Engineering of helicobacter pylori lasparaginase: Characterization of two functionally distinct groups of mutants e0117025

Article abstract of DOI:10.1371/journal.pone.0117025

Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively). Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy.

Full text of DOI:10.1371/journal.pone.0117025

RESEARCH ARTICLE
Engineering of Helicobacter pylori L-
Asparaginase: Characterization of Two
Functionally Distinct Groups of Mutants
Maristella Maggi1,3‡, Laurent R. Chiarelli2‡, Giovanna Valentini3, Claudia Scotti1*
1 Department of Molecular Medicine, Unit of Immunology and General Pathology, University of Pavia, Pavia,
Italy, 2 Department of Biology and Biotechnologies “Lazzaro Spallanzani”, Laboratory of Molecular
Microbiology, University of Pavia, Pavia, Italy, 3 Department of Biology and Biotechnologies “Lazzaro
Spallanzani”, Laboratory of Protein Biochemistry, University of Pavia, Pavia, Italy
‡ These authors contributed equally to this work.
* claudia.scotti@unipv.it
Abstract
Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though
presenting, along with their therapeutic efficacy, several side effects due to their bacterial
origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II
L-asparaginase possesses interesting features, among which a reduced catalytic efficiency
for L-GLN, compared to the drugs presently used in therapy. In the present study, we de-
scribe some enzyme variants with catalytic and in vitro cytotoxic activities different from the
wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) de-
plete the enzyme of both its catalytic activities, once more underlining the essential role of
such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs
L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly,
this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single
mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respec-
tively). Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and
halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cyto-
toxic effect comparable to, or higher than, the one of the wild type enzyme when similar as-
paraginase units were used. These findings may be relevant to determine the role of
glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed
light on how to engineer the best asparaginase/glutaminase combination for an ever im-
proved, patients-tailored therapy.
OPEN ACCESS
Citation: Maggi M, Chiarelli LR, Valentini G, Scotti C
(2015) Engineering of Helicobacter pylori L-
Asparaginase: Characterization of Two Functionally
Distinct Groups of Mutants. PLoS ONE 10(2):
e0117025. doi:10.1371/journal.pone.0117025
Academic Editor: Ashley M. Buckle, Monash
University, AUSTRALIA
Received: August 19, 2014
Accepted: December 18, 2014
Published: February 9, 2015
Copyright: © 2015 Maggi et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: MM’s salary comes from an Istituto
Universitario di Studi Superiori fellowship. The
salaries for LRC, GV and CS come from Ministero
dell ’ Istruzione, Universitàe Ricerca. The funds of
Regione Lombardia Ricerca.tissimi prize was used to
buy the reagents needed for this work. The funders
had no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript.
Introduction
L-asparaginases (L-ASNase) are amidohydrolases (EC 3.5.1.1) that primarily catalyze
L-asparagine (L-ASN) deamination leading to the formation of L-aspartate (L-ASP) and am-
monia. L-ASNases also exhibit the capability of carrying out L-glutamine (L-GLN) hydrolysis,
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Helicobacter pylori L-Asparaginase Mutants
Competing Interests: The authors have declared
that no competing interests exist.
although the efficiency toward this substrate greatly depends on the enzyme source [1]. On the
basis of their affinity toward L-ASN, cellular localization and glutaminolytic activity, bacterial
L-ASNases are divided into two groups. Type I L-ASNases are cytoplasmatic enzymes, consti-
tutively expressed, present low affinity vs L-ASN (in the order of mM) and are also active to-
ward L-GLN. Type II L-ASNases are periplasmic, have anaerobiosis-dependent expression,
display high affinity vs L-ASN (in the order of μM) and low to negligible activity toward
L-GLN [2].
For over 4 decades Echerichia coli and Erwinia chrysantemi type II L-ASNases (EcAII and
ErA, respectively) have been used in the treatment of acute lymphoblastic leukaemia (ALL),
above all in childhood [3, 4, 5, 6]. Their therapeutic efficacy can be primarily attributed to the
reduction of serum L-ASN, which is essential for survival of L-asparagine synthetase (ASNS)
knock-out (or down regulated) lymphoblastic tumoral cells [7, 8]. Systemic depletion of extra-
cellular L-ASN prevents protein biosynthesis in malignant lymphoblasts which ultimately can
result in tumor cell cycle inhibition or apoptosis [9].
Among the cytotoxic agents employed in regimens for acute lymphoblastic leukemia (ALL),
L-ASNase II is one of the most effective and with relatively mild side effects [10]. Nevertheless,
some of the latter, such as hypersensitivity, coagulative disorders, impaired liver function, pan-
creatitis and neurological dysfunctions hamper their clinical application [11]. Apart from
hypersensitivity, all other side effects are most likely linked to the secondary L-glutaminase
(L-GLNase) activity of the enzyme [12, 13, 14, 15]. In fact, massive depletion of L-GLN, the
major transport form of amino nitrogen in the blood and donor of amino group for many bio-
synthetic reactions, can be responsible for loss of cellular functions. Several studies, however,
show that this activity participates in inducing the cytotoxic effect of L-ASNase, most of all in
ASNS-positive tumoral cells [12, 16, 17]. This suggests that leukemia cells environment and
their genetic profile must be considered in order to obtain an effective treatment strategy [18]
and that enzymes with variable activity and affinity toward L-GLN are expected to become
more and more useful to devise finely patient-tailored treatments [19].
Helicobacter pylori CCUG 17874 type II L-asparaginase (HpASNase) displays a strong pref-
erence for L-ASN over L-GLN, good heat stability, and, most of all, high cytotoxicity [20, 21].
Interestingly, the higher S0.5 toward L-GLN exhibited by HpASNase with respect to the en-
zymes used as drugs from E. coli and E. chrisantemi could help to reduce the side effect mani-
festations linked to excessive L-GLN depletion in blood, especially when L-GLNase activity is
not essential [19]. In general, bacterial L-ASNases exhibit a hyperbolic response toward sub-
strates, except two enzymes [2, 22, 23], E. coli type I and HpASNase, which exhibit cooperativ-
ity vs L-ASN [23] and L-GLN [21], respectively. The basis of these enzymatic features are still
to be clarified and must be searched in the structure of the molecule.
Bacterial L-asparaginases are 140–150 kDa homotetramers more accurately described as di-
mers of intimate dimers, each defined by A/C and B/D monomeric interactions. Each mono-
mer is organized into two well distinct domains, a large N-terminal domain and a smaller
C-terminal domain, connected by a linker region [24]. Four independent catalytic sites are lo-
cated at the intersubunits interface of the intimate dimers with amino acid residues contributed
by both monomers [24, 25].
The precise reaction mechanism of bacterial L-ASNses is still unknown, although it has
been compared to the two-step ping-pong mechanism of classic serine proteases [25], with the
involvement of two catalytic triads (Thr12-Tyr25-Glu283, and Thr89-Asp90-Lys162, respec-
tively, in E. coli type II L-ASNase), and the formation of an acylenzyme as intermediate. More-
over, it implies the motion of the so-called flexible loop (EcAII residues 13–40) for correct
arrangement of the active site and substrate allocation [24, 25, 26].
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Helicobacter pylori L-Asparaginase Mutants
In order to explore the role of specific residues in the shaping of the catalytic site, site-directed
mutants were expressed in recombinant form and characterized from the biochemical point of
view. The most relevant ones were also tested for cell cytotoxicity and a serendipitous, double
mutant was helpful in defining a critical region able to modulate GLNase activity.
Materials and Methods
Rationale for site direct mutagenesis
The rational engineering of HpASNase was performed starting from structural and catalytic
data available from other bacterial L-ASNases, mainly from the crystallographic structure of a
H. pylori L-ASNase (HpA, PDB ID: 2WT4, 2WLT [25]) which displays 92% of sequence identi-
ty with H. pylori strain CCUG 178174. Two groups of mutants, namely, catalytic triads mu-
tants (T16D, T95E) and substrates binding and stabilization mutants (Q63E, M121C/T169M,
M121C, T169M), were planned (Table 1).
Generation, expression and purification of HpASNase mutants
In order to obtain enzymatic variants of H. pylori CCUG 17874 L-ASNase, the pDC1 plasmid,
a derivative of pET101 D-TOPO (Invitrogen) containing the ansB wild type gene [21] was sub-
jected to site direct mutagenesis using Quick Change XL Site-directed Mutagenesis Kit
(Stratagene).
The sense and antisense mutagenic oligonucleotides used are reported in Table 2. All the
mutants were checked by insert sequencing. E. coli BL21(DE3) pLysS cells transformed with
pDC1 derivatives were grown in a ZYP-5052 medium [27] containing 100 μg/ml ampicillin
and protein expression performed by auto-induction. For enzyme purification, cells were col-
lected by centrifugation from a 1 l culture, resuspended in 50 mM Na-phosphate pH 8.0, 300
mM NaCl, 10 mM imidazole (ImOH) (buffer A), added with 1 mM PMSF, sonicated and cen-
trifuged (8000 g, 20 min, 4°C). After an additional centrifugation at 150,000 g (30 min, 4°C),
the supernatant was applied to a HisTrap 5 ml column (GE Healthcare), equilibrated in buffer
A, and protein elution was performed by an ImOH discontinuous gradient (25, 50, 100, 250,
500 mM). HpASNase eluted at 250 mM ImOH. The fractions containing HpASNase were
pooled together, concentrated and applied onto a HiLoad-Superdex S200 16/60 column (GE
Healthcare) equilibrated in Na-phosphate 50 mM, pH 7.5 (buffer B). After elution, the frac-
tions were checked by 12% SDS-PAGE and those containing a single band of 37 kDa were
pooled together. Protein concentration was determined according to Lowry et al. [28].
Table 1. Site directed mutants.
Residue
number
Original amino
acid
Amino acid
replacement
Role in the wild type enzyme
Aim
16
63
T
D
E
Catalysis and substrates binding
Substrate accommodation
Active site study
Q
Increase affinity to L-ASN; reduction of
L-GLNase activity
95
T
E
Catalysis and substrates binding
L-GLN binding
Active site study
121
169
M
T
C
M
Removal of L-GLNase activity
Random mutation*
Unclear, maybe involved in intersubunits
interactions
* Note: this is a serendipitous mutation introduced by PCR during the generation of the M121C mutant. Both the M121C/T169M double mutant and the
single mutants were characterized.
doi:10.1371/journal.pone.0117025.t001
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Helicobacter pylori L-Asparaginase Mutants
Table 2. Sense and antisense oligonucleotides used for site direct mutagenesis.
Mutations
Oligonucleotides
T16E
Sense
50-GACAGGGGGGGAGATTGCAGGGAGTG-30
Anti-sense
Sense
50-CTGTCCCCCCCTCTAACGTCCCTCAC-30
Q63E
T95E
50-GGTTTCTAATATCGGCTCAGAAGACATGAATGAAG-30
50-CCAAAGATTATAGCCGAGTCTTCTGTACTTACTTC-30
50-GTCATCACGCATGGCGATGACACTTTAGAAGAG-30
50-CAGTAGTGCGTACCGCTACTGTGAAATCTTCTC-30
50-CTAGTGGGAGCGTGTCGTAACGCTGCTTC-30
50-GAAGCAGCGTTACGACACGCTCCCACTAG-30
50-GAGAGGTAGTTAAAATGCACACCACCCACAC-30
50-GTGTGGGTGGTGTGCATTTTAACTACCTCTC-30
Anti-sense
Sense
Anti-sense
Sense
M121C
T169M
Anti-sense
Sense
Anti-sense
The underlined and bold letters indicate the mutated bases.
doi:10.1371/journal.pone.0117025.t002
Circular dichromism
Far-UV circular dichroism (CD) measurements were carried out using a Jasco J-700 spectropo-
larimeter (Jasco-Europe, Cremella, Italia) with a 1 mm path cell. Scans were performed be-
tween 190 and 250 nm at a speed of 20 nm min-1 with a spectral band of 2 nm and a sensitivity
of 20 millidegrees. Measurements were performed in 10 mM Na-phosphate pH 7.5 at 25°C.
The protein concentration was 5 μM. The Dichroweb platform (http://dichroweb.cryst.bbk.ac.
uk/html/home.shtml) was used for data analysis obtained from at least 10 different scans.
Activity assays and kinetic analyses
Both L-ASNase and L-GLNase activities were determined at 37°C with L-ASN or L-GLN, re-
spectively, as a substrate, by the L-glutamic dehydrogenase-coupled spectrophotometric assay
[29], in the same conditions adopted for the wild type enzyme [21]. Briefly, the standard reac-
tion mixture contained 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)
buffer, pH 7.5, 1 mM α-ketoglutarate (α-KG), 0.24 mM NADH, 20 U L-glutamic dehydroge-
nase and 5 mM L-ASN or 150 mM L-GLN, in a final volume of 0.5 ml. The reaction was started
by adding the enzyme solution (5–10 μg) and NADH consumption was monitored at 340 nm
with a Jasco V-550 UV/VIS spectrophotometer (Jasco-Europe).
For steady-state kinetic studies, enzyme activity was assayed in triplicate under the above
conditions using at least 10 different L-ASN or L-GLN concentrations (0–10 mM L-ASN or
0–400 mM L-GLN). The reaction was started adding the substrate to the reaction mixture con-
taining 5–15 μg enzyme. The kinetic parameters Vmax and Km were determined by Lineweaver-
Burk plot, while S0.5 and nH values were determined by Hill plot using, in all cases, the Enzyme
Kinetic Module 1.1 for Sigma Plot (SPSS Inc.).
Kcat, or turn-over number, is the number of catalytic events per second. Km is the substrate
concentration at which the reaction rate is half maximal. S0.5 is the Km equivalent value for a co-
operative enzyme. kcat/Km is an index of enzyme catalytic efficiency at sub-saturating substrate
concentrations. Hill coefficient (nH) is an empirical parameter related to the degree of coopera-
tivity; values larger than unity indicate positive cooperativity among ligand binding sites.
Thermal stability assay
T50 is the temperature at which an enzyme loses 50% of its activity in a given period of time. To
evaluate the T50 of HpASNase variants, enzyme solutions (0.5 mg ml-1) were incubated at
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Helicobacter pylori L-Asparaginase Mutants
different temperatures for 10 min in 50 mM Na-phosphate, pH 7.5. After incubation, the en-
zyme solutions were chilled on ice and assayed for activity. The residual activity was expressed
as the percentage of activity of heat-treated enzyme with respect to the untreated one.
Cell culture and viability assay
Human promyeloblast cells (HL-60) were grown at 37°C in RPMI-1640 medium containing
100 IU ml-1 penicillin and 100 μg ml-1 streptomycin and added with 10% FBS and 2 mM
L-GLN in a 5% CO2 humidified atmosphere. For the viability tests, 50000 cells/well were seed-
ed in a 96-well plate and incubated at 37°C for 24 h in the presence of different concentrations
(0.03–3.3- U ml-1) of purified recombinant H. pylori wild type, T16D, Q63E and M121C/
T169M L-ASNase variants in phosphate buffered saline (Sigma Aldrich). As for the wild type
and Q63E variants, the viability vs L-GLNase activity (0.03–3.8 U ml-1) was also determined.
Each treatment was performed at least three times in different days and in triplicate wells for
each condition.
Cell survival was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) reduction assay. The viability value was expressed as a percentage of living cells vs
non treated sample and the values obtained at different enzyme concentrations were compared
by ANOVA and Tukey test. A p-value<0.05 was considered significant. For the dose-response
analysis as EC50 value, the data were plotted using the four parameter logistic function. The
EC50 value indicates L-ASNase concentration inducing 50% of the maximal cytotoxic effect.
Results
Cloning, expression and purification of HpASNase mutants
Four mutants (T16D, T95E, Q63E, M121C) of HpASNase were initially designed and generat-
ed starting from the cloned ansB gene by site-direct mutagenesis. In the case of the insert
planned to encode the M121C mutant, an additional PCR-derived mutation at nucleotide posi-
tion 506 C>T lead to a T169M substitution in the protein molecule. After a preliminary bio-
chemical characterization of the resulting M121C/T169M enzyme, the T169M and M121C
variants were also generated.
All the mutants (T16D, T95E, Q63E, M121C, T169M and M121C/ T169M) were expressed
in E. coli BL21(DE3) pLysS cells and purified to homogeneity (Fig. 1) following the procedure
reported in the Material and Methods section.
Kinetic properties
The mutants were subjected to kinetic analysis using either L-ASN or L-GLN as a substrate.
At least ten different concentrations of L-ASN or L-GLN were used for each kinetics (Fig. 2A
and B).
All of them, except T16D and T95E, resulted to be active toward L-ASN, although to a dif-
ferent extent (Fig. 2A and Table 3). Circular dichroism and analytical gel filtration experiments
indicated that also these inactive mutants were properly folded and correctly arranged as tetra-
mers (data not shown).
Only the Q63E mutant preserved the hyperbolic kinetic vs. L-ASN of the wild type enzyme;
the M121C, M121C/T169M and T169M mutants showed a slightly cooperative kinetic toward
L-ASN (nH, 1.5, 1.7 and 1.8, respectively).
The double mutant was the less affected in catalytic activity (kcat, nearly 70% with respect to
that of the wild type) and its catalytic efficiency was even comparable to that of the wild type
enzyme (kcat/S0.5, 60.73 s-1 mM-1 vs 66.4 s-1 mM-1, respectively), owing to a slightly higher
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Helicobacter pylori L-Asparaginase Mutants
Figure 1. Example of SDS-PAGE of the purified HpASNase double mutant. The enzyme was run in
parallel with molecular mass standards on a 12% gel and stained with Coomassie Blue R-250. Lane 1:
molecular mass markers (97, 66, 45, 31, 21.3 and 14.4 kDa). Lanes 2 and 5: empty lanes. Lane 3 and 4:
250 mM ImOH Nichel fractions. Lane 6: gel filtration, pooled fractions.
doi:10.1371/journal.pone.0117025.g001
Figure 2. Activity dependence on substrate concentrations. Steady state kinetics of recombinant HpASNase wild type and variants as a function of
L-ASN (panel A) and as a function of L-GLN (panel B). All experiments were performed at 37°C as reported in the Materials and Methods section. Symbols:
black circle: wild type; black triangle pointing up: T16D; black square: T95E; diamond: Q63E; empty square: M121C; empty circle: T169M; black triangle
pointing down: M121C/T169M (double mutant).
doi:10.1371/journal.pone.0117025.g002
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Helicobacter pylori L-Asparaginase Mutants
Table 3. Apparent kinetic constants of HpASNase wild type and mutant forms.
L-asparagine
L-glutamine
Vmax (U mg-1) kcat (s-1)
S0.5 (mM) kcat/S0.5 (s-1 mM-1) nH
Vmax (U
mg-1)
k
cat (s-1)
S0.5 (mM)
kcat/S0.5
nH
(s-1 mM-1)
wild type
T16D
31.22 0.90
19.26 0.56 0.29 0.03 66.4
1.0 0.06 35.8 2.25
22.10 1.39 46.4 4.02
0.47 0.10 2.0 0.10
-
-
-
-
-
-
-
-
-
-
Q63E
10.58 0.39
-
6.53 0.24
-
0.10 0.01 65.3
1.0 0.05 23.57 1.72 14.5 0.84
76.45 3.67 0.19 1.39
2.3 0.16
T95E
-
-
-
-
-
-
-
-
-
-
-
-
-
M121C/
T169M
22.6 0.28
13.95 0.17 0.23
0.007
60.73
1.5 0.09
M121C
T169M
8.23 0.13
4.83 0.08
7.40 0.22
0.25 0.02 30.20
0.25 0.02 29.6
1.7 0.21
1.8 0.20
-
-
-
-
-
-
-
-
-
-
11.99 0.36
Results are means ( SE) for 3 determinations from at least 3 protein preparations.
doi:10.1371/journal.pone.0117025.t003
affinity toward L-ASN (S0.5 for M121C/T169M: 0.23 vs wt: 0.29 mM). The M121C and T169M
mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme), but the
Q63E mutant, in spite of its 3-fold lower catalytic rate (kcat for Q63E: 6.53 sec-1 vs wild type:
19.26 sec-1), displayed a catalytic efficiency superimposable to that of the wild type enzyme,
thanks to its 3-fold higher affinity (S0.5 for Q63E: 0.10 mM vs wild type: 0.29 mM).
As for L-GLN, only the Q63E mutant showed the capability to hydrolyze this substrate.
Moreover, it had a cooperative kinetic like the wild type enzyme (nH, 2.3 vs. 2.0 of the wild
type, Fig. 2B and Table 3), though with a 2.5 times reduced catalytic efficiency.
Figure 3. Protein stability of recombinant HpASNase wild type and variants. Plots of the residual
activities at 10 min incubation vs. temperature. The enzyme solutions were subjected to heat inactivation in a
given range of temperatures. Residual activities are expressed as a percentage of initial activity. Black circle:
wild type; diamond: Q63E; empty square: M121C; empty circle: T169M; black triangle pointing down: M121C/
T169M (double mutant).
doi:10.1371/journal.pone.0117025.g003
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Helicobacter pylori L-Asparaginase Mutants
Table 4. Thermal stability of recombinant HpASNase wild type and variants.
T50 (°C)
wild type
T16D
53
-
Q63E
56
-
T95E
M121C/T169M
M121C
T169M
61
60
58
doi:10.1371/journal.pone.0117025.t004
Protein stability
Protein stability was evaluated incubating the variants in a wide range of temperatures (40–70°
C) within 10 min and calculating T50 values after evaluation of their residual activities, as re-
ported in the Materials and Methods section. All the enzymes endowed with L-ASNase activity
resulted to be very thermostable (Fig. 3) with T50 values 3–8°C higher than the wild type en-
zyme (Table 4). Particularly, the M121C/T169M double mutant showed a T50 value 7°C higher
than the wild type (61°C vs. 53°C, respectively). This high thermostability would be helpful for
both industrial and therapeutic purposes.
L-asparaginases cytotoxicity
The cytotoxic effect of variable concentrations of the wild type, T16D, Q63E and M121C/
T169M L-asparaginases on the human promyeloid HL-60 cells is reported in Fig. 4A and B
and Table 4. For the mutant enzymes, the protein dosage was scaled to match the wild type ac-
tivity (U ml-1) both toward L-ASN and L-GLN. In the case of the inactive T16D mutant, the
amount of protein as mg ml-1 was considered. Mutants M121C and T169M were not tested on
cells because they showed no GLNase activity and half the ASNase catalytic efficiency of the
wild-type and were therefore considered not very informative.
The wild type enzyme halved the viability of the cells when used at 1.58 U ml-1 (Table 5).
The T16D enzyme, devoid of any catalytic activity, did not exhibit any cytotoxic effect. The
double mutant, devoid of GLNase activity, exhibited a negligible and non-ASNase dose-depen-
dent cytotoxic effect, which was not significant if compared to the inactive T16D mutant
(p>0.05). In fact, viability reduction was significant only if compared with the negative control
(cells treated in the absence of enzyme, p>0.05). The Q63E mutant, the only enzyme exhibiting
both L-ANSase and L-GLNase activities, although partly reduced, showed a cytotoxic effect
comparable with or higher than the wild type enzyme if the same concentration (U ml-1) of
ASNase was used (EC50; Q63E:, 1.37 U ml-1 vs wild type: 1.58 U ml-1, Fig. 4A and Table 5).
When the treatment was performed with similar U ml-1 of GLNase activity, Q63E showed a re-
duced cytotoxic effect (EC50 wt: 1.58 U ml-1 and Q63E: 1.73 U ml-1, Table 5).
Discussion
The rational engineering of six HpASNases here described included two groups of mutants,
namely, catalytic triads mutants (T16D, T95E) and substrates binding and stabilization mu-
tants (Q63E, M121C/T169M, M121C, T169M) (Fig. 5).
The T16D and T95E replacements, which have never been experimented before in ASNase,
were performed to verify the role of HpASNase threonines in substrate binding and catalysis.
L-ASNases carry out a ping-pong like catalysis mechanism that involves transitory and
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Helicobacter pylori L-Asparaginase Mutants
Figure 4. Cell respiration inhibition on HL-60 by wild type HpASNase and its variants as a function of different enzyme units. Panel A: correlation
between asparaginase activity and cell respiration inhibition. Inset: low asparaginase activity concentration respiration inhibition. Panel B: correlation between
glutaminase activity and cell respiration inhibition. Symbols: black circle: wild type; black triangle pointing up: T16D; diamond: Q63E; black triangle pointing
down: M121C/T169M (double mutant). Empty symbols indicate significance vs the negative control (p<0.05). * indicate significance (p<0.05) between values
for wild type and Q63E mutant. Panel C: L-glutamine consumption analysis for the wild type (straight black line) and Q63E mutant (straight blue line) using U of
glutaminase activity (mmol l-1 min-1) corresponding to the protein amount used for analysis at 3.3 U ml-1 of asparaginase activity. Ammonia production is as well
reported in the figure (wild type: dashed black line; Q63E: dashed blue line). Analysis was performed using the COPASI 4.12 software. The time course
analysis was done according to the Hill cooperativity function and using the following kinetic parameters: wild type: rate 3.81 mmol l-1 min-1, S0.5 46.4 mM, nH 2;
Q63E: rate 7.35 mmol l-1 min-1, S0.5 76.45 mM, nH 2.3. Initial glutamine concentration: 2 mM.
doi:10.1371/journal.pone.0117025.g004
sequential nucleophilic attacks resolved in the release of ammonia and aspartic or glutamic
acid. The two threonines of the triads (Thr12 and Thr89 in EcAII and the corresponding
Thr16 and Thr95 in HpASNase) and several water molecules are reported to be involved in
this mechanism [24].
Crystallographic data show that Thr16 acquires hydrogen bonds through its OG1 atom, ex-
clusively from elements of the side chain of the substrate (CA, CB, CG, OD1, OD2), while the
Thr95 OG1 interacts also with the O of the carboxylic group in α of the main chain [25]. We
replaced Thr16 with an Asp residue to increase the charge in the active site region maintaining
the steric occupancy, thus possibly increasing the affinity of the enzyme for the L-ASN
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Helicobacter pylori L-Asparaginase Mutants
Table 5. EC50 values for HpASNase wild type and variants (T16D, Q63E, M121C/T169M).
EC50 (U ml-1)
Asparaginase activity
Glutaminase activity
wild type
T16D
1.58
ND
1.58
ND
Q63E
1.37
ND
1.73
ND
M121C/T169M
ND: value not detectable. In the case of the T16D and M121C/T169M the data points did not fit the four
parameter logistic curve.
doi:10.1371/journal.pone.0117025.t005
substrate. Replacement of Thr95 with a Glu, which led to the extension of the side chain of the
residue, had the purpose of reducing the active site space thus blocking L-GLN accommodation
and allowing, at the same time, the interaction of the L-ASN main chain with the Glu carboxyl-
ic function. Each replacement on both triad threonines (Thr16 and Thr95), however, resulted
to be deleterious for enzyme activity, which is consistent with the characteristics of a previously
reported EcAII T89V mutant [30] and with their expected essential role in catalysis [24, 25].
Regarding the second group of mutants, amino acid replacement concerned the Gln63 resi-
due that, with Asn255 and Glu289, is located around the active site and is involved in substrate
stabilization and in modulating the accessibility of the binding pocket [25]. Specifically, Gln63
makes hydrogen interactions (OE1) with the α-NH3+ of the substrate (L-ASN or L-GLN). Its
replacement with a Glu leads to the introduction of a carboxylic group at position 63 of the
polypeptide chain, resulting in a locally increased negative charge, which should allow a better
interaction with L-ASN as a substrate. In fact, the characterization of the Q63E mutant con-
firmed the residue role in both substrates binding and catalysis. The variant had a catalytic effi-
ciency (kcat/S0.5: 65.3 s-1 mM-1) vs L-ASN similar to the wt enzyme (kcat/S0.5, 66.4 s-1 mM-1),
with a reduction of catalytic activity (nearly 3-fold), but increased affinity (nearly 3-fold), and
halved glutaminase efficiency (kcat/S0.5: 0.19 vs0.47 s-1 mM-1) due to the reduction of both cata-
lytic activity and affinity. Several mutants on the corresponding EcAII residue (Q59) have been
characterized to have perturbed ASNase and GLNase activities [16, 19].
In order to discuss the M121C mutant, we need to consider that, beside the flexible loop of
the active site (HpASNase residues 19–46), at least two other highly conserved loops play a key
role in active site stabilization: a left-handed crossover β4-β5 loop (HpASNase residues 119–
153) and the active site forming β13–14 loop (HpASNase residues 286–297) [25]. Residue
M121 is highly conserved in the structure of over 500 bacterial L-ASNases [31] and plays two
main roles: stabilization of the left-handed cross-over loop on the active site and creation of a
hydrophobic environment for the stabilization of catalytic Thr95. M121 is one of the few resi-
dues that differentially interacts with L-ASN and L-GLN [32]. Indeed, it creates hydrogen
bonds with the L-GLN NE through the sulphur atom of its side chain but does not interact
with L-ASN. The M121C replacement introduces a shortened hydrophilic residue that should
lose the capability of interacting with L-GLN, leaving the affinity vs L-ASN unchanged.
During the mutagenesis experiments to produce the M121C mutant, an unwanted, addi-
tional random mutation was introduced, leading to the formation of the M121C/T169M mu-
tant, namely a double mutant. Very little is known about the Thr169 residue, though the
corresponding EcAII residue (Thr163) is reported to be involved in intersubunits interactions
[4, 23]. To further investigate the role of T169, we also built the single T169M mutant. In the
structure of the HpASNase monomer, Thr169 is involved in hydrogen bonds with the O of the
PLOS ONE | DOI:10.1371/journal.pone.0117025 February 9, 2015
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Helicobacter pylori L-Asparaginase Mutants
Figure 5. Residues involved in substrate binding/stabilization, and/or catalysis. L-ASN is reported as a
ball and stick model. Red/tan residues belong to the A chain, blue residues belong to the C chain. Red and
blue residues belong to the catalytic triads. Elements from each subunity of an intimate dimer participate to
substrate binding/stabilization and catalysis. Met121 is located nearby the catalytic Thr95 residue and
participate to create the hydrophobic environment necessary for Thr95 activation; Gln63 (subunity A)
stabilize the catalytic Glu289 (subunity C) of the close monomer.
doi:10.1371/journal.pone.0117025.g005
Thr176 and with two water molecules. None of these structural elements seem to be directly in-
volved in enzyme catalysis.
Replacements of either Met121, or Thr169, or both (Met121/Thr169) killed the enzyme
GLNase activity leaving the affinity toward L-ASN unchanged. Interestingly, in all the three
cases mutants displayed positive cooperativity vs L-ASN, underlining alterations within en-
zyme subunits interactions.
Both mutants carrying each single mutation (M121C or T169M) had a reduced ASNase ac-
tivity, but the double mutant had an activity quite similar to the wild type enzyme. Its preserved
activity toward L-ASN most likely depends on a complementary action of the two mutations
affecting residues located in two critical regions of the enzyme structure: residue Met121, near
the active site region, and Thr169, in the intersubunit one (Fig. 6). The distance of Thr169
from the active site suggests the possibility to interfere with the enzyme activity even tackling
regions seemingly unrelated to the catalytic mechanism.
The cytotoxic effect of the T16D, Q63E and M121C/T169M mutants was assayed on HL-60
cells. The inactive T16D mutant did not exert any cytotoxic effect, proving that the anti-
proliferative efficacy of HpASNase is related only to its enzymatic activity and is independent
from unknown features of the protein or from the recombinant nature of the enzyme carrying
the N-terminal His-tag. The double mutant, completely devoid of GLNase activity, had a very
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Helicobacter pylori L-Asparaginase Mutants
Figure 6. Residue M121 and T169 localization. L-ASN, Met121 and Thr169 are represented as spheres;
the subunits A (red) and C (light blue) are represented as ribbons. Thr169 locates in a loop shared by
intersubunits regions and, in E. coli, it resulted to interact with residues of the near subunit in the intimate
dimer. Residue Met121 locates far from the Thr169 residue. Both residues do not seem to interact with
each other.
doi:10.1371/journal.pone.0117025.g006
low cytotoxic effect that was not dependent on the enzyme dose, an effect confirmed by Trypan
blue exclusion in slightly different conditions [33].
Previous work on EcAII [16] has shown that GLNase activity is essential for cell cytotoxicity
only when ASNS is expressed. In the model proposed by the Authors, L-GLN, one of the sub-
strates of ASNS, is removed by the secondary GLNase activity of ASNase, thus contributing to
enhance cytotoxicity. HL-60 cells are known to express ASNS [34], thus explaining the effects
we observed at comparable units of ASNase. In the absence of GLNase activity (M121C/
T169M mutant), the effect on cell viability was similar to the one exerted by the T16D mutant,
devoid of any activity. In the presence of intermediate GLNase units (wild-type), cytotoxicity
became relevant. It is interesting to note that, doubling GLNase units (Q63E mutant) com-
pared to the wild-type, the further gain in cytotoxicity is only about 10% and this, because of
the subsaturating L-GLN concentrations in which the experiments were performed (2 mM, as
initially added to the culture medium), is probably related not to the lower affinity of Q63E for
L-GLN with respect to the wild-type, but to its roughly halved GLNase efficiency (kcat/S0.5 vs
L-GLN, wt: 0.47 and Q63E, 0.19). In fact, mathematical modelling by COPASI of the relative
GLNase activity for the wild type and Q63E mutant, respectively, at similar ASNase units
(Fig. 4C) shows both a slightly quicker consumption of L-GLN and accumulation of ammoni-
um in the mutant.
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Helicobacter pylori L-Asparaginase Mutants
In EcAII mutants [16], affinity for GLNase has been engineered to remove its side effects
and this is particularly relevant for ASNS negative leukemia clones. On the other hand, for leu-
kemia clones expressing ASNS, modulation of GLNase catalytic efficiency could be relevant to
optimize its therapeutic index.
All in all, the data obtained from our cytotoxicity tests revealed that GLNase activity is abso-
lutely needed for the HpASNase anti-proliferative effect on HL-60 cells and that its modulation
can contribute to reach the enzyme maximal cytotoxic effect.
Conclusions
The biochemical characterization of the six variants of HpASNase, whose proper folding and
quaternary structure were systematically verified, showed that substitution of the catalytic tri-
ads residues completely impairs the enzyme capability to carry out the reaction, underlining
their essential role. On the other hand, substitutions in the region of substrates binding and sta-
bilization resulted in a reduction of the catalytic rate toward L-ASN in all cases except one
(M121C/T169M), and in a reduction (Q63E) or complete removal of the glutaminolytic activi-
ty, indicating that co-participation of other structural elements besides the triads elements is
relevant for the catalytic cycle.
The production of ASNase variants with reduced or null glutaminolytic activity has allowed
to verify the importance of GLNase activity in the cytotoxicity exterted on HL-60 cells, con-
firming that modulation of the GLNase kinetic parameters of ASNase is a relevant issue to con-
sider in order to optimize the outcome of its therapeutic applications.
Supporting Information
S1 Fig. Wild type and T16D, Q63E, T95E, M121C, T169M, M121C/T169M mutants amino
acids alignment. The amino acids sequences derived from each mutant nucleotide sequencing
are aligned to the one of the wild type enzyme. The wild type derived secondary structure is as
well reported. α-helices, 3 -helices and π-helices are displayed as medium, small and large
squiggles respectively. β-strands are rendered as arrows, strict β-turns as TT letters and strict
α-turns as TTT. (ESPript -http://espript.ibcp.fr). Robert, X. and Gouet, P. (2014) “Deciphering
key features in protein structures with the new ENDscript server”. Nucl. Acids Res. 42(W1),
W320-W324. doi: 10.1093/nar/gku316 (freely accessible online).
(TIF)
Acknowledgments
We are grateful to Vassilios Avramis and Steven Mittelman for useful discussion during
data collection.
Author Contributions
Conceived and designed the experiments: MM LRC GV CS. Performed the experiments: MM
LRC. Analyzed the data: MM LRC GV CS. Contributed reagents/materials/analysis tools: GV
LRC GV CS. Wrote the paper: MM LRC GV CS.
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