 8.0, supplemented with 20 mM MgCl2 and 150 mM KCl
(buffer C) was selected for the following studies on the ATPase
activity of rPhGshA II, a choice allowing the comparison with the
data referred to GshAs from other organisms.
To investigate the afnity of rPhGshA II for each substrate
involved in reaction (1), LineweavereBurk plots of the ATPase ac-
tivity were obtained at 15  C in buffer C using three different
experimental conditions; in particular, the concentration of one
among the three substrates ATP, L-Glu or L-Cys was varied, whereas
that of the remaining ones was kept xed and almost saturating.
Three straight lines were obtained, extrapolating to an almost
identical y-axis intercept, a nding indicating that a similar
maximum rate of ATP hydrolysis (Vmax) was reached in the three
different experimental conditions (Fig. 4). This kinetic parameter
was then converted into the catalytic constant (kcat) of rPhGshA II
and the values obtained for ATP, L-Glu and L-Cys were 4.1, 3.2 and
3.6 s1, respectively. The LineweavereBurk plots of Fig. 4 allowed
the determination of the different afnity of rPhGshA II for its three
substrates and the calculated KM values for ATP, L-Glu and L-Cys
were 0.093 mM, 2.8 mM and 0.050 mM, respectively. Therefore, the
catalytic efciency of rPhGshA II, evaluated through the kcat/KM
ratio referred to the substrate ATP, was 44 s1 M1, a value higher
than that reported for the ATPase activity of rPhGshB (7.1 s1 M1)
[38]. A summary of the kinetic parameters of rPhGshA II and the
comparison with the corresponding data from rPhGshB is reported
in Table 1.
Previous reports indicated that glutathione, in its reduced GSH
or oxidised GSSG form, was able to affect functioning of the en-
zymes involved in its biosynthesis [21,38,51,53]. The level of these
compounds in P. haloplanktis cells was measured. The amount of
GSH and GSSG in a culture of this psychrophile grown at 5  C in the
exponential phase (0.98 A600) was 11.1 0.2 and 1.19 0.06 nmol
per million cells, respectively. Similar values, i.e. 11.8 0.3 and
1.37 0.05 nmol per million cells, for GSH and GSSG respectively,
were measured when the culture was grown up to stationary phase
 A. Albino et al. / Biochimie 104 (2014) 50e60 55

Fig. 4. LineweavereBurk plots for the three substrates of rPhGshA. (A) Afnity for ATP.
The reaction mixture contained 0.16 mM rPhGshA II, 10 mM L-Glu and 2.5 mM L-Cys in
250 mL nal volume of buffer C. The reaction started with the addition of the indicated
concentration of [g-32P]ATP (specic radioactivity 6.2e552.4 cpm pmol1). (B) Afnity
for L-Glu. The reaction mixture contained 0.16 mM rPhGshA II, 2.5 mM L-Cys and the
indicated concentration of L-Glu in 250 mL nal volume of buffer C. The reaction started
with the addition of 2 mM [g-32P]ATP (specic radioactivity 3.3 cpm pmol1). (C)
Afnity for L-Cys. The reaction mixture contained 0.16 mM rPhGshA II, 10 mM L-Glu and
the indicated concentration of L-Cys in 250 mL nal volume of buffer C. The reaction
started with the addition of 2 mM [g-32P]ATP (specic radioactivity 3.9 cpm pmol1). Fig. 5. Inhibition prole of rPhGshA II by GSH and GSSG. The reaction mixture for
All the reactions were carried out at 15  C and were followed kinetically through the measuring the rPhGshA II ATPase in the presence of the indicated concentrations of
analysis of the amount of 32Pi released from aliquots withdrawn at appropriate times. GSH (C) or GSSG (-) contained 0.16 mM rPhGshA II, 10 mM L-Glu and 2.5 mM L-Cys in
The values of v (mol ATP hydrolysed mol rPhGshA II1 min1) were calculated from the 250 mL nal volume of buffer C. The reaction, carried out at 15  C, started with the
slope of linear kinetics (r2 values ranging between 0.93 and 0.99) and then reported in addition of 2 mM [g-32P]ATP (specic radioactivity 2.7 cpm pmol1) and was followed
the LineweavereBurk plots. The r2 values of double reciprocal plots were 0.97 (panel kinetically. Care was taken to add GSH or GSSG just before the addition of [g-32P]ATP.
A), 0.94 (panel B) and 0.66 (panel C), whereas the p-values were lower than 0.0001 The ATPase activity, evaluated form the slope of linear kinetics (r2 values ranging
(panels A and B) or lower than 0.001 (panel C). between 0.97 and 0.99) was expressed as a percentage of that measured in the absence
of the glutathione forms.

(7.0 A600). Interestingly, the molar GSH/GSSG ratio in P. haloplanktis,

ranging between 9.3 and 8.6, was roughly ten-fold lower than that of the reduction of Vmax and KM observed in the presence of GSH,
usually reported in other bacteria [9]. To investigate the possible the average value of the calculated Ki for GSH was 10.8 mM; an
regulation of the biochemical properties of rPhGshA II by GSH or approximately two times higher value was estimated for GSSG.
GSSG, the effect of increasing concentrations of these compounds Table 1 also reports the comparison of the inhibition constants of
on the ATPase activity of the psychrophilic enzyme was assayed rPhGshA II and rPhGshB.
(Fig. 5). A typical dose-dependent inhibition prole was observed in It is known that rPhGshB, the enzyme involved in the second
the presence of GSH and the concentration leading to 50% reduc- step of GSH biosynthesis in P. haloplanktis, was the target of a co-
tion of the activity (IC50) was above 10 mM. Concerning GSSG, the valent modication by GSSG and that the S-glutathionylation re-
reduction of activity caused by this compound was not very action caused the irreversible inhibition of the enzyme [38]. To
different from that of GSH, even though the inhibition prole search for the occurrence of a potential covalent modication of
observed in this case was somehow biphasic. The mechanism of rPhGshA II by GSH or GSSG, an aliquot of this enzyme was analysed
inhibition by GSH and GSSG was also studied through kinetic by ESI/Q-TOF mass spectrometry before and after its incubation for
measurements of the activity of rPhGshA II in the absence or in the
presence of each inhibitor (Fig. 6). The effect of the inhibitors on the
Vmax and KM for ATP is shown in Fig. 6A. Either GSH or GSSG caused
a reduction of the Vmax of the reaction, thus excluding that these
glutathione forms could act as competitive inhibitors of the sub-
strate ATP; moreover, GSH and GSSG also led to a decrease of the KM
for this substrate, and therefore the binding of these compounds to
the ATP-bound form of the enzyme could be excluded. The effect of
GSH and GSSG on the Vmax and KM for L-Glu is shown in Fig. 6B and
also in this case both inhibitors caused a reduction of the kinetic
parameters. This behaviour seems to exclude that GSH or GSSG
could compete with L-Glu for the binding to rPhGshA II. On the basis
Fig. 6. Effect of GSH and GSSG on the LineweavereBurk plots of the rPhGshA II ATPase.
Table 1 (A) Afnity for ATP. The reaction mixture contained 0.16 mM rPhGshA II, 2.5 mM L-Cys,
Kinetic and inhibition parameters of the ATPase catalysed by rPhGshA II; comparison 10 mM L-Glu, without (B) or with 10 mM GSH (C) or 10 mM GSSG (-) in 250 mL nal
with rPhGshB. volume of buffer C. The reaction started with the addition of the indicated concen-
tration of [g-32P]ATP (specic radioactivity 1.3e106.7 cpm pmol1). (B) Afnity for L-
Enzyme Substrate Kinetic parameter at 15  C Inhibitor Ki (mM) Reference
Glu. The reaction mixture contained 0.16 mM rPhGshA II, 2.5 mM L-Cys, the indicated
kcat KM kcat/KM concentration of L-Glu, without (B) or with 10 mM GSH (C) or 10 mM GSSG (-) in
(s1) (mM) (s1 mM1) 250 mL nal volume of buffer C. The reaction started with the addition of 2 mM [g-32P]
ATP (specic radioactivity 1.3 cpm pmol1). All the reactions were carried out at 15  C
rPhGshA II ATP 4.1 0.093 44 GSSG n.d. This work
and were followed kinetically through the analysis of the amount of 32Pi released from
L-Glu 3.2 2.8 1.1 GSH 10.8
aliquots withdrawn at appropriate times. The values of v (mol ATP hydrolysed
L-Cys 3.6 0.050 72
mol rPhGshA II1 min1) were calculated from the slope of linear kinetics (r2 values
rPhGshB ATP 1.85 0.26 7.1 GSSG 10.7 [38]
ranging between 0.93 and 0.99) and then reported in the LineweavereBurk plots. The
g-Glu-Cys 1.93 0.25 7.7 GSH n.d.
r2 values of double reciprocal plots ranged between 0.96 and 0.99 and the p-values
Gly 2.03 0.75 2.7
were all lower than 0.005.
56
15 min at 15  C with 3.6 mM GSH or GSSG. The molecular mass of
untreated rPhGshA II (57662.50 Da) remained essentially un-
changed after the treatment with GSH (57662.82 Da). On the con-
trary, an ion signal with a molecular mass of 57967.50 Da was
detected following the incubation of rPhGshA II with GSSG, corre-
sponding to a mono-glutathionylated form of rPhGshA II. Indeed,
the 305 Da extra mass was in agreement with that expected if the
enzyme formed one mixed disulde with glutathione, thus indi-
cating that rPhGshA II was subjected to S-glutathionylation reaction
by GSSG. Interestingly, the mono-glutathionylated form of rPhGshA
II was detected even when incubation with GSSG was performed in
the presence of 10 mM L-Glu and 2.5 mM L-Cys (not shown).
3.3. Identication of the Cys residue target of the S-
glutathionylation reaction in rPhGshA II
Three cysteinyl residues are present in the rPhGshA II sequence
at positions 107, 357 and 386. To identify the Cys residue target of
the S-glutathionylation reaction, a comparative MALDI-TOF MS
mapping of tryptic peptides obtained from rPhGshA II samples
incubated in the absence or in the presence of GSH or GSSG was
carried out. The two signals at m/z 689.3 and 1630.8 were clearly
identied in spectra of the tryptic digests of untreated rPhGshA II
and rPhGshA II incubated with GSH (see solid arrows in Fig. 7).
These ions correspond to tryptic peptides with sequence positions
380e385 (theoretical [M H]: 689.36) and 386e400 (theoretical
[M H]: 1630.81), respectively. Following incubation with GSSG,
these peaks disappeared, whereas an additional ion signal with m/
z 2606.2, was specically detected in this spectrum (dotted arrow
in Fig. 7). This ion signal corresponds to the glutathionylated
tryptic peptide 380e400 (theoretical [M H]: 2606.15) with a
missing tryptic cleavage on Arg 385, likely occurring as a conse-
quence of the S-glutathionylation of the neighbouring Cys residue
at position 386.
Fig. 7. Magnication of MALDI-TOF spectra of tryptic digests of rPhGshA II incubated without or with GSH or GSSG, as described in the text. Signal ions of the unmodied tryptic
peptides in the untreated or GSH-treated sample are indicated by solid arrows; sequence of these peptides is also reported. The tryptic peptide diagnostic of the S-glutathionylation
on Cys 386 in the GSSG-treated sample is indicated by a dotted arrow.
A. Albino et al. / Biochimie 104 (2014) 50e60
3.4. Effect of temperature on the biochemical properties of rPhGshA
II
The cold adaptation of rPhGshA II was investigated by analysing
the thermodependence of activity and stability exhibited by the
psychrophilic enzyme. To this aim, the ATPase reaction catalysed by
rPhGshA II was studied in the 10e35  C interval, in order to
determine the effect of temperature on the kinetic parameters Vmax
and KM for ATP. The temperature dependence of the Vmax shown in
Fig. 8 indicates that rPhGshA II was already active at 10  C; however,
the Vmax moderately increased up to 28  C, and then decreased
above this temperature. Also the KM for ATP showed a similar
moderate increase with temperature (not shown). To better analyse
the thermodependence of the reaction velocity, the values of Vmax
were converted into kcat and then analysed by the Arrhenius
equation. As shown in the inset to Fig. 8, the plot was linear in the
10e28  C interval and the calculated low value of the energy of
activation (Ea, 42.3 kJ mol1) was in agreement with the modest
effect of temperature on enzyme activity.
The signicant decrease of the Vmax between 30 and 35  C, as
emerged from the study of the thermodependence of the rPhGshA
II activity, could suggest the onset of a heat inactivation process of
the psychrophilic enzyme in this temperature range. A heat inac-
tivation prole realised on rPhGshA II samples incubated for 10 min
at different temperatures indicated that the enzyme was rather
sensitive to heat, because its half-inactivation occurred at nearly
35  C (not shown). Therefore, the thermal stability of rPhGshA II
was analysed through heat inactivation kinetics carried out at
temperatures ranging between 25 and 41  C. At each temperature
the kinetics was linear, when analysed according to the equation of
a rst-order kinetics, and this allowed the calculation of the cor-
responding inactivation rate constants (kin). The values of kin were
then treated according to the Arrhenius equation to determine the
energetic parameters of the heat inactivation process. As shown in
 A. Albino et al. / Biochimie 104 (2014) 50e60
Fig. 8. Effect of temperature on the ATPase activity of rPhGshA II. At each indicated
temperature the kinetic parameters of the ATPase were determined essentially as
indicated in the legend to Fig. 4A. In particular, the reaction started with the addition of
ve different concentrations of [g-32P]ATP (specic radioactivity
8.9e157.5 cpm pmol1) within the 11e201 mM interval. Values of Vmax were extrap-
olated from LineweavereBurk plots and reported as a function of temperature. After
conversion of the Vmax into kcat (s1) values, an Arrhenius plot was built and the linear
t of the data was drawn in the 10e28  C interval (inset panel, r2 value 0.98, p-value
lower than 0.0001).
Fig. 9, the Arrhenius plot was linear in the interval of temperature
analysed and the calculated value of Ea (296.9 kJ mol1) was rather
high for a psychrophilic enzyme.
4. Discussion
This work enlarges the information on the enzyme system for
GSH biosynthesis in P. haloplanktis, to investigate the role played by
this cellular thiol in a cold-adapted organism. Under this concern,
the production of this thiol in P. haloplanktis cells, although already
hypothesised by the presence of glutathionylated proteins in a
growing culture of this bacterium [17], was directly demonstrated.
Furthermore, the lower GSH/GSSG ratio found in this psychrophile
with respect to other bacterial sources could be related to the
adaptation of P. haloplanktis at a constant mild oxidant growth
condition. Previous studies described the characterisation of a re-
combinant form of glutathione synthetase (rPhGshB), the enzyme
involved in reaction (2) leading to GSH formation in this psychro-
phile [37,38]; now, also the enzyme activity catalysing reaction (1),
g-glutamyl-cysteine ligase, has been studied in this organism. The
work reports the molecular properties and functioning of rPhGshA
II, a recombinant product from the Ph-gshA II gene located in
chromosome II of P. haloplanktis.
When assessing the best experimental conditions for the pro-
duction of rPhGshA II, some differences were noted with the het-
erologous production of rPhGshB, in spite of the usage of a similar
IPTG-inducible expression system. In particular, growth of the
culture for obtaining rPhGshA II was carried out at 30  C, a lower
temperature compared to that of the rPhGshB production, and no
IPTG induction was required. Furthermore, the presence of the
reducing agent TCEP during purication and storage of rPhGshA II
was essential for obtaining the enzyme in an active form. Indeed, if
TCEP was omitted during the purication procedure, the puried
sample of rPhGshA II was poorly active; however, its activity could
be partially restored upon the addition of a reducing agent, such as
dithiothreitol, in the assay buffer. Finally, in some cases, the
57
Fig. 9. Arrhenius plot of the heat inactivation process of rPhGshA II. The values of the
rate constant of heat inactivation (kin, s1) were obtained from inactivation kinetics
carried out within the 25e41  C interval. To this aim, a 1.0 mM solution of rPhGshA II in
20 mM Tris$HCl, pH 7.8 was incubated at each temperature and at selected times,
aliquots were withdrawn from the incubation and immediately chilled on ice for
30 min. The residual ATPase activity of these enzyme samples was measured kineti-
cally at 15  C in buffer C supplemented with 10 mM L-Glu and 2.5 mM L-Cys essentially
as indicated in the legend to Fig. 3 (specic radioactivity [g-32P]ATP, 1.7 cpm pmol1).
After data analysis according to rst-order kinetics (r2 values ranging between 0.93
and 0.99), the kin values were treated according to the Arrhenius equation (r2 value
0.99, p-value lower than 0.0001).
preparation of the protein sample was complicated by the precip-
itation of the heterologous product either during expression or
purication. By studying the molecular properties of rPhGshA II, it
emerged that the enzyme had the expected size and acted as a
monomer, similarly to what was reported for EcGshA [51]. Differ-
ently from EcGshA, containing nine cysteines with two of them
forming a disulde bridge [52], the active form of rPhGshA II con-
tains three cysteine residues, all present as free thiols. The pair-wise
alignment between the amino acid sequence of PhGshA II and
EcGshA revealed that only two cysteines are conserved between
PhGshA II and EcGshA and none of them corresponded to the res-
idues forming the disulde bridge in EcGshA.
The functional properties of rPhGshA II were studied through the
ATPase reaction coupled to the synthesis of g-Glu-Cys from L-Glu
plus L-Cys (reaction (1)). Indeed, the radioisotopic method chosen
allowed a direct evaluation of the effect of various molecules and
physico-chemical parameters on the psychrophilic enzyme. Con-
cerning the substrate requirement for triggering the ATPase of
rPhGshA II, the results showed that this activity occurred only when
both L-Glu and L-Cys were present in the reaction mixture; however,
the substrate L-Cys could be replaced by L-2-aminobutyric acid, a
nding already reported for EcGshA and the corresponding
mammalian enzyme [21,51]. Concerning the best pH and ionic
conditions for triggering the ATPase activity of rPhGshA II, the
enzyme was full active in a moderately alkaline buffer and required a
divalent cation. In particular, the study revealed the high selectivity
of the psychrophilic enzyme for the divalent cation Mg, added at
high concentration for reaching high levels of activity, because
Mn was essentially unable to replace Mg in the stimulation of
the activity. Therefore, compared to the corresponding enzyme from
other bacteria or eukarya, where a possible Mg/Mn replace-
ment in the activity was observed [28e30], the psychrophilic
rPhGshA II appeared to be more selective for the requirement of
58 A. Albino et al. / Biochimie 104 (2014) 50e60
Fig. 10. Three-dimensional model of rPhGshA II obtained by homology modelling. The
residue Cys 386 located at the protein surface and identied as glutathionylated
following treatment with GSSG is indicated by the red arrow.
Mg. Finally, compared to that reported for rPhGshB [38], the
higher Mg concentration for triggering the rPhGshA II activity
cannot be required for complexing the substrate ATP. The present
results do not exclude that Mg ions could have an effect on
conformation and dynamics of the enzyme.
The natural habitat of P. haloplanktis is the Antarctic coastal sea
water and therefore, this microorganism is adapted to live at tem-
peratures near to 0  C. However, most of the kinetic data in this work
were obtained at 15  C, a temperature commonly used for
measuring and comparing the activity of enzymes from
P. haloplanktis [32,34,36,38], because it represents the optimum
growth temperature of this psychrophile [54]. In any case, rPhGshA II
displayed a well measurable activity even at 10  C, a nding in
accordance with functioning of this enzyme in the specic cold
habitat of P. haloplanktis. In other organisms it was reported that
reaction (1) is the rate-limiting step of the whole process of GSH
biosynthesis [26]. However, this seems not to be the case of
P. haloplanktis, because the catalytic efciency of reaction (1) was
nearly 6-fold higher than that calculated for reaction (2), measured
in the same experimental conditions of temperature and ATP con-
centration [38]. The apparent discrepancy with the other organisms
could be also related to the different method for measuring the ac-
tivity of the corresponding enzymes and/or to the fact that a sub-
optimal g-glutamyl-cysteine ligase activity could be measured in
other organisms when using the substrate L-2-aminobutyrate in
place of L-Cys. The differences could also derive from the specic
ionic requirement for maximum activity of the two enzymes, as for
instance observed for the diverse Mg optimum for rPhGshA II or
rPhGshB. The different afnity of rPhGshA II for its three substrates
Table 2
Energy of activation and related thermodynamic parameters of the enzymes involved in the biosynthesis of glutathione in Pseudoalteromonas haloplanktis.
Process Enzyme Ea (kJ mol1)
ATPase activity rPhGshA II 42.3
rPhGshB 75.0
Heat inactivation rPhGshA II 296.9
rPhGshB 208.0
a
Values calculated at 15  C for both enzymes for the parameters referred to ATPase activity; values calculated at 35  C or 50  C for rPhGshA II or rPhGshB, respectively, for
parameters referred to heat inactivation.
was evaluated through the KM values for ATP, L-Glu and L-Cys. Indeed,
the psychrophilic enzyme had an afnity for L-Glu signicantly
lower than that for L-Cys or ATP, a nding similar to what reported
for the corresponding enzyme from E. coli or kidney rat [22,51].
The study on the inhibitory properties of GSH and GSSG on
rPhGshA II was prompted by previous reports, pointing to the effect
of these glutathione forms on enzymes involved in GSH biosyn-
thesis. Indeed, GSH exerted a feedback inhibition on the activity of
g-glutamyl-cysteine ligase from E. coli or kidney rat [21,27,51]; the
data also suggested the possible involvement of free cysteine res-
idues in the inhibition of the eukaryal enzyme, as well as the pro-
tective role displayed by L-Glu. Concerning the second enzyme
involved in GSH biosynthesis, it was demonstrated that the activity
of glutathione synthetase from E. coli or P. haloplanktis was
inhibited by GSSG [38,53]. Furthermore, in the case of that psy-
chrophilic enzyme, the formation of a mixed disulde bridge with
glutathione was demonstrated [38]. The inhibition prole of the
ATPase activity of rPhGshA II by GSH and GSSG showed that also
this enzyme was inhibited by both glutathione forms. However,
none of them could be considered as a strong inhibitor of rPhGshA
II, and among the two inhibitors, only GSH gave a typical dose-
dependent inhibition prole. An insight in the mechanism of in-
hibition by GSH and GSSG was realised through LineweavereBurk
plots and the results obtained seemed to exclude that these com-
pounds acted as competitive inhibitors of rPhGshA II. In fact, either
GSH or GSSG caused a decrease of the Vmax of the reaction, while
reducing also the KM for ATP or L-Glu. The possible reactivity of
rPhGshA II towards these glutathione forms was considered. Under
this concern, the ESI/Q-TOF mass spectrometry data clearly indi-
cated that GSSG caused the covalent modication of rPhGshA II,
whereas GSH was ineffective. Interestingly, a complete mono-
glutathionylation of the enzyme was obtained after its incubation
with a GSSG concentration, that otherwise caused only a low
inhibitory effect on the enzyme. A similar result was obtained even
when the GSSG treatment was realised in the presence of L-Glu and
L-Cys, thus excluding that these substrates could protect rPhGshA II
from glutathionylation. The MALDI-TOF MS mapping of the cys-
teinyl residue target of the covalent modication allowed its
identication as Cys 386. To have an information on the location of
this residue in the enzyme structure, a three-dimensional model of
rPhGshA II, obtained by homology modelling using EcGshA as a
template, was constructed (Fig. 10). The picture shows that Cys 386
is located within a loop region at the protein surface, thus sup-
porting the higher accessibility of this cysteinyl residue to GSSG.
Furthermore, this residue is located in a region apparently far from
the core of the molecule containing the active site, a nding
explaining the minimum effect on enzyme activity. Considering
that only GSSG covalently modied the enzyme and that the
presence of a mixed disulde caused only a slight reduction of the
catalytic properties of rPhGshA II, the atypical dose-dependent in-
hibition prole by GSSG could be explained by the combination of
two aspects. In the presence of a low GSSG concentration, a dose-
dependent inhibition prole was observed, because the modica-
tion of the enzyme was probably understoichiometric, also
considering the minimum reaction time required for evaluating the
DH*a (kJ mol1) DS*a (J mol1 K1) DG*a (kJ mol1) Reference
39.9 95.4 67.4 This work
72.6 11.3 69.3 [38]
294.3 651.2 93.6 This work
205.3 332.1 98.0 [38]
 A. Albino et al. / Biochimie 104 (2014) 50e60
inhibition; at high GSSG concentration, the covalent modication of
the enzyme was complete and therefore, the dose-dependence was
lost. On the other hand, GSH appeared to be a typical reversible
inhibitor of rPhGshA II, as it gave a normal dose-dependent inhi-
bition prole. Indeed, GSH did not cause the covalent modication
of the enzyme and its inhibitory properties were due to a non co-
valent enzymeeinhibitor interaction, probably regarding a binding
pocket different from the active site of the enzyme.
Cold-adapted microorganisms possess enzymes adapted to
function at low temperatures, a fruitful property for assessing the
possible biotechnological application of these macromolecules. For
instance, enzymes isolated from psychrophilic sources usually
adjust their kinetic parameters to function in the cold, a nding
extremely important for the optimisation of a biotechnological
process and for lowering the related energetic costs [55e57].
Indeed, several studies reported that the cold-adaptation of psy-
chrophilic enzymes could imply an increase of the local exibility in
their catalytic sites and/or a decrease of the overall protein stability
[35,36,58e64]. The studies on the effect of temperature on the
rPhGshA II activity indicated that this enzyme possessed a typical
cold adaptation. Indeed, rPhGshA II was already active at 10  C, and
when studying the thermodependence of its kinetic parameters,
only a modest effect of temperature was observed. Indeed, both the
Vmax and the KM of rPhGshA II moderately increased with temper-
ature between 10 and 28  C and, as a consequence, the catalytic
efciency of the psychrohilic enzyme remained approximately
constant up to 28  C; above this temperature, the enzyme activity
started to decrease, probably for the onset of a heat-inactivation
process. The moderate thermodependence of rPhGshA II was also
proved by the low value of Ea (42.3 kJ mol1) determined for the
enzyme activity. This behaviour is dissimilar from that reported for
rPhGshB, endowed with a signicantly higher value of Ea
(75.0 kJ mol1) [38]. The difference between these enzymes is also
evident from the comparison of their thermodynamic parameters
reported in Table 2. Indeed, the lower enthalpic barrier of the ATPase
catalysed by rPhGshA II was counteracted by a signicantly unfav-
ourable entropic factor compared to that of rPhGshB. On the other
hand, the two enzymes possess similar values of DG*. All these
features make rPhGshA II more similar to a typical psychrophilic
enzyme, usually endowed with a low thermophilic behaviour.
The studies on the heat inactivation of rPhGshA II conrm the
cold-adaptation of this enzyme. Indeed, rPhGshA II was rather
sensitive to a modest heat treatment, because 50% of enzyme ac-
tivity was lost after an incubation of rPhGshA II for 12.6 min at
35  C. Conversely, rPhGshB had a signicantly higher heat resis-
tance, because the activity of this enzyme was halved after an in-
cubation for 11.4 min at 50  C (not shown). These results conrm
that the decrease of enzyme activity observed for rPhGshA II at
temperatures higher than 28  C could be due to the onset of a heat
inactivation process. The low heat resistance of this enzyme was
also investigated through the determination of the energetic pa-
rameters of the heat inactivation process. Surprisingly, the calcu-
lated value of Ea (296.9 kJ mol1) was rather high for a
psychrophilic enzyme, even when compared with the corre-
sponding value of 208.0 kJ mol1 reported for rPhGshB, endowed
with a higher heat resistance [38]. The comparison of the thermal
stability between rPhGshA II and rPhGshB was extended to the
other thermodynamic parameters of the heat inactivation process
(Table 2). In this case, the higher enthalpic barrier of the heat
inactivation process of rPhGshA II compared to that of rPhGshB was
counteracted by a more favourable entropic factor. However, in
spite of the great variations observed in temperature range for
observing heat inactivation, as well as in the corresponding
enthalpic and entropic factors, the two enzymes involved in GSH
biosynthesis of P. haloplanktis possessed similar values of DG*.
59
5. Conclusions
Glutathione biosynthesis occurs through a two-step reaction
system conserved among bacteria and eukarya and usually
requiring two distinct activities, g-glutamyl-cysteine ligase and
glutathione synthetase. In aerobic cold adapted microorganisms,
such as P. haloplanktis, GSH plays a crucial role in the regulation of
growth and survival of the microorganism. Together with the
previous characterisation on glutathione synthetase (rPhGshB), the
present study on a g-glutamyl-cysteine ligase activity from
P. haloplanktis (rPhGshA II) extends the knowledge on the rst
enzyme system leading to GSH biosynthesis in a cold-adapted or-
ganism. The investigation regarded the pH and ionic conditions for
triggering the activity, substrate specicity and afnity, inhibitors,
thermodependence and heat stability. The comparison with the
data related to rPhGshB revealed the different features of the cold-
adaptation of these enzymes. However, P. haloplanktis possesses
another putative g-glutamyl-cysteine ligase activity (PhGshA I) and
it will be interesting to evaluate if the presence of a redundant
activity in a psychrophilic organism is useful for facing different
and specic requirements occurring during the growth under
extreme conditions.
Conict of interest
The Authors declare no conict of interest.
Acknowledgements
This work was nancially supported by grants awarded by MIUR
(PRIN 2009, prot. 2009P2HZZ7_001 to E. De Vendittis; PRIN 2009,
prot. 2009P2HZZ7_002 to M. Masullo; PRIN 2009, prot.
2009P2HZZ7_003 to G. Raimo), Rome, Italy. V. Severino was sup-
ported by a post-doctoral fellowship awarded from the Institute of
Biostructures and Bioimaging, CNR, Naples, Italy.
References
[1] A. Meister, M.E. Anderson, Glutathione, Annu. Rev. Biochem. 52 (1983)
711e760.
[2] R.C. Fahey, G.L. Newton, B. Arrick, T. Overdank-Bogart, S.B. Aley, Entamoeba
histolytica: a eukaryote without glutathione metabolism, Science 224 (1984)
70e72.
[3] M.E. Anderson, Glutathione: an overview of biosynthesis and modulation,
Chem. Biol. Interact. 111e112 (1998) 1e14.
[4] R.C. Fahey, W.C. Brown, W.B. Adams, M.B. Worsham, Occurrence of gluta-
thione in bacteria, J. Bacteriol. 133 (1978) 1126e1129.
[5] R.C. Fahey, R.M. Bushbacher, G.L. Newton, The evolution of glutathione
metabolism in phototrophic microorganisms, J. Mol. Evol. 25 (1987) 81e88.
[6] R.C. Fahey, A.R. Sundquist, Evolution of glutathione metabolism, Adv. Enzy-
mol. Relat. Areas Mol. Biol. 64 (1991) 1e53.
[7] M.J. Penninckx, M.T. Elskens, Metabolism and functions of glutathione in
microorganisms, Adv. Microb. Physiol. 34 (1993) 239e301.
[8] G.L. Newton, K. Arnold, M.S. Price, C. Sherrill, S.B. Delcardayre, Y. Aharonowitz,
G. Cohen, J. Davies, R.C. Fahey, C. Davis, Distribution of thiols in microorgan-
isms: mycothiol is a major thiol in most acinomycetes, J. Bacteriol. 178 (1996)
1990e1995.
[9] L. Masip, K. Veeravalli, G. Georgiou, The many faces of glutathione in bacteria,
Antioxid. Redox Signal. 8 (2006) 753e762.
[10] G.L. Newton, R.C. Fahey, G. Cohen, Y. Aharonowitz, Low-molecular-weight
thiols in streptomycetes and their potential role as antioxidants, J. Bacteriol.
175 (1993) 2734e2742.
[11] I. Dalle Donne, R. Rossi, D. Giustarini, R. Colombo, A. Milzani, S-gluta-
thionylation in protein redox regulation, Free Radical Biol. Med. 43 (2007)
883e898.
[12] I. Dalle Donne, R. Rossi, G. Colombo, D. Giustarini, A. Milzani, Protein S-glu-
tathionylation: a regulatory device from bacteria to humans, Trends Biochem.
Sci. 34 (2009) 85e96.
[13] M.L. Circu, T.Y. Aw, Glutathione and apoptosis, Free Radical Res. 42 (2008)
689e706.
[14] F.V. Pallardo
, J. Markovic, J.L. Garcia, J. Vin
~ a, Role of nuclear glutathione as a
key regulator of cell proliferation, Mol. Asp. Med. 30 (2009) 77e85.
60 A. Albino et al. / Biochimie 104 (2014) 50e60
[15] J.A. Thomas, B. Poland, R. Honzatko, Protein sulfhydryls and their role in the
antioxidant function of protein S-thiolation, Arch. Biochem. Biophys. 319
(1995) 1e9.
[16] P. Klatt, S. Lamas, Regulation of protein function by S-glutathiolation in
response to oxidative and nitrosative stress, Eur. J. Biochem. 267 (2000)
4928e4944.
[17] I. Castellano, M.R. Ruocco, F. Cecere, A. Di Maro, A. Chambery, A. Michniewicz,
G. Parlato, M. Masullo, E. De Vendittis, Glutathionylation of the iron super-
oxide dismutase from the psychrophilic eubacterium Pseudoalteromonas
haloplanktis, Biochim. Biophys. Acta 1784 (2008) 816e826.
[18] B.E. Janowiak, O.W. Grifth, Glutathione synthesis in Streptococcus agalactiae.
One protein accounts for gamma-glutamylcysteine synthetase and gluta-
thione synthetase activities, J. Biol. Chem. 280 (2005) 11829e11839.
[19] S. Gopal, I. Borovok, A. Ofer, M. Yanku, G. Cohen, W. Goebel, J. Kreft,
Y. Aharonowitz, A multidomain fusion protein in Listeria monocytogenes cat-
alyzes the two primary activities for glutathione biosynthesis, J. Bacteriol. 187
(2005) 3839e3847.
[20] B. Vergauwen, D. De Vos, J.J. Van Beeumen, Characterization of the bifunc-
tional g-glutamate-cysteine ligase/glutathione synthetase (GshF) of Pasteur-
ella multocida, J. Biol. Chem. 281 (2006) 4380e4394.
[21] P.G. Richman, A. Meister, Regulation of gamma-glutamyl-cysteine synthetase
by nonallosteric feedback inhibition by glutathione, J. Biol. Chem. 250 (1975)
1422e1426.
[22] G.F. Seelig, A. Meister, Glutathione biosynthesis; gamma-glutamylcysteine
synthetase from rat kidney, Methods Enzymol. 113 (1985) 379e390.
[23] L. Oppenheimer, V.P. Wellner, O.W. Grifth, A. Meister, Glutathione synthe-
tase. Purication from rat kidney and mapping of the substrate binding sites,
J. Biol. Chem. 254 (1979) 5184e5190.
[24] C.S. Huang, W. He, A. Meister, M.E. Anderson, Amino acid sequence of rat
kidney glutathione synthetase, Proc. Natl. Acad. Sci. U. S. A. 92 (1995)
1232e1236.
[25] R.R. Gali, P.G. Board, Sequencing and expression of a cDNA for human gluta-
thione synthetase, Biochem. J. 310 (1995) 353e358.
[26] O.W. Grifth, R.T. Mulcahy, The enzymes of glutathione synthesis: gamma-
glutamylcysteine synthetase, Adv. Enzymol. Relat. Areas Mol. Biol. 73 (1999)
209e267.
[27] S.R. Soltaninassab, K.R. Sekhar, M.J. Meredith, M.L. Freeman, Multi-faceted
regulation of gamma-glutamylcysteine synthetase, J. Cell. Physiol. 182 (2000)
163e170.
[28] J.J. Abbott, J. Pei, J.L. Ford, Y. Qi, V.N. Grishin, L.A. Pitcher, M.A. Phillips,
N.V. Grishin, Structure prediction and active site analysis of the metal binding
determinants in gamma-glutamylcysteine synthetase, J. Biol. Chem. 276
(2001) 42099e42107.
[29] B.S. Kelly, W.E. Antholine, O.W. Grifth, Escherichia coli gamma-
glutamylcysteine synthetase. Two active site metal ions affect substrate and
inhibitor binding, J. Biol. Chem. 277 (2002) 50e58.
[30] M. Orlowski, A. Meister, Isolation of highly puried gamma-glutamylcysteine
synthetase from rat kidney, Biochemistry 10 (1971) 372e380.
[31] C. Medigue, E. Krin, G. Pascal, V. Barbe, A. Bernsel, P.N. Bertin, F. Cheung,
S. Cruveiller, S. D'Amico, A. Duilio, G. Fang, G. Feller, C. Ho, S. Mangenot,
G. Marino, J. Nilsson, E. Parrilli, E.P. Rocha, Z. Rouy, A. Sekowska, M.L. Tutino,
D. Vallenet, G. von Heijne, A. Danchin, Coping with cold: the genome of the
versatile marine Antarctica bacterium Pseudoalteromonas haloplanktis
TAC125, Genome Res. 15 (2005) 1325e1335.
[32] P. Falasca, G. Evangelista, R. Cotugno, S. Marco, M. Masullo, E. De Vendittis,
G. Raimo, Properties of the endogenous components of the thioredoxin sys-
tem in the psychrophilic eubacterium Pseudoalteromonas haloplanktis TAC
125, Extremophiles 16 (2012) 539e552.
[33] H.O. Po rtner, L. Peck, G. Somero, Thermal limits and adaptation in marine
Antarctic ectotherms: and integrative view, Philos. Trans. R. Soc. Lond. B Biol.
Sci. 362 (2007) 2233e2258.
[34] I. Castellano, A. Di Maro, M.R. Ruocco, A. Chambery, A. Parente, M.T. Di Mar-
tino, G. Parlato, M. Masullo, E. De Vendittis, Psychrophilic superoxide dis-
mutase from Pseudoalteromonas haloplanktis: biochemical characterization
and identication of a highly reactive cysteine residue, Biochimie 88 (2006)
1377e1389.
[35] A. Merlino, I. Russo Krauss, I. Castellano, E. De Vendittis, B. Rossi, M. Conte,
A. Vergara, F. Sica, Structure and exibility in cold-adapted iron superoxide
dismutases: the case of the enzyme isolated from Pseudoalteromonas hal-
oplanktis, J. Struct. Biol. 172 (2010) 343e352.
[36] R. Cotugno, M.R. Ruocco, S. Marco, P. Falasca, G. Evangelista, G. Raimo,
A. Chambery, A. Di Maro, M. Masullo, E. De Vendittis, Differential cold-
adaptation among protein components of the thioredoxin system in the
psychrophilic eubacterium Pseudoalteromonas haloplanktis TAC 125, Mol.
BioSyst. 5 (2009) 519e528.
[37] A. Merlino, I. Russo Krauss, A. Albino, A. Pica, A. Vergara, M. Masullo, E. De
Vendittis, F. Sica, Improving protein crystal quality by the without-oil
microbatch method: crystallization and preliminary X-ray diffraction anal-
ysis of glutathione synthetase from Pseudoalteromonas haloplanktis, Int. J. Mol.
Sci. 12 (2011) 6312e6319.
[38] A. Albino, S. Marco, A. Di Maro, A. Chambery, M. Masullo, E. De Vendittis,
Characterization of a cold-adapted glutathione synthetase from the psy-
chrophile Pseudoalteromonas haloplanktis, Mol. BioSyst. 8 (2012)
2405e2414.
[39] M. Masullo, P. Arcari, B. de Paola, A. Parmeggiani, V. Bocchini, Psychrophilic
elongation factor Tu from the antarctic Moraxella sp. Tac II 25: biochemical
characterization and cloning of the encoding gene, Biochemistry 39 (2000)
15531e15539.
[40] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: a Laboratory Manual,
second ed., Cold Spring Harbor Laboratory Press, New York, 1989.
[41] S. Marco, R. Rullo, A. Albino, M. Masullo, E. De Vendittis, M. Amato, The thi-
oredoxin system in the dental caries pathogen Streptococcus mutans and the
food-industry bacterium Streptococcus thermophilus, Biochimie 95 (2013)
2145e2156.
[42] G. Sander, R.C. Marsh, J. Voigt, A. Parmeggiani, A comparative study of the 50S
ribosomal subunit and several 50S subparticles in EF-T- and EF-G-dependent
activities, Biochemistry 14 (1975) 1805e1814.
[43] C.C. White, H. Viernes, C.M. Krejsa, D. Botta, T.J. Kavanagh, Fluorescence-based
microtiter plate assay for glutamate-cysteine ligase activity, Anal. Biochem.
318 (2003) 175e180.
[44] I. Castellano, F. Cecere, A. De Vendittis, R. Cotugno, A. Chambery, A. Di Maro,
A. Michniewicz, G. Parlato, M. Masullo, E.V. Avvedimento, E. De Vendittis,
M.R. Ruocco, Rat mitochondrial manganese superoxide dismutase: amino acid
positions involved in covalent modications, activity, and heat stability, Bio-
polymers 91 (2009) 1215e1226.
[45] M.M. Bradford, A rapid and sensitive method for the quantitation of micro-
gram quantities of protein utilizing the principle of protein-dye binding, Anal.
Biochem. 72 (1976) 248e254.
[46] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head
of bacteriophage T4, Nature 227 (1970) 680e685.
[47] R. Dosi, A. Di Maro, A. Chambery, G. Colonna, S. Costantini, G. Geraci,
A. Parente, Characterization and kinetics studies of water buffalo (Bubalus
bubalis) myoglobin, Comp. Biochem. Physiol. B Biochem. Mol. Biol. 145 (2006)
230e238.
[48] T.E. Creighton, Disulphide Bonds Between Cysteine Residues, in:
T.E. Creighton (Ed.), Protein Structure A Practical Approach, Oxford University
Press, Oxford, UK, 1989, pp. 155e167.
[49] M. Gelzo, G. Granato, F. Albano, A. Arcucci, A. Dello Russo, E. De Vendittis,
M.R. Rocco, G. Corso, Evaluation of cytotoxic effects of 7-dehydrocholesterol
on melanoma cells, Free Radical Biol. Med. 70 (2014) 129e140.
[50] A. Roy, A. Kucukural, Y. Zhang, I-TASSER: a unied platform for automated
protein structure and function prediction, Nat. Protoc. 5 (2010) 725e738.
[51] C.S. Huang, W.R. Moore, A. Meister, On the active site thiol of g-gluta-
mylcisteine synthetase: relationships to catalysis, inhibition, and regulation,
Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 2464e2468.
[52] T. Hibi, H. Nii, T. Nakatsu, A. Kimura, H. Kato, J. Hiratake, J. Oda, Crystal
structure of gamma-glutamylcysteine synthetase: insights into the mecha-
nism of catalysis by a key enzyme for glutathione homeostasis, Proc. Natl.
Acad. Sci. U. S. A. 101 (2004) 15052e15057.
[53] H. Gushima, T. Miya, K. Murata, A. Kimura, Purication and characterization of
glutathione synthetase from Escherichia coli B, J. Appl. Biochem. 5 (1983)
210e218.
[54] M.M. Corsaro, R. Lanzetta, E. Parrilli, M. Parrilli, M.L. Tutino, S. Ummarino,
Inuence of growth temperature on lipid and phosphate contents of surface
polysaccharides from the antarctic bacterium Pseudoalteromonas haloplanktis
TAC 125, J. Bacteriol. 186 (2004) 29e34.
[55] R. Papa, E. Parrilli, F. Sannino, G. Barbato, M.L. Tutino, M. Artini, L. Selan, Anti-
biolm activity of the Antarctic marine bacterium Pseudoalteromonas hal-
oplanktis TAC125, Res. Microbiol. 164 (2013) 450e456.
[56] M. Giuliani, E. Parrilli, C. Pezzella, V. Rippa, A. Duilio, G. Marino, M.L. Tutino,
A novel strategy for the construction of genomic mutants of the Antarctic
bacterium Pseudoalteromonas haloplanktis TAC125, Methods Mol. Biol. 824
(2012) 219e233.
[57] E. Parrilli, R. Papa, M.L. Tutino, G. Sannia, Engineering of a psychrophilic
bacterium for the bioremediation of aromatic compounds, Bioeng. Bugs 1
(2010) 213e216.
[58] G. Feller, C. Gerday, Psychrophilic enzymes: hot topics in cold adaptation, Nat.
Rev. Microbiol. 1 (2003) 200e208.
[59] S. D'Amico, T. Collins, J.C. Marx, G. Feller, C. Gerday, Psychrophilic microor-
ganisms: challenges for life, EMBO Rep. 7 (2006) 385e389.
[60] K.S. Siddiqui, R. Cavicchioli, Cold-adapted enzymes, Annu. Rev. Biochem. 75
(2006) 403e433.
[61] E. De Vendittis, I. Castellano, R. Cotugno, M.R. Rocco, G. Raimo, M. Masullo,
Adaptation of model proteins from cold to hot environments involves
continuous and small adjustments of average parameters related to amino
acid composition, J. Theor. Biol. 250 (2008) 156e171.
[62] I. Ruggiero, G. Raimo, M. Palma, P. Arcari, M. Masullo, Molecular and func-
tional properties of the psychrophilic elongation factor G from the Antarctic
eubacterium Pseudoalteromonas haloplanktis TAC 125, Extremophiles 11
(2007) 699e709.
[63] G. Evangelista, P. Falasca, I. Ruggiero, M. Masullo, G. Raimo, Molecular and
functional characterization of polynucleotide phosphorylase from the Ant-
arctic eubacterium Pseudoalteromonas haloplanktis, Protein Pept. Lett. 16
(2009) 999e1005.
[64] A. Merlino, I. Russo Krauss, I. Castellano, M.R. Ruocco, A. Capasso, E. De Ven-
dittis, B. Rossi, F. Sica, Structural and denaturation studies of two mutants of a
cold adapted superoxide dismutase point to the importance of electrostatic
interactions in protein stability, Biochim. Biophys. Acta 1844 (2014) 632e640.