γ-Glutamyl transpeptidase architecture: Effect of extra sequence deletion on autoprocessing, structure and stability of the protein from Bacillus licheniformis

Article abstract of DOI:10.1016/j.bbapap.2014.09.001

γ-Glutamyl transpeptidases (γ-GTs, EC 2.3.2.2) are a class of ubiquitous enzymes which initiate the cleavage of extracellular glutathione (γ-Glu-Cys-Gly, GSH) into its constituent glutamate, cysteine, and glycine and catalyze the transfer of its γ-glutamy

Full text of DOI:10.1016/j.bbapap.2014.09.001

Biochimica et Biophysica Acta 1844 (2014) 2290–2297
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Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbapap
γ-Glutamyl transpeptidase architecture: Effect of extra sequence
deletion on autoprocessing, structure and stability of the protein from
Bacillus licheniformis
b,c,
Meng-Chun Chi a, Yi-Hui Lo a, Yi-Yu Chen a, Long-Liu Lin a, , Antonello Merlino
⁎
⁎
a
Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 600, Taiwan
b
Department of Chemical Sciences, University of Naples Federico II, Napoli, Italy
c
Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 July 2014
Received in revised form 30 August 2014
Accepted 2 September 2014
Available online 10 September 2014
γ-Glutamyl transpeptidases (γ-GTs, EC 2.3.2.2) are a class of ubiquitous enzymes which initiate the cleavage of
extracellular glutathione (γ-Glu-Cys-Gly, GSH) into its constituent glutamate, cysteine, and glycine and catalyze
the transfer of its γ-glutamyl group to water (hydrolysis), amino acids or small peptides (transpeptidation).
These proteins utilize a conserved Thr residue to process their chains into a large and a small subunit that then
form the catalytically competent enzyme. Multiple sequence alignments have shown that some bacterial
γ-GTs, including that from Bacillus licheniformis (BlGT), possess an extra sequence at the C-terminal tail of the
large subunit, whose role is unknown. Here, autoprocessing, structure, catalytic activity and stability against
both temperature and the chemical denaturant guanidinium hydrochloride of six BlGT extra-sequence deletion
mutants have been characterized by SDS-PAGE, circular dichroism, intrinsic fluorescence and homology
modeling. Data suggest that the extra sequence has a crucial role in enzyme activation and structural stability.
Our results assist in the development of a structure-based interpretation of the autoprocessing reaction of
γ-GTs and are helpful to unveil the molecular bases of their structural stability.
Keywords:
Protein stability
γ-Glutamyl transpeptidase
Ntn hydrolase
Site-directed mutagenesis
Deletion mutants
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
problems [7,8], hypertension [9], diabetes [10] and cancer [11,12]. hGT
inhibition is a strategy under development for treatment of various tu-
γ-GT catalyzes the transfer of GSH γ-glutamyl group and related γ-
glutamyl amides to water (hydrolysis) or to amino acids and peptides
(transpeptidation), according to the following scheme [1–4]:
mors [13]. It has been shown that inhibiting γ-GT prior to chemothera-
py or radiation would sensitize γ-GT-positive tumors to treatment [14]
and that inhibition of γ-GT activity blocked cisplatin-induced nephro-
toxicity in mice [15]. Clinical inhibitors of hGT may be therapeutic also
for other diseases, for example for asthma [16].
γ-GluCONHR + H2O = Glu + NH2R (hydrolysis)
In bacteria, γ-GTs are found in the cytosol or in the periplasmic space
and can be heterodimeric [1,2,17] or heterotetrameric [18,19]. Bacterial
γ-GTs have attracted much interest because of their numerous biotech-
nological applications [1,2,20]; indeed, these proteins can be used as
cephalosporin acylases [21] and as glutaminases [22] and can be in-
volved in the enzymatic production of theanine [23], glutamyl phenyl
hydrazine analogs [24] and various pharmacological agents [2,20].
Both mammalian and microbial proteins utilize a conserved Thr
residue to process its chain into a large and a small subunit that then
assemble to form a catalytically competent (generally heterodimeric)
enzyme [25–28]. Although it has been shown that precursor protein is
more stable than the mature enzyme towards both temperature and
chemical denaturation [29,30] and that it can be found in vivo, a physi-
ological role for the precursor is not known.
γ-GluCONHR + R1NH2 = γ-GluCONHR1 + RNH2 (transpeptidation).
In humans, the expression of γ-GT (hGT) is restricted predominantly
to the apical surface of ducts and glands where fluids leave the body [5].
The highest concentration of hGT is in the kidney tubules [6], where it
prevents excretion of GSH into the urine by cleaving GSH present
in the glomerular filtrate. Aberrant expression and localization of
hGT are associated with many diseases, including cardiovascular
⁎
Correspondence to: A. Merlino, Department of Chemical Sciences, University of Naples
Federico II, Complesso Universitario di Monte Sant' Angelo, Via Cintia, I-80126 Napoli,
Italy. Tel.: +39 081674276; fax: +39 081674090. L.-L. Lin, Department of Applied
Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 600, Taiwan. Tel:
+886 52717969; fax: +886 52717901.
E-mail addresses: llin@mail.ncyu.edu.tw (L.-L. Lin), antonello.merlino@unina.it
(A. Merlino).
Multiple sequence alignments have shown that γ-GTs share some fea-
tures: the catalytic Thr, which is responsible for both the autoprocessing
and enzymatic activity, and a Gly-Gly dyad, which is involved in the
http://dx.doi.org/10.1016/j.bbapap.2014.09.001
1570-9639/© 2014 Elsevier B.V. All rights reserved.

M.-C. Chi et al. / Biochimica et Biophysica Acta 1844 (2014) 2290–2297
2291
stabilization of the intermediate that forms during the catalytic process
(Fig. S1, A and B) [1,27,28]. On the other hand, some relevant differences
have been also underlined. Mammalian γ-GTs are extensively glycosylat-
ed [31–33], whereas many microbial γ-GTs lack the lid loop [18,19,34],
the region which extends over the active site, and possess an extra
sequence at the C-terminal tail of the large subunit [35,36] (Fig. 1A).
Here we have investigated the way in which the protein from
Bacillus licheniformis (BlGT) responds to change in the length of the
extra sequence. The autoprocessing, structure, stability and catalytic
activity of six BlGT deletion mutants (Fig. 1B) have been studied.
purified by affinity chromatography with a Ni2+-NTA agarose column
under native conditions. The eluted fractions (total volume for each
preparation = ~5 mL) were pooled and dialyzed overnight against
500 mL of 50 mM Tris–HCl buffer (pH 8.0) through a 10-kDa cutoff
membrane to remove salts. Protein purity was assessed by Coomassie
Blue staining of SDS–polyacrylamide gels. Protein concentrations were
determined by the Bio-Rad protein assay reagent using bovine serum
albumin as a standard.
2.2. Autoprocessing of the enzymes
2. Materials and methods
Autocatalytic processing studies on BlGT and deletion mutants
(1 mg mL−1) were performed by incubating the proteins in 50 mM
Tris–HCl buffer (pH 8.0) at 4 °C up to 56 days. Aliquots were denatured
by boiling in SDS loading buffer for 5 min. Samples were analyzed by
SDS-PAGE 12%; the gel was stained using Coomassie Brilliant Blue
R-250. The degree of autocatalytic processing was evaluated by measur-
ing the intensity of the protein bands in the gels with a computerized
densitometer using CP ATCAS 2.0 program (http://www.lazarsoftware.
com).
2.1. Site-directed mutagenesis and protein analyses
A previously constructed plasmid pQE-BlGGT [35], which contains a
1.758-kb insert of B. licheniformis genomic DNA with the entire BlGT
gene, was used as a template for site-directed mutagenesis. Two over-
lapping complementary primers with the desired nucleotide changes
were designed for each deletion (Table S1). Six deletion mutants
(ΔY385–I387, ΔY385–V388, ΔY385–P391, ΔY385–K394, ΔY385–I396
and ΔY385–E398) were constructed by a QuikChange II site-directed
mutagenesis kit (Stratagene) according to the manufacturer's instruc-
tions. Clones of the recombinant plasmids that had the desired muta-
tions were identified by DNA sequencing with the dideoxynucleotide
chain termination method.
2.3. Enzyme activity assay and determination of kinetic parameters
The enzyme activity of BlGT and its deletion mutants towards
substrate analog L-γ-glutamyl-p-nitroanilide (GpNA) was determined
by measuring the amount of p-nitroaniline (pNA) released from GpNA
using the method assay by Tate and Meister (1974) [37]. Briefly,
the concentration of pNA was measured spectrophotometrically
BlGT and mutant enzymes were over-expressed in recombinant
Escherichia coli M15 cells as described previously [36]. Proteins were
Fig. 1. (A) Multiple sequence alignment of bacterial γ-GTs. The amino acid sequence of BlGT was aligned with those of B. pumilus, B. subtilis, B. amyloliquefaciens, Thiobacillus denitrificans,
E. coli, Labrenzia aggregate, Helicobacter pylori, Pseudomonas aeruginosa, and Geobacillus thermodenitrificans. The conserved catalytic Thr responsible for autoprocessing of the enzyme is
marked with a star and the extra sequence evidenced. (B) Particular of the sequence of the BlGT deletion mutants that have been analyzed here.

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M.-C. Chi et al. / Biochimica et Biophysica Acta 1844 (2014) 2290–2297
following the adsorption spectrum at 412 nm. Catalytic activity was also
measured at different temperatures and as a function of pH, using the
following buffers: 50 mM acetate–HCl buffer (pH 3–6), 50 mM Tris–
HCl buffer (pH 7–9) and 50 mM glycine–NaOH buffer (pH 10–12).
To evaluate kinetic constants, dependence of the reaction on the
substrate concentration was evaluated using fixed amounts of purified
BlGT (or of its deletion mutants) incubated with increasing concentra-
tions of GpNA from 1 to 1000 μM. Values of KM and kcat were determined
from the non-linear least squares fit of the initial velocity data to the
Michaelis–Menten equation.
and calculate the root mean square deviations. The figures have been
done with Pymol [42].
3. Results and discussion
3.1. Autoprocessing and structure of BlGT variants
The γ-GTs structurally characterized until now share the same
folding pattern. Their overall structure mainly consists of four layers of
α-helices and β-sheets (αββα motif) assembled in a kidney shape
(Fig. 2). The main structural differences between the diverse γ-GTs are
located on the large subunit, on surface loops and at the subunit termi-
nal ends. An interesting feature of some bacterial γ-GTs is the presence
of an extra sequence on the C-terminal tail of the large subunit (residues
Y385–E398 in the protein from B. licheniformis) (Fig. S1). The X-ray
structure of BlGT, recently solved [40], suggests that in this enzyme
the extra sequence could play an important role in both autoprocessing
and enzymatic activity. In particular, the different flexibility of this
region in the presence of the substrate highlighted by the comparison
between the structure of ligand-free protein and that of its complex
with L-Glu has suggested that this region could play a key role in
catalysis [40].
In order to verify these hypotheses, autoprocessing, structure, stabil-
ity and catalytic activity of six new BlGT deletion mutants (ΔY385–I387,
ΔY385–V388, ΔY385–P391, ΔY385–K394, ΔY385–I396, and ΔY385–
E398) have been investigated. The monitoring of the autocatalytic pro-
cessing of these BlGT variants has been carried out by incubating the
proteins at 4 °C in 50 mM Tris–HCl buffer (pH 8.0) for about 2 months.
Aliquots of the deletion mutants have been withdrawn six times in the
first two weeks and then once a week up to 56 days and analyzed by
SDS-PAGE (Fig. 3A). With the notable exception of ΔY385–E398,
which mainly remains as precursor protein even after two months, all
the variants are able to autoprocess themselves, leading to the forma-
tion of a small and a large subunit with masses of about 41 kDa and
22 kDa, respectively, in about two weeks. However, the ΔY385–P391,
2.4. Circular dichroism (CD) studies
Far-UV CD spectra of the protein (0.2 mg mL−1) in 50 mM Tris–HCl
buffer, pH 8.0, were measured at 25 °C using a JASCO J-815 spectropolar-
imeter equipped with a Peltier block arrangement (PTC-423S/15), a
quartz cuvette of 1 cm path length, and a spectral band pass of 4 nm.
Raw ellipticity data were converted to mean residue ellipticity using
the formula [θ] = [θraw × 100 × MRW] / c × l, where MRW is the mean
residue weight for BlGT, c is the concentration of BlGT in mg mL−1 and
l is the path length in cm. Thermal unfolding curves were registered by
monitoring the changes in mean residue ellipticity (at 222 nm) as a
function of temperature, for BlGT and its deletion mutants, using a scan
rate of 2 °C/min. Deconvolution of CD spectra for secondary structure
amount has been performed using CDNN software [38].
2.5. Fluorescence spectroscopy
The fluorescence emission spectra for BlGT and its deletion mutants
were recorded at 25 °C using a Jasco FP-6500 spectrophotometer.
Spectra were registered between 310 and 450 nm, using a protein con-
centration of 0.05 mg mL−1 in 50 mM Tris–HCl buffer (pH 8.0), upon
excitation using λexc = 280 nm or λexc = 295 nm. The chemical
unfolding was evaluated by measuring the emission upon mixing
1 mL guanidinium hydrochloride (GdnHCl) (from 1 to 6 M) with 1 mL
of protein (0.05 mg mL−1). Fluorescence was recorded at 25 °C with
protein concentration of 0.05 mg mL−1 and a final concentration of
0.5–3.0 M GdnHCl in 50 mM Tris–HCl buffer (pH 8.0) using 1 cm quartz
cell. The two-state unfolding model (Eq. (1)) was used to calculate the
thermodynamic parameters [39].
ꢀ
ꢁ
h
ꢀ
ꢁ.
i
2O
yN þ mf ½Dꢀ þ ðyU þ mu½DꢀÞ ꢁ exp − ΔGHN−U−m½Dꢀ RT
h
ꢀ
ꢁ.
i
yobs
¼
: ð1Þ
1 þ exp − ΔGHN−2 U−m½Dꢀ RT
O
Then, [GdnHCl]0.5,N − U can be determined from the equation below:
ΔGHN−2 U
m
O
½GdnHClꢀ0:5;N−U
¼
ð2Þ
where yobs represents the observed biophysical signal, yN and yU
represent the intercepts, mf and mu represent the slopes of the pre-
and post-transition baselines, T is the temperature, R is the universal
O
gas constant, [D] is the concentration of GdnHCl, ΔGHN−2 U represents
the free energy change for the unfolding process, and m is a measure
of the dependence of ΔG on GdnHCl concentration.
2.6. Molecular modeling
The models of the precursor and mature forms of the BlGT deletion
mutants were obtained using the structures of the T393A variant of
E. coli γ-GT [26] and of the mature form of BlGT [40] as starting models
and the SWISS-MODEL server (http://swissmodel.expasy.org/). The
program DeepView-Swiss-PdbViewer [41] was used to build the assem-
bly of the heterodimeric mature enzymes, which was assumed to be
similar to that of mature BlGT, to minimize the energy of the structures
Fig. 2. Ribbon diagram of the structure of BlGT in complex with L-Glu [40]. L-Glu atoms
are shown as gray spheres. Small and large subunits are colored in yellow and cyan,
respectively. Extra sequence is colored in red.

M.-C. Chi et al. / Biochimica et Biophysica Acta 1844 (2014) 2290–2297
2293
Fig. 3. Autoprocessing and formation of active heterodimeric mature form of BlGT and its deletion mutants evidenced by Coomassie stained SDS-PAGE (A) and following the specific
activities (B) as a function of time. In the SDS-PAGE, the following legend has been used: 1. BlGT; 2. ΔY385–I387; 3. ΔY385–V388; 4. ΔY385–P391; 5. ΔY385–K394; 6. ΔY385–I396;
7. ΔY385–E398. Specific activity was determined by monitoring catalysis of GpNA. Each point represents the mean of at least three independent measurements. Error bars represent
one standard deviation of uncertainty, calculated on the basis of three experimental measurements.
ΔY385–K394, ΔY385–I396 mutants process themselves more slowly
than the other variants. This indicates that the deletion of residues
389–391 (Pro89, Gln390 and Pro391) somewhat affects the enzyme
autoprocessing, whereas the whole extra sequence is needed for its
proper maturation. In this respect, it should be interesting to note that
Gln390 and Pro391 are conserved in BlGT, BpGT, BsGT and BaGT,
which are the only γ-GTs with an extra sequence constituted of four-
teen residues (Fig. 1A). To study the autoprocessing of the BlGT deletion
mutants in more detail, the percentage of processing has been evaluated
for each sample, as reported in the Materials and methods section, and
plotted as a function of time (Fig. S2). Similar results have been obtained
following the time evolution of the specific activity (Fig. 3B). In this case,
it is interesting to note the unusual behavior of the Y385–I387 mutant
which shows a dramatic increase in the activity between 7 and
14 days, when it is almost completely processed (Fig. S2).
Nevertheless, altogether these data further support the idea that
deletion of residues Y385–E398 significantly affects the autoprocessing
of the protein.
Since the differences in the rate of autoprocessing observed for the
BlGT variants could be linked to conformational variations induced by
the deletion, the structure of the six variants has been monitored by an-
alyzing far-UV CD (Fig. 4A) and fluorescence spectra (Fig. 4B) collected
at 25 °C. CD spectra of the deletion mutants are almost superimposable
to that of BlGT, indicating that the deletions have no significant effect on

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M.-C. Chi et al. / Biochimica et Biophysica Acta 1844 (2014) 2290–2297
residues Y385–P391, Y385–K394 and Y385–I396 significantly increases
the accessibility of the active site region (Fig. S3C). These findings could
well explain the catalytic changes observed for these mutants (see
below). More fine perturbations of the structure and dynamics of the
protein must be invoked to explain the significant changes in
the catalytic activity observed for the ΔY385–I387 and ΔY385–V388
mutants (see below).
3.2. Catalytic activity and structural stability of BlGT deletion mutants
Specific activities of the BlGT variants towards the synthetic
substrate GpNA have been also studied in comparison to that of BlGT.
The results of these experiments are reported in Table 1. All deletion
variants exhibit a specific activity significantly altered when compared
to BlGT. ΔY385–E398 is inactive, but this is not surprising since it mainly
exists as an unprocessed inactive enzyme. ΔY385–P391, ΔY385–K394
and ΔY385–I396 are slightly less active than BlGT, whereas ΔY385–
I387 and ΔY385–V388 are the most active variants and are even more
active than the wild-type protein. These findings suggest that the
deletions introduce minor structural or dynamic changes that affect
the catalysis. The relative activity of the deletion mutants has been
also characterized as a function of pH and of temperature (Fig. 5A and
B, respectively). All the mutants, like BlGT, present their maximum
activity at pH 9.0. Optimum temperature of the mutants is between 60
and 72 °C.
The conformational stability of the deletion variants against both
temperature and presence of guanidinium hydrochloride (GdnHCl)
has been also analyzed by collecting CD spectra as a function of temper-
ature (Fig. 6A) and fluorescence spectra at increasing concentration of
GdnHCl (Fig. 6B), respectively. Apparent melting temperature (Tm)
and the values of denaturant concentration at half-completion of
the transition characterizing the chemical-induced denaturation (C1/2
)
of each analyzed system have been reported in Tables 2 and 3,
respectively.
As it clearly emerges from Fig. 6A which shows the temperature-
dependence of loss of secondary structure content in BlGT and its
mutants, the protein deletion mutants show unfolding curves with
different midpoints (Tm values). In particular, the Tm is between 45.3
and 47.9 °C for ΔY385–P391, ΔY385–K394, ΔY385–I396 and ΔY385–
E398 whereas it is N60 °C for ΔY385–I387 and ΔY385–V388 and BlGT.
Evidently the deletion of the Pro389, Gln390, and Pro391 has resulted
in a significant lowering of BlGT apparent melting temperature. These
results are in line with those collected measuring the relative activity
of the protein as a function of temperature (Fig. S4). The results obtain-
ed by denaturation experiments carried out in the presence of GdnHCl
are only partly in line with those found denaturing the proteins by
Fig. 4. (A) Far-UV CD spectra of BlGT and its deletion mutants (concentration 0.2 mg mL−1
in 50 mM Trsi–HCl buffer, pH 8.0, recorded at 25 °C. The proteins exhibit a typical α-helical
CD profile with minimal peaks at 208 nm and 222 nm. (B) Intrinsic fluorescence spectra of
BlGT and its deletion mutants (concentration 0.05 mg mL−1) in 50 mM Tris–HCl buffer,
pH 8.0, recorded at 25 °C. Emission fluorescence λmax for ΔY385–I387, ΔY385–V388,
ΔY385–P391, ΔY385–K394, ΔY385–I396, and ΔY385–E398 is reported in Table S3. Spectra
have been registered using an excitation wavelength of 280 nm.
)
the secondary structure of the protein. Deconvolution of the spectra
reveals that the variants have similar secondary structure content,
i.e. 35–40% of alpha helices and 15–20% of beta strands, in close agree-
ment with the X-ray structure of BlGT [40] (Table S2). The comparison
of intrinsic fluorescence spectra of the deletion variants reveals that
there are just minor shifts in λmax (Table S3), indicating that Trp residues
are similarly exposed to the solvent in BlGT and its deletion mutants.
Overall these data suggest that the deletions do not elicit major
structural changes on the overall structure of BlGT. To further investi-
gate the consequences of the extra sequence cuttings on the structure
of BlGT, the three-dimensional models of both precursor and mature
form of the six protein variants have been built, using the X-ray struc-
tures of the precursor-like T393A variant of E. coli γ-GT [26] and of the
mature form of BlGT [40] as templates (Fig. S3). As expected, the overall
structures of precursors are very similar to each other (Cα root mean
square deviations calculated between the model of ΔY385–I387 and
the other five models are in the range 0.44–0.51 Å). The main structural
differences are located in the loop regions and in the deletion
sites, which adopt different conformations in the various mutants
(Fig. S3A). Similarly, the structures of the mature form present signifi-
cant variations in the deletion regions (Fig. S3B). In this case, the
comparison between the structures suggests that the deletion of
Table 1
Specific activity and kinetic parameters of the purified BlGT and deletion mutant
enzymesa.
Enzyme
KM (mM)
kcat (s−1
)
kcat/KM (mM−1 s−1
)
BlGT
0.37
0.81
0.57
0.76
0.74
1.06
–
0.06b
12.45
55.11
24.41
4.74
3.01
2.09
–
1.23b
2.74
1.46
0.41
0.28
0.27
27.22b
67.92
43.03
6.2
4.09
1.97
–
ΔY385–I387
ΔY385–V388
ΔY385–P391
ΔY385–K394
ΔY385–I396
ΔY385–E398
0.07
0.04
0.05
0.05
0.06
a
The kinetic parameters were determined for BlGT and the deletion mutants exactly in
the same experimental conditions and maturation process time. The parameters were
determined from the initial reaction rates at 25 °C and at pH 8.0, in the presence of
1–1000 μM GpNA, as previously described. KM and kcat are the averages of three
determinations
standard deviations.
b
We have recalculated the values of KM and kcat also for BlGT in order to perform a more
meaningful comparison with the deletion mutants. These values do not match completely,
but they are in general agreement, with those that we have previously reported [40].
The small differences in the parameters are probably due to small differences in the
maturation process of different batch of BlGT preparations.

M.-C. Chi et al. / Biochimica et Biophysica Acta 1844 (2014) 2290–2297
2295
Fig. 5. (A) Relative activity of BlGT and its deletion mutants evaluated as a function of pH
(A) and temperature (B). The activity was expressed as a percentage of the highest activity
measured over the pH and temperature range examined. Each point represents the mean
of at least three independent measurements. Error bars represent one standard deviation
of uncertainty.
Fig. 6. (A) Thermal unfolding of BlGT and its mutants as followed by CD spectroscopy at
222 nm. (B) GdnHCl-induced denaturation of BlGT and its mutants as followed by
monitoring the average emission wavelength at increasing concentrations of guanidine
hydrochloride.
thermal unfolding: BlGT, ΔY385–I387, ΔY385–V388 and ΔY385–P391
show a C1/2 between 2.6 and 2.9 M, whereas the ΔY385–K394,
ΔY385–I396 and ΔY385–E398 variants unfold at 2.0 M, 1.0 M and
1.1 M, respectively (Fig. 6B). Such results suggest that the chemical de-
naturation of BlGT deletion mutants follows a mechanism different from
that used by thermal unfolding, as observed in other protein systems
(see for example [43–45]), including members of γ-GT superfamily
[29,30]. In the present case, deletion of Pro389, Gln390, and Pro391
does not significantly alter the protein stability against the denaturant,
whereas the removal of residues 392–398 has an important role in
this process. In this respect, it is interesting to note that this protein re-
gion contains three charged residues (Glu392, Asp396, and Lys397)
which probably contribute to the chemical stability of BlGT against
GdnHCl, in agreement with the well-accepted idea that charged resi-
dues play a role in the protein stability against GdnHCl [45,46].
B. licheniformis. Data reveal that deletion of residues Y385–E398 leaves
the protein folding unaffected, but hampers the precursor to process it-
self in the large and small subunits. Deletion of Y385–I387 results in a
large increase in the specific activity of BlGT. Removal of Pro389,
Gln390, and Pro391 results in a significant lowering of BlGT apparent
melting temperature, whereas removal of chain segment constituted
by residues 392–398, where three charge residues are located
(Glu392, Asp396, and Lys397), significantly alters the structural stability
Table 2
Summary of the thermal stability of BlGT and its deletion mutants.
Enzyme
Tm (°C)
ΔHm
ΔSm
ΔG (25 °C)
(kcal mol−1
)
(cal mol−1 K−1
)
(kcal mol−1 K−1
)
BlGT
64.7
0.5a
1.0
2.3
0.7
1.0
79.8
54.0
37.5
62.3
63.2
12.6 236.1
11.9 160.4
12.1 111.9
13.4 195.9
19.8 197.4
29.4 337.3
25.9 302.9
3.0
3.9
5.8
4.5
9.6
12.5
10.8
3.7
2.7
2.1
1.4
1.4
3.5
2.2
ΔY385–I387 63.6
ΔY385–V388 61.8
ΔY385–P391 45.3
ΔY385–K394 47.0
ΔY385–I396 46.8
ΔY385–E398 47.9
4. Conclusions
Despite the extensive interest in the study of γ-GTs, there is a limited
knowledge on the role of specific regions of their sequence on the
structure and stability of these proteins. In particular, there is still no
paper characterizing the role of the extra sequence on the maturation
process of bacterial γ-GTs. Here, we have reported the results of cutting
experiments of the extra sequence of the protein from the bacterium
0.6 107.9
0.6 97.2
a
We have re-measured the thermal stability of BlGT in order to perform a more
meaningful comparison with the deletion mutants. The experiments have been per-
formed exactly in the same conditions for BlGT and the deletion mutants (0.2 mg mL−1
protein in 50 mM Tris–HCl buffer, pH 8.0).

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[14] J.B. King, M.B. West, P.F. Cook, M.H. Hanigan, A novel, species-specific class of un-
Table 3
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Values of denaturant concentration at half-completion of the transition
(C1/2) characterizing the chemical-induced denaturation of BlGT and its
mutants.
Enzyme
[GdnHCl]0.5,N − U (M)
BlGT
2.6
2.9
2.9
2.6
2.0
2.0
1.1
0.2a
0.1
0.1
0.1
0.1
0.1
0.1
ΔY385–I387
ΔY385–V388
ΔY385–P391
ΔY385–K394
ΔY385–I396
ΔY385–E398
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a
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Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
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This work was supported by a research grant (NSC-100-2313-B-
415-003-MY3) from the National Science Council of Taiwan.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.bbapap.2014.09.001.
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