Low resolution X-ray structure of γ-glutamyltranspeptidase from Bacillus licheniformis: Opened active site cleft and a cluster of acid residues potentially involved in the recognition of a metal ion

Article abstract of DOI:10.1016/j.bbapap.2014.04.016

γ-Glutamyltranspeptidases (γ-GTs) cleave the γ-glutamyl amide bond of glutathione and transfer the released γ-glutamyl group to water (hydrolysis) or acceptor amino acids (transpeptidation). These ubiquitous enzymes play a key role in the biosynthesis and

Full text of DOI:10.1016/j.bbapap.2014.04.016

BBAPAP-39345; No. of pages: 7; 4C: 3, 4
Biochimica et Biophysica Acta xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
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1
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Low resolution X-ray structure of γ-glutamyltranspeptidase from Bacillus
licheniformis: Opened active site cleft and a cluster of acid residues
potentially involved in the recognition of a metal ion
4Q1 Long-Liu Lin a, Yi-Yu Chen a, Meng-Chun Chi a, Antonello Merlino b,c,
⁎
a
5
6
7
Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 600, Taiwan
Department of Chemical Sciences, University of Naples Federico II, Napoli, Italy
Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy
b
c
OOF
8
a r t i c l e i n f o
a b s t r a c t
9
Article history:
γ-Glutamyltranspeptidases (γ-GTs) cleave the γ-glutamyl amide bond of glutathione and transfer the released 22
γ-glutamyl group to water (hydrolysis) or acceptor amino acids (transpeptidation). These ubiquitous enzymes 23
10
11
12
13
Received 25 March 2014
Received in revised form 12 April 2014
Accepted 21 April 2014
Available online xxxx
play a key role in the biosynthesis and degradation of glutathione, and in xenobiotic detoxification.
24
Here we report the 3 Å resolution crystal structure of Bacillus licheniformis γ-GT (BlGT) and that of its complex 25
with L-Glu. X-ray structures confirm that BlGT belongs to the N-terminal nucleophilic hydrolase superfamily 26
and reveal that the protein possesses an opened active site cleft similar to that reported for the homologous en- 27
zyme from Bacillus subtilis, but different from those observed for human γ-GT and for γ-GTs from other microor- 28
ganisms. Data suggest that the binding of L-Glu induces a reordering of the C-terminal tail of BlGT large subunit 29
and allow the identification of a cluster of acid residues that are potentially involved in the recognition of a metal 30
ion. The role of these residues on the conformational stability of BlGT has been studied by characterizing the 31
autoprocessing, enzymatic activity, and chemical and thermal denaturation of four new Ala single mutants. 32
The results show that replacement of Asp568 with an Ala affects both the autoprocessing and structural stability 33
1Q4 3 Keywords:
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Protein structure
γ-Glutamyltranspeptidase
γ-Glutamyltransferase
Ntn hydrolase
X-ray structure
Enzyme chemistry
Structure–activity relationship
of the protein.
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© 2014 Published by Elsevier B.V.
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1. Introduction
microbial proteins undergo an autoproteolytic post-translational cleav- 57
age (autoprocessing) into a heavy and a light chain [19], usually called 58
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45
46
47
48
49
50
51
52
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γ-Glutamyltranspeptidases (γ-GTs, EC 2.3.2.2) are enzymes belong-
ing to the class of N-terminal nucleophilic (Ntn) hydrolases which initi-
ate the breakdown of extracellular glutathione (γ-Glu-Cys-Gly, GSH)
into its constituent amino acids, glutamate, cysteine, and glycine and
catalyze the transfer of its γ-glutamyl moiety to water, amino acids or
small peptides [1–5]. They are widely distributed in nature from mam-
mals to bacteria and involved in many crucial cellular events such as
aging, senescence and drug detoxification [6,7]. In humans, γ-GT has
been associated with several physiological disorders related to oxidative
stress, such as Parkinson's disease and diabetes [8,9]; it is currently used
as a marker of liver diseases [10], cardiovascular diseases [11,12] and
cancer [13–16].
large and small subunits, respectively [20].
59
The catalytic cycle of mature γ-GTs follows a ping-pong mechanism 60
[1–3,20]. In the first step, the γ-glutamyl moiety of the donor substrate 61
(e.g., GSH or GSH conjugate) reacts with the hydroxyl group of a 62
conserved Thr in the active site to generate a tetrahedral intermediate 63
[1,3,20]. In the case of the human enzyme (hGT), the essential catalytic 64
residue is Thr380. Subsequent release of the free amine donor 65
(e.g., Cys–Gly) from the intermediate produces a covalent γ-glutamyl 66
enzyme intermediate. The cycle is finished when the γ-glutamyl moiety 67
is transferred to an acceptor substrate, such as water (hydrolysis) or a 68
dipeptide (transpeptidation), releasing glutamate or a γ-glutamyl di- 69
peptide, respectively. Both hydrolysis and transpeptidation reactions 70
are believed to have physiological importance, although the role of 71
transpeptidation in bacteria is not clear [1,18]. Proteins from different 72
sources are characterized by a broad substrate specificity, which in- 73
UNCORRECTED PR
In mammalians, γ-GTs are membrane heterodimeric glycoproteins
that are localized at the cell surfaces (mainly in the kidney); in bacteria,
γ-GTs are found in the cytosol or in the periplasmic space and can be
heterodimeric [1,7] or heterotetrameric [18]. Both mammalian and
cludes a large variability of acceptors.
74
Beyond their physiological function, γ-GTs can be employed for the 75
biosynthesis of various γ-glutamyl compounds, with great potential for 76
pharmaceutical and biotechnological applications [1,5,21,22]. In fact, it 77
has been shown that these proteins can be used as glutaminase in 78
food industry [23,24], as cephalosporinacylases [25], in the production 79
⁎
Corresponding author at: 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.
E-mail address: antonello.merlino@unina.it (A. Merlino).
http://dx.doi.org/10.1016/j.bbapap.2014.04.016
1570-9639/© 2014 Published by Elsevier B.V.
Please cite this article as: L.-L. Lin, et al., Low resolution X-ray structure of γ-glutamyltranspeptidase from Bacillus licheniformis: Opened active
site cleft and a cluster of ..., Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.016

2
L.-L. Lin et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
80
of theanine [26], and in the biosynthesis of flavor enhancers and medical
The atomic coordinates and structure factors for BlGT (with PDB ID: 142
4OTT) and its complex with L-Glu (PDB ID: 4OTU) were deposited in the 1Q483
8Q1 4 compounds [27,28].
82
83
84
85
86
87
Although γ-GTs have been characterized from many different
Protein Data Bank (http://www.rcsb.org/).
144
sources, crystal structures of just five members of their family have
been solved [20,29,31–33]; two out of these have not been described
in literature and one has been deposited in the Protein Data Bank
when this manuscript was in preparation [33]. Biochemical studies
and X-ray structures of γ-GTs have provided insights on the mecha-
2.2. Site-directed mutagenesis
145
Mutant proteins were constructed using a template plasmid pQE- 146
BlGGT that expresses an N-terminal His6-tagged form of BlGT. 147
Mutations were created by a QuikChange site-directed mutagenesis 148
kit obtained from Stratagene (La Jolla, CA, USA). The kit essentially em- 149
ploys the high fidelity PfuTurbo DNA polymerase and appropriate for- 150
ward and reverse primers (Table S2) to replicate both strands of the 151
supercoiled plasmid containing the γ-GT gene. Following the thermal 152
cycling, the reaction mixture was treated with DpnI endonuclease that 153
digests the hemimethylated template leaving behind the nicked DNA 154
with the mutation of interest. After transformation of the digested mix- 155
ture into Eschericchia coli XL-1 blue supercompetent cells, single colony 1Q596
was picked up to verify and confirm the mutations by DNA sequencing. 157
8Q8 5 nisms of autoprocessing and hydrolysis reaction [19,20,30,34] and on
89
90
91
92
93
94
95
96
97
98
99
their structural stability [35,36].
As a further step in the structural characterization of γ-GTs, here we
report the crystal structure of the protein from Bacillus licheniformis
(BlGT) at 2.98 Å resolution, and that of its complex with L-Glu at 3.02
Å resolution. Data demonstrate that the protein possesses an opened ac-
tive site cleft and show that the binding of L-Glu induces a reordering of
the C-terminal tail of the large subunit of the protein. The results allow
the identification of a cluster of acid residues that are potentially in-
volved in the recognition of a metal ion. The role of these residues on
the structure, stability and catalytic activity of the protein has been
studied using alanine-scanning mutagenesis. The substitution of
2.3. Protein expression and purification
158
100 Asp568 significantly affects the biophysical behavior of the protein.
For biochemical and biophysical analyses, wild-type and mutant 159
BlGTs were expressed in E. coli M15 (pRep4) as previously described 160
[37,48]. Target proteins were purified by affinity chromatography with 161
a Ni2+–NTA agarose column under native conditions. The eluted frac- 162
tions (total volume for each preparation = ~5 mL) were pooled and di- 163
alyzed overnight against 500 mL of 10 mM Tris–HCl buffer (pH 8.0) 164
101 2. Materials and methods
102 2.1. Crystallization, structure determination and refinement
1Q036
Protein preparation and materials and methods of crystallization
through a 10-kDa cutoff membrane to remove salts.
165
104 were already described [37]. Briefly, the best crystals of BlGT (UNIPROT
105 code A9YTT0) have been grown using hanging drop vapor diffusion
106 method, protein concentration 20 mg mL−1 and a precipitant solution
107 consisting of 20% (w/v) PEG 3350, 0.2 M MgCl2 hexahydrate, 0.1 M
108 Tris–HCl (pH 8.2) [37]. Crystals of the complex between BlGT and L-
109 Glu have been obtained by performing a screening around the crystalli-
110 zation conditions previously used to grow crystals of BlGT. The best
111 crystals appeared in drops consisting of 22% (w/v) PEG 3350, 0.2 M
112 MgCl2 hexahydrate, 0.1 M Tris–HCl (pH 8.0), protein solution at a con-
113 centration of 10 mg mL−1 and 8.5 mM L-Glu. Crystals were flash-
114 cooled in liquid nitrogen at 100 K. X-ray diffraction data were collected
115 at the Institute of Biostructures and Bioimages, CNR, Naples, Italy using a
116 Rigaku MicroMax-007 HF generator producing Cu Kα radiation (45 kV
117 and 60 mA) and equipped with a Saturn 944 CCD detector. The
118 crystal-to-detector distance was set to 70 mm, with all frames collected
119 at 100 K. Diffraction data were recorded over a 350° rotation of the crys-
120 tal with a width of 0.5° per image. Diffraction data were processed using
121 the program HKL2000 [38] and CCP4 program suite [39]. Data collection
122 statistics and scaling results are listed in Table S1. Crystals diffract at
123 about 3 Å resolution and belong to the space group P212121. Since the
124 known structures of γ-GTs have been determined at high resolution
125 (b2 Å), many attempts have been done to improve the diffraction
126 power of BlGT crystals, but unfortunately, all these trials failed. Different
127 cryoprotection strategies, including the crystal dehydration [40] and the
128 flash-cooling in the absence of cryoprotectant [41] have been used, but
129 in all cases we are not able to improve the resolution of the collected
130 data sets. Since the BlGT crystals are very fragile, they do not resist to
131 the transport to synchrotrons.
Protein purity was confirmed by sodium dodecyl sulfate–polyacryl- 166
amide gel electrophoresis (SDS-PAGE) carried out on 12% acrylamide 167
slabs using the buffer system of Laemmi [49]. Before electrophoresis, 168
the samples were heated to 100 °C for 5 min in dissociating buffer con- 169
taining 2% SDS and 5% 2-mercaptoethanol. Protein concentration was 170
measured with a protein assay kit (Bio-Rad Laboratories) using bovine 171
serum albumin as the standard.
172
2.4. Autoprocessing of the enzymes
173
Autocatalytic processing studies on BlGT and its mutants 174
(1 mg mL−1) were performed by incubating the proteins in 20 mM 175
Tris–HCl, pH 8.0 at 45 °C up to two days. Aliquots were denatured 176
by boiling in SDS loading buffer for 5 min. Samples were analyzed 177
by SDS-PAGE 12%, and the gel was stained using Coomassie Brilliant 178
Blue.
179
2.5. Enzyme activity assay and determination of kinetic parameters
180
The enzyme activity of wild-type and BlGT variants towards sub- 181
strate analog L-γ-glutamyl-p-nitroanilide (GpNA) was determined 182
spectrophotometrically by monitoring the p-nitroaniline released from 183
GpNA at 412 nm using the method assay by Tate and Meister [50]. 184
One unit of γ-GT activity is defined as the amount of enzyme that pro- 185
duced 1 μmol of p-nitroaniline per min under the assay conditions. Cat- 186
alytic activity was measured in 50 mM Tris–HCl buffer, pH 8.0, and 187
containing 1.25 mM GpNA, 30 mM Gly–Gly, 1 mM MgCl2, and 20 μL of 1Q8180
enzyme solution at a suitable dilution, and enough distilled water to 189
bring the final volume to 1 mL. The reaction mixtures were incubated 190
at 25 °C for 10 min. The reaction was stopped by the addition of 191
132
Initial phases were determined by a molecular replacement
133 method with the program Phaser [42], using the structure of the pro-
134 tein from Bacillus subtilis (BsGT) [32], which shares about 70% of se-
135 quence identity with BlGT, as search model. The structures were
136 refined using the programs Refmac5.7 [43] and Coot [44]. Mg2+
1Q377 ions were introduced in the model in correspondence with peaks
138 above 5σ in the Fo–Fc electron density map. The number of residues
139 in the most favored regions of a Ramachandran plot [45] was deter-
140 mined by the program PROCHECK [46]. Cavities were identified
141 using Fpocket [47].
100 μL of 3.5 N acetic acid.
192
To evaluate kinetic constant, dependence of the reaction on the sub- 193
strate concentration was evaluated using fixed amounts of purified BlGT 194
(or of its mutants) incubated with increasing concentrations of GpNA 195
from 1 to 1000 μM. Saturation kinetics were observed, and data were 196
fit to the Michaelis–Menten equation to obtain KM and Vmax for the cat- 197
alyzed reaction.
198
Please cite this article as: L.-L. Lin, et al., Low resolution X-ray structure of γ-glutamyltranspeptidase from Bacillus licheniformis: Opened active
site cleft and a cluster of ..., Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.016

L.-L. Lin et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
3
199 2.6. Circular dichroism (CD) studies
200
Far-UV CD spectra were acquired on a JASCO J-815 spectropolarim-
2Q0111 eter using a cuvette path length of 2 mm, and a spectral band pass of
202 4 nm. Raw ellipticity data were converted to mean residue ellipticity
203 using the formula [θ] = [θraw × 100 × MRW] / c × l, where MRW is
204 the mean residue weight for BlGT, c is the concentration of BlGT in
205 mg mL−1 and l is the path length in cm. Changes in mean residue ellip-
206 ticity (at 222 nm) as a function of temperature were monitored, for BlGT
207 and its mutants, using constant temperature ramping rates of 1 °C/min,
208 using the spectropolarimeter's Peltier block arrangement (PTC-423S)
2Q0912 and a cuvette path length of 1.0 cm.
210 2.7. Fluorescence spectroscopy
211
212 spectrophotometer with a protein concentration of 0.05 mg mL−1 in
213 10 mM Tris–HCl buffer (pH 8), using λexc = 280 nm or λexc
Protein intrinsic fluorescence was measured using a Jasco FP-6500
=
214 295 nm. Emission was collected between 310 and 450 nm. For chemical
215 denaturation experiments, 1 mL of guanidinium hydrochloride
216 (GdnHCl) (from 1 to 6 M) was mixed with 1 mL of protein
217 (0.1 mg mL−1). Fluorescence was recorded at room temperature with
218 a constant protein concentration of 0.05 mg mL−1 and a final concentra-
219 tion of 0.5–3.0 M GdnHCl in 20 mM Tris–HCl buffer (pH 8.0) using 1 cm
220 quartz cell.
OOF
Fig. 1. Overall structure of BlGT shown as a cartoon. Small and large subunits are colored in
red and gray, respectively.
structure of hGT, recently solved, has suggested that in human enzyme 259
the lid loop has greater flexibility than in HpGT or EcGT [33]. The differ- 260
ent flexibility of the lid loop would allow hGT to bind a wider range of 261
substrates compared to the microbial enzyme [33]. The absence of the 2Q6123
lid loop in BlGT (and in BsGT) could allow to accommodate larger sub- 2Q6134
221 3. Results and discussion
222 3.1. Overall structure of BlGT
strate, like the polymeric material γ-polyglutamic acid [51].
264
223
The structure of BlGT was solved by the molecular replacement
224 method using the coordinates of the heterodimeric γ-GT from
225 B. subtilis (PDB ID: 3A75) [32] as a search model and refined against
226 data extending to 2.98 Å resolution to an R-factor of 19.0% and a R-
227 free of 25.1%. A full list of the refinement statistics is reported in
228 Table S3. The refined model consists of two polypeptide chains (resi-
229 dues Asp35–Pro391 form the large subunit and residues Thr399–
230 Ile580 form the small subunit) which constitute the heterodimeric
231 molecule, of six water molecules and two Mg2+ ions. The His-tag N-
232 terminal region (see [37] for details) and C-terminal residues of both
233 large and small subunits (residues Glu392–Glu398 and Lys581–
234 Lys585, respectively) are not defined in the electron density. There are
235 4 outliers in the Ramachandran plot (Lys54, Glu99, Lys155, Glu419).
236 Four peptides (Ser53–Lys54, Gly98–Glu99, Lys161–Met162 and
237 Pro286–Pro287) adopt the cis conformation.
3.2. γ-Glutamyl binding site
265
The catalytic site of BlGT was unambiguously identified by solving 266
the structure of its complex with L-glutamate. The structure of the com- 267
plex (BlGT-L-Glu hereafter), which includes 6 residues more than that 268
observed in the structure of the wild-type protein at the C-terminal 269
tail of the large subunit (residues Glu392–Gly397), refines to an R- 270
factor of 0.201 (R-free 0.288) using data up to 3.02 Å resolution. Details 271
of the refinement statistics are reported in Table S3. L-Glu (occupancy 272
0.5, average B-factor about 16.7 Å2) forms hydrogen bonds with back- 273
bone atoms of residues Ser461 and Gly481 and with side chain atoms 274
of Asp441 (Fig. S1). The binding of this substrate occurs in a concave 275
site of the structure lined by residues Thr399, Glu438, Ser460, Ser461, 276
Gly481, Gly482, and Arg109 (Fig. S2); due to the lack of the lid loop, 2Q7175
this region is exposed to solvent (“opened”), as suggested for the γ-GT 278
from Geobacillus thermodenitrificans (GthGT) [18] and observed in the 279
238
BlGT structure agrees in all general features with B. subtilis γ-GT,
239 which was used for phasing. As all γ-GT structures determined so far,
240 it adopts the conserved fold of proteins belonging to the Ntn hydrolase
241 family (Fig. 1). Briefly, the large subunit consists of a globular domain
242 comprised of 14 α-helices, six small 310 helices and 11 β-strands,
243 whereas the small subunit consists of 3α-helices, two small 310 helices
244 and 11 β-strands. Both large and small subunits provide strands to a
245 nearly flat β-sheet that constitutes the core of the heterodimeric ar-
246 rangement (Fig. 1).
case of BsGT [32] (Fig. 2).
280
The active site channel is formed by two extensive consecutive 281
pockets lined mainly by residues His49, Thr80, Glu81, Met83, Met84, 282
Val182, Leu183, Ala186, Thr399, Glu419, Gln420, Leu421, Pro480, 283
Gly481, Gly482, Arg521, Asp547, Gly549, Asn550, Ser570, Gly573, and 284
Asn572 and residues Asn78, Pro82, Ser85, Gly86, Ile87, Leu221, 285
Thr224, Phe225, Ile418, Tyr431, Gly432, Leu433, and Phe434, respec- 286
tively (Fig. S2). The two pockets have preponderance of hydrophobic 287
amino acids and are within the intersubunit interface of the protein. 288
The total volume of the two pockets is about 1400 Å3. Multiple sequence 289
alignments show that the only conserved residues constituting the 290
channel are Gly481 and Gly482, which form the oxyanion hole and 291
are known to be involved in stabilizing the formation of intermediate 292
(Fig. S3). The observed variability of residues in the active site channel 293
likely contributes to the differences in transpeptidase activity found 294
UNCORRECTED PR
247
Comparison of BlGT structure with that of hGT [33], BsGT [32], EcGT
248 [29] and HpGT [19] results with average root mean square deviations of
249 1.29 Å (437 atoms), 0.62 Å (538 atoms), 1.25 Å (425 atoms) and 1.21 Å
250 (443 atoms), respectively. Main structural difference among BlGT, hGT,
251 BsGT, EcGT and HpGT was observed at the level of active site (see
252 below). In particular, BlGT lacks the loop comprising residues 438–449
253 of EcGT (lid loop), which is supposed to regulate the access of the sub-
254 strate to the active site cleft of HpGT [20] and EcGT [29,31] (Fig. 2). It
255 has been proposed that when the γ-glutamyl binding site is occupied
256 by a substrate (or inhibitor) the lid loop has a well-defined position
257 and shields the catalytic pocket from the solvent, otherwise, when the
258 pocket is empty, the lid loop is highly flexible [20,29,31]. The X-ray
when proteins from different sources are compared [1,2,20,52].
The comparison between the structure of BlGT and that of its com- 296
plex with L-glutamate reveals a significant structural variation: the 297
295
Please cite this article as: L.-L. Lin, et al., Low resolution X-ray structure of γ-glutamyltranspeptidase from Bacillus licheniformis: Opened active
site cleft and a cluster of ..., Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.016

4
L.-L. Lin et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
Fig. 2. Structures of BlGT (green), BsGT (violet), and BsGT in complex with L-Glu (cyan) and EcGT (orange). The active site channel is evidenced.
298 binding of L-Glu induces an ordering of six residues at the C-terminal tail
299 of the large subunit (Fig. 3). On the other hand, the position of side
300 chains of residues at the active site channel entry is only slightly affect-
301 ed. Notably, only a small structural variation in the position of active site
302 channel residues has been observed comparing the structure of ligand
303 free BsGT with that of its complex with L-Glu (Fig. S4).
residues, which in the structure have been interpreted to be Mg2+, be- 307
cause 100 mM MgCl2 was added to the protein during the crystalliza- 308
tion. The first peak is located in the small subunit, close to a cluster of 309
carboxylates (Glu508, Glu523, Asp547, and Asp568) (Fig. S5). The oc- 310
currence of four carboxylates in this region implies that the site is well 311
designed to accommodate a cation. These residues are conserved in 312
BsGT (Fig. S3). In the structure of the latter enzyme, a water molecule 313
with B-factor of 9 Å2 is located approximately in the same position occu- 314
pied by the Mg2+ ion in the BlGT structure. This water forms a well- 315
defined network of hydrogen bonds with the carboxylates and with 316
other solvent molecules [32]. Interestingly, the same position is occu- 317
pied by the guanidinium group of an Arg (Arg571 in BlGT and Arg575 318
304 3.3. Potential metal ion binding sites
305
After the building of the BlGT molecule, inspection of the Fo–Fc elec-
306 tron density maps reveals the presence of two N5σ peaks close to acid
in BsGT) in the protein from E. coli [29].
319
A second putative metal ion binding site is located at the interface 320
between the large and the small subunit. In this site, the ion is coordi- 321
nated to Glu498 and His277. Position 498 is occupied by negatively 322
charged residues in other γ-GTs (Fig. S3), whereas His277 is a not con- 323
served residue. This site seems to be unoccupied in the BlGT-L-Glu 324
structure.
325
The magnesium ion is a divalent metal cation found abundantly in 326
protein structures. Most magnesium ions are coordinated by four to 327
six residues, but there are few examples of other coordination numbers 328
(i.e. 2, 3 or 7). The most frequent atoms observed in the coordination 329
sphere of Mg ions are the N atom of imidazole, and O atom of carboxyl 330
groups of Asp or Glu. The average distances between the Mg and 331
coordinating atoms are 2.14
2.13 0.19 Å for the carboxyl O atom [53]. These data indicate that 333
Mg2+ coordination in BlGT adopts features typically observed in 334
proteins. 335
0.20 Å for the imidazole N atom, and 332
It is possible that the binding of a cation to one or both the putative 336
metal binding sites of BlGT could regulate the protein enzymatic activi- 337
ty. In fact, catalytic activity of BlGT increases in the presence of Mg2+, al- 338
beit inhibited by Hg2+, Zn2+, Pb2+ and Ni2+ [47]. The presence of a 339
Fig. 3. γ-GT active site. Comparison between the structure of BlGT (green) and that of its
complex with L-Glu (violet).
Please cite this article as: L.-L. Lin, et al., Low resolution X-ray structure of γ-glutamyltranspeptidase from Bacillus licheniformis: Opened active
site cleft and a cluster of ..., Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.016

L.-L. Lin et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
5
340 metal ion binding site could be a feature shared by members of γ-GT su-
341 perfamily. In fact, other γ-GTs, including EcGT [54] and BsGT [55] have
342 been found to increase their transpeptidase or their hydrolase activity
343 in the presence of Mg2+, whereas the γ-GT from shiitake mushroom
344 is slightly inhibited by Mg2+, but presents a catalytic activity promoted
345 by Ca2+ [56].
346
Interestingly, other two metal ion binding sites have been identified
347 in the solved structures of γ-GTs: one for a Sm3+, used for phasing, and
348 the other for a Ca2+ ion [29,30]. The former ion is located close to OD1
349 and OD2 atoms of Asp433 and to OD1 atom of Asn411 [29,30]. The lat-
350 ter, observed in the X-ray structure of EcGT in complex with L-glutamate
351 is bound to carboxylic oxygens of Asp575, carbonyl oxygens of Asp569
352 and Ser572 and three water molecules [29].
353 3.4. Mg2+ binding to BlGT: spectroscopic studies
354
In order to investigate the influence of Mg2+ ions on the structure of
355 BlGT, we performed experiments using far-UV CD and fluorescence
356 analyses in the presence and in the absence of these ions. As previously
357 described, BlGT exhibits a typical α-helical CD profile with minimal
358 peaks at 208 nm and 222 nm [48]. The binding of Mg2+ does not signif-
359 icantly alter the secondary structure content of the protein (data not
360 shown). On the contrary, upon addition of Mg2+ a significant change
361 in emission fluorescence intensity is observed (Fig. S6). In particular, a
362 decreasing in fluorescence intensity is observed with increasing concen-
363 trations of Mg2+. The quenching is not associated with a shift in the
364 maximum emission wavelength. These findings suggest that the ions
365 bind the protein without affecting significantly its overall fold.
OOF
366 3.5. Site-directed mutagenesis studies
367
The role played by the four acid residues (Glu508, Glu523, Asp547,
368 Asp568) that form the conserved cluster potentially involved in the rec-
369 ognition of a metal ion on the structure, stability and catalytic activity of
370 BlGT has been studied. Alanine mutants were prepared by PCR-based
371 mutagenesis, expressed in E. coli as recombinant proteins containing a
372 N-terminal six-histidine tag (His6-tag) and purified by affinity chroma-
373 tography on nickel-nitrilotriacetic acid agarose column, as described in
374 the Materials and methods. In order to monitor the autocatalysis of
375 the precursors to their mature forms, the mutants were incubated at
376 45 °C for 48 h in 20 mM Tris–HCl buffer (pH 8.0). Aliquots of the
377 mutants were withdrawn and analyzed by SDS-PAGE. The proteins are
378 able to autoprocess themselves, leading to the formation of a small
379 and a large subunit with masses of about 41 kDa and 22 kDa, respective-
380 ly. The unprocessed D568A mutant processes itself more slowly than
381 the unprocessed precursors of the other mutants. This indicates
382 that the substitution of Asp568 somewhat affects the enzyme
383 autoprocessing.
Fig. 4. (A) Far-UV CD spectra of BlGT and mutant enzymes. As previously described [47],
BlGT exhibits a typical α-helical CD profile with minimal peaks at 208 nm and 222 nm.
(B) Intrinsic tryptophan fluorescence spectra of wild-type and mutant enzymes. Emission
fluorescence λmax for BlGT, E508A, E523A, D547A, and D568A was 343.2, 342.8, 343.2,
342.4, and 343.0 nm, respectively. Spectra have been registered using an excitation wave-
length of 280 nm.
CD spectra as function of temperature (Fig. 5A) and fluorescence spectra 400
at increasing concentration of GdnHCl (Fig. 5B), respectively. The results 401
of these biophysical studies are summarized in Tables 2 and 3, where 402
apparent melting temperature (Tm) and the values of denaturant con- 403
centration at half-completion of the transition (C1/2) characterizing 404
the chemical-induced denaturation of each analyzed system have 405
been reported.
406
384
All mutants correctly fold as suggested by far-UV CD (Fig. 4A) and
As it clearly emerges from Fig. 5A which shows the temperature- 407
dependence of loss of secondary structural content in BlGT and its 408
mutants, the proteins show unfolding curves with different midpoints 409
(apparent Tm values). In particular, the apparent Tm is ~60 °C for the 410
385 fluorescence spectra (Fig. 4B): CD spectra are virtually superimposable,
386 indicating that the mutations have no detectable effect on the protein's
387 folded structure; furthermore, the comparison of intrinsic fluorescence
388 spectra of the mutants does not show a significant transition or shift
389 in λmax, indicating that tryptophan residues are similarly exposed to sol-
UNCORRECTED PR
390 vent in the buffer.
Table 1
t1:1
Specific activity and kinetic parameters of the purified BlGT and mutant enzymes.
t1:2
391 3.6. Catalytic activity and structural stability of BlGT mutants
Enzyme
Specific activity
(U/mg)
KM (mM)
kcat (s−1
)
kcat/KM
(mM s−1
)
t1:3Q2
392
At first, specific activities of the purified mature wild-type and mu-
BlGT
7.79
7.86
8.27
9.56
3.11
0.48
0.16
1.03
0.03
0.05
0.320
0.389
0.433
0.528
0.323
0.03
10.32
11.36
14.08
16.87
5.00
1.45
32.21
29.22
32.52
31.93
15.47
t1:4
t1:5
t1:6
t1:7
t1:8
393 tated enzymes towards the synthetic substrate GpNA were examined
394 using the standard enzymatic assay. The results of these experiments
395 are reported in Table 1. The mutants exhibit a specific activity compara-
396 ble to BlGT, thus suggesting that these residues do not play an important
397 role in the catalysis.
E508A
E523A
D547A
D568A
0.09
0.01
0.07
0.02
0.97
101
2.21
0.11
The kinetic parameters were determined from the initial reaction rates at 25 °C and at
t1:9
398
The conformational stability of the mutants against both tempera-
pH 8.0, in the presence of 1–1000 μM GpNA, as previously described. KM and kcat are the
t1:10
t1:11
399 ture and presence of denaturant has been also analyzed by collecting
averages of three determinations
standard deviations.
Please cite this article as: L.-L. Lin, et al., Low resolution X-ray structure of γ-glutamyltranspeptidase from Bacillus licheniformis: Opened active
site cleft and a cluster of ..., Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.016

6
L.-L. Lin et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
Table 3
t3:1
t3:2
t3:3
t3:4
t3:5
Values of denaturant concentration at half-
completion of the transition (C1/2) characterizing
the chemical-induced denaturation of BlGT and its
mutants.
Protein
C1/2 (M)
t3:6
BlGT
t3:7
2.82
2.51
2.38
2.79
2.01
0.13
0.12
0.14
0.02
0.25
t3:8
E508A
E523A
D547A
D568A
t3:9
t3:10
t3:11
Here we have reported the low resolution structure of the γ-GT from 423
the bacterium B. licheniformis and its complex with L-Glu. Data reveal 424
the existence of an opened active site cleft and of a cluster of carboxyl- 425
ates that could constitute a potential metal ion binding site. In this re- 426
spect it should be recalled that the Mg2+ ion is necessary for the 427
crystallization of BlGT. We could not generate crystals of the protein in 428
the absence of magnesium ion. The comparison between the structure 429
of BlGT and that of its complex with L-Glu suggests that the binding of 430
the molecule induces a reordering of six residues at the C-terminal tail 431
of the large subunit. This structural modification could play a role in en- 432
zymatic activity.
433
The role of the four acid residues potentially involved in the recogni- 434
tion of a metal ion on the autoprocessing, catalytic activity and structur- 435
al stability of BlGT has been also evaluated. We have found that 436
mutation of Asp568, which is conserved in microbial γ-GTs, to Ala sig- 437
nificantly affects the structural stability of the enzyme.
438
5. Uncited reference
4Q3197
[17]
440
Acknowledgements
441
We are grateful to Giovanna di Martino for her help in the crystalli- 442
zation trials and to Giosuè Sorrentino and Maurizio Amendola for the 443
collection of diffraction data. This work was partly supported by a 444
research grant (NSC-100-2313-B-415-003-MY3) from the National Sci- 445
Fig. 5. (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 moni-
toring the average emission wavelength at increasing concentrations of guanidine
hydrochloride.
ence Council Taiwan.
4Q4168
4Q1116 wild-type enzymes, E508A and D547A, whereas it is ~56 °C for E523A
412 and D568A. Evidently the deletion of the E523 and D568 side chains
413 has resulted in a significant lowering of its apparent melting tempera-
414 ture. Chemical denaturation experiments are in line with these results:
415 the wild type BlGT shows a [GdnHCl]1/2 = 2.8 M in comparison with 2.0
416 M for D568A and with 2.4 M for E523A. D547A and E508A did not show
417 a significant shift in C1/2. Such results confirm that D568A and E523A
418 have a role in the structural stability of the protein.
Appendix A. Supplementary data
447
Supplementary data to this article can be found online at http://dx. 448
doi.org/10.1016/j.bbapap.2014.04.016.
449
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Please cite this article as: L.-L. Lin, et al., Low resolution X-ray structure of γ-glutamyltranspeptidase from Bacillus licheniformis: Opened active
site cleft and a cluster of ..., Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.016

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Please cite this article as: L.-L. Lin, et al., Low resolution X-ray structure of γ-glutamyltranspeptidase from Bacillus licheniformis: Opened active
site cleft and a cluster of ..., Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.016