PH-Dependent hydrolase, glutaminase, transpeptidase and autotranspeptidase activities of Bacillus subtilis γ-glutamyltransferase

Article abstract of DOI:10.1111/febs.12591

γ-Glutamyltransferases (γ-GTs) are heterodimeric enzymes that catalyze the transfer of a γ-glutamyl group from a donor species to an acceptor molecule in a transpeptidation reaction through the formation of an intermediate γ-glutamyl enzyme. In our search for a γ-GT from a generally recognized as safe microorganism suitable for the production of γ-glutamyl derivatives with flavor-enhancing properties intended for human use, we cloned and overexpressed the γ-GT from Bacillus subtilis. In this study, we report the behavior of B. subtilis γ-GT in reactions involving glutamine as the donor compound and various acceptor amino acids. The common thread emerging from our results is a strong dependence of the hydrolase, transpeptidase and autotranspeptidase activities of B. subtilis γ-GT on pH, also in relation to the pK_a of the acceptor amino acids. Glutamine, commonly referred to as a poor acceptor molecule, undergoes rapid autotranspeptidation at elevated pH, affording oligomeric species, in which up to four γ-glutamyl moieties are linked to a single glutamine. Moreover, we found that d-glutamine is also recognized both as a donor and as an acceptor substrate. Our results prove that the B. subtilis γ-GT-catalyzed transpeptidation reaction is feasible, and the observed activities of γ-GT from B. subtilis could be interpreted in relation to the known ability of the enzyme to process the polymeric material γ-polyglutamic acid.

Full text of DOI:10.1111/febs.12591

pH-Dependent hydrolase, glutaminase, transpeptidase and
autotranspeptidase activities of Bacillus subtilis
c-glutamyltransferase
Carlo F. Morelli1,2, Cinzia Calvio3, Marco Biagiotti1 and Giovanna Speranza1,2,4
1 Department of Chemistry, University of Milan, Italy
2 Italian Biocatalysis Center (IBC), c/o Dipartimento di Scienza del Farmaco, University of Pavia, Italy
3 Department of Biology and Biotechnology, University of Pavia, Italy
4 Istituto di Scienze e Tecnologie Molecolari (INSTM), CNR, c/o Department of Chemistry, University of Milan, Italy
Keywords
c-Glutamyltransferases (c-GTs) are heterodimeric enzymes that catalyze the
transfer of a c-glutamyl group from a donor species to an acceptor molecule
in a transpeptidation reaction through the formation of an intermediate
Bacillus subtilis; enzyme catalysis
poly-c-glutamic acid; transpeptidation
reaction; c-glutamyltransferase
c-glutamyl enzyme. In our search for a c-GT from a generally recognized as
safe microorganism suitable for the production of c-glutamyl derivatives
Correspondence
with flavor-enhancing properties intended for human use, we cloned and
overexpressed the c-GT from Bacillus subtilis. In this study, we report the
behavior of B. subtilis c-GT in reactions involving glutamine as the donor
compound and various acceptor amino acids. The common thread emerging
from our results is a strong dependence of the hydrolase, transpeptidase
and autotranspeptidase activities of B. subtilis c-GT on pH, also in relation
to the pKa of the acceptor amino acids. Glutamine, commonly referred to
as a poor acceptor molecule, undergoes rapid autotranspeptidation at
elevated pH, affording oligomeric species, in which up to four c-glutamyl
moieties are linked to a single glutamine. Moreover, we found that
D-glutamine is also recognized both as a donor and as an acceptor sub-
strate. Our results prove that the B. subtilis c-GT-catalyzed transpeptida-
tion reaction is feasible, and the observed activities of c-GT from B. subtilis
could be interpreted in relation to the known ability of the enzyme to
process the polymeric material c-polyglutamic acid.
C. F. Morelli, Department of Chemistry, via
Golgi, 19, 20133 Milano, Italy
Fax: +39 2 50314072
Tel: +39 2 50314099
E-mail: carlo.morelli@unimi.it
Website: http://www.chimica.unimi.it
(Received 5 September 2013, revised 25
October 2013, accepted 28 October 2013)
doi:10.1111/febs.12591
Introduction
c-Glutamyltransferases (c-GTs, also known as c-glut-
amyltranspeptidases; EC 2.3.2.2) [1] are heterodimeric
enzymes that are widely distributed, from bacteria
[2–5] to plants [6] and mammals [7], belonging to the
N-terminal nucleophile hydrolase superfamily [8]. The
two subunits originate from autocatalytic cleavage of a
single peptide chain [9].
enzyme intermediate through the oxygen atom of the
conserved, catalytically active N-terminal threonine of
the small subunit [10]. The c-glutamyl moiety can then
be transferred to an acceptor nucleophile. If the nucleo-
phile is a water molecule, hydrolysis of the c-glutamyl
enzyme intermediate occurs, and the net result is the
release of free glutamic acid (Scheme 1, path a) [11]; if
the nucleophile is the amino group of an amino acid or
of a small peptide, a transpeptidation reaction ensues,
with the formation of a new c-glutamyl compound
c-GTs cleave the amide bond involving the
c-carboxyl group of glutamic acid in a c-glutamyl
derivative, usually glutathione, affording a c-glutamyl
Abbreviations
DbsCl, dabsyl chloride; GPNA, c-glutamyl-p-nitroanilide; c-GT, c-glutamyltransferase; c-PGA, poly-c-glutamic acid.
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C. Calvio et al.
B. subtilis c-glutamyltransferase activities
Scheme 1. c-GT-catalyzed reactions. Path a: hydrolysis. Path b: transpeptidation. Path c: autotranspeptidation.
(Scheme 1, path b) [12–15]. In the presence of a high
concentration of the c-glutamyl donor, an autotransp-
eptidation reaction is also possible, in which the same
compound behaves both as the donor and as the accep-
tor molecule (Scheme 1, path c) [11].
breakdown of the c-glutamyl bond of poly-D-glutamic
acid, transferring the polymeric chains to the peptido-
glycan cell wall, thus forming the covalently bound
poly-D-glutamic acid capsule of the bacterial cells
[26,27].
Whereas the biological significance of mammalian c-
GT in glutathione metabolism [7] is well defined, the
physiological roles of bacterial c-GTs are often less
clear. Bacterial c-GTs are soluble proteins found in
the periplasmic [3] or extracellular [4] space.
Even though the involvement of c-GT in the bio-
synthesis of poly-c-glutamic acid (c-PGA) produced
and secreted by B. subtilis (natto) was initially sug-
gested [28], it is more likely to be involved in c-PGA
hydrolysis for energy supply under carbon-limiting
conditions [29] rather than in its biosynthesis.
Among the bacterial c-GTs, those from Bacillus spe-
cies have attracted interest [16–19], owing to some
peculiar features.
As B. subtilis c-GT showed transpeptidase activity
towards some acceptor amino acids [4], its use as a bi-
ocatalyst for the enzymatic synthesis of c-glutamyl
compounds was proposed.
The gene encoding c-GT in Bacillus subtilis was
identified in 1996 [20]. It encodes a 587-residue pro-
tein, carrying a 28-residue N-terminal portion signaling
the extracellular localization. It shares 41% sequence
homology with Escherichia coli c-GT.
This possibility was, indeed, sometimes suggested by
analytical results, and only for a few c-GTs from the
genus Bacillus it was experimentally demonstrated at a
preparative level [30,31]. When c-GT-catalyzed reac-
tions were carried out at a preparative level, the reac-
tion conditions, such as pH optimum, temperature,
and reactant molar ratio, usually had to be optimized
for each acceptor substrate in an ad hoc way, despite
the fact that the specificities for acceptor substrates
have been previously determined with standardized
methods.
Comparison of the amino acid sequence of B. subtil-
is c-GT with that of other known bacterial and mam-
malian c-GTs showed that it lacks the lid loop
covering the glutamate-binding site, and has an extra
sequence at the C-terminus of the large subunit [21].
The absence of the lid loop is a structural feature
shared by c-GTs from other Bacillus species, such as
Bacillus licheniformis [22] and also by the c-GTs from
Geobacillus thermodenitrificans [23], Deinococcus radio-
durans, and Thermus thermophilus [24]. CapD from
Bacillus anthracis [25] is another member of the N-
terminal nucleophile hydrolase superfamily lacking
the lid loop. It shares sequence similarity with both
mammalian and bacterial c-GTs, and catalyzes the
The transpeptidation activities of c-GTs were usu-
ally evaluated at fixed pH (8.0 or 8.5) in the presence
of a large excess of the acceptor compounds, up to
~ 70-fold with respect to the donor, taking the release
of the chromogenic p-nitroaniline from c-glutamyl-p-
nitroanilide (GPNA), used as the donor substrate, as a
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B. subtilis c-glutamyltransferase activities
C. Calvio et al.
measure of the enzyme-catalyzed reaction. The accep-
tor propensities of the tested amino acids were also
evaluated with respect to the hydrolysis reaction (i.e.
the same reaction carried out in the absence of an
acceptor substrate), taking advantage of the faster
transpeptidation reaction in the presence of a good
acceptor compound with respect to the hydrolysis of
the donor [32]. Moreover, the ability of a compound
to act as an acceptor was evaluated with respect to
glycylglycine, considered to be the best acceptor.
The aim of this study was to investigate the behav-
ior of B. subtilis c-GT in transpeptidation reactions, in
view of its possible use as a biocatalyst for preparative
purposes.
pH-activity profile
pH-rate profiles for the hydrolase and the transpepti-
dase activities were determined with the standard
method, with GPNA as the donor and glycylglycine as
the acceptor [13,32,41]. We found that the hydrolase
activity reached a maximum at a pH of ~ 9, and then
decreased, giving a bell-shaped curve. In contrast, in
the presence of the acceptor glycylglycine, the activity
steadily increased up to a pH of ~ 10, and showed a
marked decrease only at the more elevated pH value
tested (Fig. 1).
Mammalian c-GT showed, instead, bell-shaped
curves for the transpeptidation reaction [43], whereas
the hydrolase activity was usually lower and much less
sensitive to pH changes [44]. Moreover, the transpepti-
dation reaction catalyzed by mammalian c-GT is up
to 180-fold faster than hydrolysis [45], whereas our
c-Glutamyl derivatives of both unmodified and
modified amino acids are naturally occurring flavor
enhancers found in many foods such as cheese [33],
commonly eaten vegetables [34], mushrooms [35] and
seasoning plants used in cuisine worldwide [36–38].
We reported recently the chemoenzymatic synthesis
of c-glutamyl derivatives of methionine and S-substi-
tuted cysteines as examples of flavor enhancers found
in plants of the genus Allium [39]. The synthesis relied
on the use of an enzyme of mammalian origin, so the
obtained products were not suitable for human use.
In continuing our work, we sought a biocatalyst
appropriate for food processing, and the c-GT derived
from B. subtilis seemed to be a good choice, being this
organism generally recognized as safe (GRAS). In this
study, the donor and the acceptor substrates were used
in the enzyme-catalyzed reactions in equimolar
amounts, usually at concentrations of 100 mM. This
approach gave us the opportunity to gain some
insights into the enzyme activity, and to find new and
unexpected behaviors with respect to those previously
reported [16–19,40].
results indicate, for B. subtilis c-GT,
a two-fold
increase in transpeptidase activity with respect to
hydrolysis at pH 10. In this respect, B. subtilis c-GT
appears to be more similar to Helicobacter pylori c-
GT [46], which showed only a modest prevalence of
the transpeptidase activity over the hydrolase activity.
pH-dependent behavior of B. subtilis c-GT
towards glutamine
Starting from the assumption that the known gluta-
minase activity of B. subtilis c-GT [4] can represent a
source of undesired glutamic acid byproduct in the
transpeptidation reactions, we started our investigation
Results and Discussion
Enzyme production and purification
Recombinant B. subtilis c-GT was purified from
E. coli as a N-terminal His-tagged protein (Fig. S1).
Its enzymatic activity, expressed as lmoles of p-nitro-
aniline liberated through the transpeptidation reaction
with GPNA as the donor and glycylglycine as the
acceptor at pH 8.5 and 22 °C [32–41], was calculated
to be 18.33 UÁmLÀ1 enzyme solution. The protein con-
tent, measured with the method of Bradford [42], was
found to be 34.4 mgÁmLÀ1. The specific activity was
therefore 529 mUÁmgÀ1 protein. The enzyme was
stored at À20 °C in aliquots, and diluted with appro-
priate buffer prior to use.
Fig. 1. pH-activity profile for B. subtilis c-GT-catalyzed hydrolysis
and transpeptidation reactions. Enzyme (43.6 mU) was added to a
0.25 mM solution of GPNA alone (hydrolysis) or in the presence of
20 mM glycylglycine as the acceptor (transpeptidation) at 22 °C.
The amount of released p-nitroaniline was monitored
spectrophotometrically at 410 nm for 2 min. The concentrations of
released p-nitroaniline were calculated by use of
a calibration
curve.
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C. Calvio et al.
B. subtilis c-glutamyltransferase activities
with a study of the behavior of the enzyme towards
glutamine at different pH values.
A
Even though the pH dependence of the glutaminase
activity of B. subtilis c-GT has been already reported
[4], we quantitatively monitored the time course of the
conversion of the substrate glutamine into glutamic
acid with HPLC. Three reactions were carried out with
100 mM solutions of glutamine, at pH 7.4, 8.2, and
9.8. At fixed time intervals (1, 2, 3, 5, 7 and 24 h),
100-lL aliquots were withdrawn from each reaction
mixture, derivatized with dabsyl chloride (DbsCl), and
analyzed with HPLC for glutamine and glutamic acid
contents.
B
At pH 7.4, a decrease in glutamine concentration in
the reaction mixture was observed with time, which
paralleled the increase in glutamic acid content (Fig. 2,
top). When the pH was increased to 8.2, a new peak in
the chromatograms appeared transiently between 1 h
and 5 h, which was attributed to c-glutamylglutamine
(Fig. 2, middle). HPLC-ESI-MS experiments confirmed
our hypothesis of an autotranspeptidation reaction,
attributing to the new peak in the chromatograms a
molecular mass of 562, corresponding to the dabsyl
derivative of c-glutamylglutamine (Fig. S2). Further
evidence came from the use of an authentic sample of
c-glutamylglutamine, deliberately synthesized and used
for the construction of a calibration curve, with the
aim of quantitative estimation in the reaction mixtures.
The most impressive result was, however, obtained at
pH 9.8 (Fig. 2, bottom), in that glutamine disappeared
very quickly within 2 h, and a series of small peaks
appeared in the chromatograms. The amounts of glu-
tamic acid and c-glutamylglutamine formed in the
same time period were insufficient to explain such a
rapid disappearance of glutamine. HPLC-ESI-MS
experiments showed that oligomeric species containing
up to four c-glutamyl residues linked to a single gluta-
mine molecule were formed (Fig. 3; Fig. S3).
C
Fig. 2. Time course of the reaction of glutamine in the presence of
B. subtilis c-GT at three different pH values. The enzyme
(43.6 mU) was added at 22 °C to three solutions of glutamine
(100 mM) at pH 7.4 (top), pH 8.2 (middle), and pH 9.8 (bottom). At
fixed time intervals, 100-lL aliquots were withdrawn from each
reaction mixture, derivatized with DbsCl, and analyzed with HPLC.
Calibration curves were constructed by the use of authentic
materials, with the aim of monitoring the concentrations of
glutamine, glutamic acid, and c-glutamylglutamine.
The same experiment was repeated with D-gluta-
mine, and identical results were obtained (Fig. S4).
In order to assess the role of substrate concentration
in the autotranspeptidation reaction, further experi-
ments were carried out with lower glutamine concen-
trations (25 mM and 50 mM) at pH 9.8 (Fig. S5). The
main reaction observed in the mixture containing
25 mM glutamine was hydrolysis (Fig. S5A). However,
low amounts of the autotranspeptidation products c-
When the concentration of the starting glutamine was
increased to 50 mM, the autotranspeptidation products
were more evident, and their amounts increased for up
to 2 h of reaction time (Figs S5B and S7). The peak
attributable to the hydrolysis product glutamic acid in
the HPLC chromatograms increased slightly more
slowly, and reached its maximum at ~ 7 h, owing to
the enzyme-catalyzed hydrolysis not only of the start-
ing glutamine, but also of the previously formed auto-
transpeptidation products.
glutamylglutamine,
c-glutamyl-c-glutamylglutamine
and c-glutamylglutamic acid were found with HPLC-
MS experiments recorded after 1 h of reaction time
(Fig. S6). These products were rapidly hydrolyzed, and
the glutamic acid concentration in the reaction mixture
reached its maximum within 5 h of reaction time.
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B. subtilis c-glutamyltransferase activities
C. Calvio et al.
donor at elevated pH, only the formation of c-glutam-
ylglutamine was reported, but no mention of the for-
mation of oligomeric compounds can be found in the
literature [47]. In the rat kidney c-GT-catalyzed reac-
tion of glutamine, the presence of c-glutamyl-c-glutam-
ylglutamine was only tentatively supposed [48]. The
ability of B. subtilis c-GT to also process oligomeric c-
glutamyl derivatives is ascribable to the possible role
of this enzyme in the homeostasis of poly-c-glutamic
acid produced by some strains of B. subtilis [49].
Third, the ability of B. subtilis c-GT to accept D-glu-
tamine as an acceptor substrate is another difference
from both mammalian and E. coli c-GTs. The latter
can indeed accept c-glutamyl compounds with the D-
configuration as donors, but amino acid acceptors
have to be in the L-configuration [3,13]. The similar
results obtained when L-glutamine and D-glutamine
were subjected to the action of B. subtilis c-GT could,
once again, be related to the involvement of the
enzyme in homeostasis of the extracellular poly-c-glu-
tamic acid, as this polymeric material is usually formed
by L-glutamic acid and D-glutamic acid units [50] in
different proportions, depending on the Bacillus strain
and environmental conditions [51,52].
Fig. 3. HPLC chromatogram derived from an HPLC-ESI-MS
experiment carried out on the sample obtained by incubating
glutamine and B. subtilis c-GT at pH 9.8 after 2 h of reaction at
22 °C. DbsNH2, dabsyl amide.
The results obtained in these experiments are, in our
opinion, noteworthy for at least three reasons. First,
they confirmed the glutaminase activity of B. subtilis
c-GT, but they also showed the ability of the enzyme
to recognize glutamine as an acceptor substrate in a
transpeptidation reaction. This is in apparent conflict
with results previously reported, in which the transpep-
tidase activity towards glutamine was reported to be
very low [4]. However, those results were obtained at a
lower pH value (8.5), below the pKa of the glutamine
amino group. It was proposed that an alkaline pH is
beneficial for the c-GT-catalyzed transpeptidation
reaction, in that it increases the concentration of un-
protonated nucleophilic amino groups [44]. In our
case, the autotranspeptidation reaction was mainly
observed at pH 9.8, which is slightly above 9.1, the
pKa of the amino group of glutamine.
Time course of the transpeptidation reaction
The time course of the transpeptidation reaction was
monitored at pH 8.5 with equimolar amounts of
100 mM glutamine as the donor and glycylglycine as
the acceptor. The reaction course monitored with
HPLC (Fig. 4) showed that the donor glutamine dis-
appeared within 24 h. In the meantime, the glutamic
Second, the enzyme can also accept as substrates
oligomeric poly-c-glutamyl derivatives, as shown by
the HPLC-ESI-MS experiments. Although the forma-
tion of oligomeric derivatives carrying more than two
c-glutamyl residues has already been proposed, and,
on the basis of those findings, the involvement of
B. subtilis c-GT in the biosynthesis of c-PGA has been
suggested [28], our results constitute the first clear evi-
dence of their presence in a c-GT-catalyzed reaction in
which only glutamine acts as the substrate. When
E. coli c-GT was used for synthetic purposes in the
presence of a high concentration of glutamine as the
Fig. 4. Time
course
of
the
B. subtilis
c-GT-catalyzed
transpeptidation reaction with glutamine as the donor and
glycylglycine as the acceptor at pH 8.5 and 22 °C. Equimolar
proportions of donor and acceptor (100 mM) were used, and the
concentrations of glutamine, glycylglycine, glutamic acid and c-
glutamylglutamine were monitored with HPLC at time intervals
after derivatization of the withdrawn samples with DbsCl.
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C. Calvio et al.
B. subtilis c-glutamyltransferase activities
acid content in the reaction mixture increased. The
expected product, c-glutamylglycylglycine, accumu-
lated during the first 5 h of reaction time, reaching an
estimated concentration of 30 mM, and the amount of
the starting compound glycylglycine decreased accord-
ingly during the same time period. Other peaks noticed
in the chromatogram were attributed to c-glutamyl-c-
glutamylglycylglycine and c-glutamylglutamine, by
means of an HPLC-MS experiment (Fig. S8). After
5 h of reaction time, the free glycylglycine concentra-
tion started to increase, while the transpeptidation
product disappeared, owing to irreversible hydrolysis
reactions. Because the hydrolysis of the donor gluta-
mine is also irreversible, within 24 h the reaction mix-
ture contained mainly the starting glycylglycine, with a
very low concentration of residual c-glutamylglycylgly-
cine and the irreversibly formed glutamic acid.
as being representative of an acceptor with a low pKa of
the nucleophilic amino group (8.2); methionine, phenyl-
alanine and serine were selected as having intermediate,
similar pKa values that are also similar to that of gluta-
mine (9.21, 9.13, 9.15, and 9.13, respectively, for methio-
nine, phenylalanine, serine, and glutamine). Alanine,
with a pKa of the amino group of 9.69, was representa-
tive of an acceptor with a slighly higher pKa.
Glycylglycine was early recognized as a very good
acceptor for c-GT-catalyzed transpeptidation reactions
[11]; it therefore became the substrate of choice and the
reference compound for such studies. This was attribut-
able to the low pKa (8.2) of its nucleophilic amino group
with respect to those of other amino acids [44]. The low
pKa of the glycylglycine amino group ensures that a
large proportion of deprotonated, reactive nucleophilic
species is present, even at those pH values at which the
amino groups of standard amino acids are mainly in the
protonated, unreactive form.
Therefore, the hydrolysis and the autotranspeptida-
tion (Fig. 2) of the donor glutamine seem to be com-
petitive reactions with respect to the expected
transpeptidation reaction at pH 8.5. Mammalian c-
GTs (e.g. equine kidney c-GT) show a different behav-
ior, owing to the very low extent of autotranspeptida-
tion and the transpeptidation reaction being favored
with respect to hydrolysis at basic pH values [39,53].
Thus, it seems to be more difficult to separate the
three enzymatic activities for B. subtilis c-GT than for
other c-GTs.
In the transpeptidation reactions with glycylglycine
as the acceptor substrate, the product concentration
appeared to be appreciable even between pH 7.5 and
pH 9.0; after that, it increased with increasing pH
(Fig. 5). However c-glutamylglutamine, derived from
the autotranspeptidation reaction, was also always
noted in the reaction mixtures, albeit in lower concen-
trations than those of the desired transpeptidation
product (data not shown). As the pH of the reaction
mixtures approximated the pKa of the donor glutamine
amino group, the concentration of the residual gluta-
mine after 1 h of reaction time decreased, owing to the
pH dependence of the transpeptidation and
autotranspeptidation reactions
The results described so far showed that the extent of
the autotranspeptidation reaction of glutamine was
clearly dependent on pH. Moreover, the hydrolase
activity of B. subtilis c-GT, unlike the activities of
other c-GTs [12,54], seems not to be inhibited at
higher pH values. On the basis of these observations,
the transpeptidation reactions between glutamine as
the donor and selected acceptor amino acids were eval-
uated at different pH values, ranging from 7.5 to 11.0.
Through this set of experiments, we tried to assess the
degree of transpeptidation with respect to the auto-
transpeptidation reaction. Usually, 100 mM solutions
of glutamine and the acceptor amino acid were incu-
bated in the presence of B. subtilis c-GT for 1 h at
22 °C, and 100-lL aliquots of the reaction mixtures
were then withdrawn, derivatized with DbsCl, and
analyzed with HPLC. Only the reaction with phenylal-
anine as the acceptor substrate was carried out at
50 mM, for solubility reasons.
Fig. 5. pH dependence of the transpeptidation reaction for
selected acceptors. Equimolar amounts (100 mM) of the donor
glutamine and the candidate acceptor were incubated for 1 h at
different pH values in the presence of a fixed amount of B. subtilis
c-GT, and 100-lL samples of each reaction mixture were then
withdrawn, derivatized with DbsCl, and analyzed with HPLC.
Results are relative to the maximum concentration of
transpeptidation product obtained (glycylglycine as the acceptor
and pH 11).
Candidate amino acids were selected on the basis of
the pKa of the amino group. Glycylglycine was chosen
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B. subtilis c-glutamyltransferase activities
C. Calvio et al.
autotranspeptidation reaction. Usually, glutamine was
hardly detectable after 1 h in reactions carried out at
pH 9.5 and above. HPLC-MS experiments on the
reaction at pH 10.5 demonstrated that, as well as
being present in the expected autotranspeptidation
product, the c-glutamyl moiety derived from the
starting glutamine also ended up in c-glutamyl-c-glut-
amylglycylglycine and c-glutamyl-c-glutamyl-c-glutam-
ylglycylglycine. Species formed by two c-glutamyl
residues bound to a single glutamine molecule were
also identified (Fig. S9).
with glutamine as acceptor substrates, provided that
the pH of the reaction medium is maintained above
the pKa of their nucleophilic amino groups.
Moreover, in the presence of a good nucleophile,
the hydrolysis reaction is minimized, as demonstrated
by the low amount of glutamic acid.
Serine was also chosen as having a pKa of the amino
group similar to that of glutamine; however, its behav-
ior in B. subtilis c-GT-catalyzed reactions was differ-
ent. It showed a very low propensity to act as an
acceptor as compared with methionine and phenylala-
nine. The transpeptidation product c-glutamylserine
was evident only at relatively high pH values and,
more interestingly, the donor substrate glutamine did
not disappear (Figs S14 and S15).
It is of note that the pH-activity profile obtained
with glutamine as the donor by monitoring the forma-
tion of the transpeptidation product by means of
HPLC is different from that obtained with GPNA as
the donor substrate and monitoring of the reaction
spectrophotometrically (Figs 1 and 5). When GPNA
was used as the donor, liberation of p-nitroaniline, and
hence only formation of the c-glutamyl enzyme inter-
mediate (Scheme 1), is detected. As stated above, at
elevated pH, autotranspeptidation and poly-glutamyla-
tion reactions occurred, so the rate of release of p-nit-
roaniline was high, owing to the rapid enzyme
turnover, which may also depend on autotranspeptida-
tion and hydrolysis reactions in addition to the
expected transpeptidation.
Similar results were obtained with alanine as the
acceptor substrate. In this case, HPLC-MS of the reac-
tion at pH 10.5 showed the presence of only unreacted
starting materials and very low amounts of transpepti-
dation and autotranspeptidation products (Fig. S17).
The low propensity of serine and alanine to act as
acceptor substrates in c-GT-catalyzed transpeptidation
reactions has already been established for B. subtilis
c-GT [4] and several related enzymes [17,19,28], but this
cannot explain the lack of the autotranspeptidation
reaction involving glutamine. Such a behavior could be
indicative of an inhibitory activity of a component of
the reaction mixture towards the enzyme. Whether the
inhibitory activity is attributable to the amino acids
themselves, or arises from the newly formed c-glutamyl
derivatives, is currently under investigation in our labo-
ratories. It is worth noting that inhibition phenomena
have been reported for alanine in E. coli c-GT-catalyzed
reactions [3], and inhibitory activity towards rat kidney
c-GT was observed for a number of amino acids [14].
However, serine and alanine have been reported to be
good acceptors in transpeptidation reactions catalyzed
by c-GTs of mammalian origin [13], albeit serine was
recognized as a competitive inhibitor of human c-GT
[11,45], and a serine–borate complex is considered to be
a transition state analog inhibitor for this enzyme [55].
Reactions in which phenylalanine and methionine
were used as acceptors showed low activity at the
lower pH values, with a steady increase in transpepti-
dation product concentration with pH (Fig. 5).
Taking the reaction with methionine as an example,
HPLC-MS analysis of the derivatized reaction mixture
after 1 h of reaction at pH 8.0 confirmed the presence
of the unreacted methionine and glutamine as the
major components (Fig. S10). The hydrolysis product
glutamic acid, the expected c-glutamylmethionine and
the autotranspeptidation product c-glutamylglutamine
were present in similar, quite low, concentrations.
After 1 h of reaction at pH 10.5, on the other hand,
the peak of the transpeptidation product c-glutamyl-
methionine was the most prominent among those of
the newly formed compounds (Fig. S11). As reported
previously, glutamine was present in very low concen-
trations, as was glutamic acid. No peaks attributable
to poly(c-glutamyl)glutamine were detected with
HPLC-MS, although low amounts of compounds
formed by up to three c-glutamyl moieties bound to a
single methionine were identified.
Use of B. subtilis c-GT as a biocatalyst
The results described so far suggest that the B. subtilis
c-GT-catalyzed transpeptidation reaction between glu-
tamine as the donor and an acceptor amino acid is
feasible, provided that the pH of the reaction medium
is basic enough to ensure a certain concentration of a
deprotonated, nucleophilic amino group of the accep-
tor molecule. It was also anticipated that, at the
required basic pH, the donor glutamine would disap-
pear rapidly from the reaction mixture, owing to the
Phenylalanine behaved similarly, and its activity as
an acceptor substrate was lower than that of methio-
nine (Figs. 5, S12 and S13).
On the basis of these results, it can be concluded
that methionine and phenylalanine compete effectively
238
FEBS Journal 281 (2014) 232–245 ª 2013 FEBS

C. Calvio et al.
B. subtilis c-glutamyltransferase activities
autotranspeptidation reaction affording c-glutamylglu-
tamine as the major byproduct, together with poly-
glutamylated glutamines in lower concentrations.
The B. subtilis c-GT-catalyzed synthesis of c-glutam-
ylmethionine [33], a known naturally occurring flavor
enhancer, was chosen in order to test this possibility.
c-GT-catalyzed synthesis of c-glutamylmethionine was
carried out at an analytical level (1 mL of a 100 mM
solution of glutamine and methionine), and monitored
with HPLC. The results are shown in Fig. 6. The con-
centration of glutamine dropped rapidly, whereas the
decrease in the methionine concentration was accompa-
nied by a parallel increase in c-glutamylmethionine up
to a calculated concentration of ~ 50 mM (50% yield)
within the first hour of reaction time, after which
their concentrations remained fairly constant for up
to ~ 2 h. The amounts of both glutamic acid and
c-glutamylglutamine remained low throughout the
reaction. Substantial amounts of glutamic acid and
hydrolysis of the formed product c-glutamylmethionine
became evident only after 24 h of reaction time (data
not shown).
which the autotranspeptidation and poly-c-glutamyla-
tion reactions of glutamine, occurring mainly at the
beginning of the reaction, reduce the molar concentra-
tions of the donor species, so that the acceptor sub-
strate methionine will be in molar excess with respect
to the donors. The transfer of the c-glutamyl moieties
from the newly formed donor species to methionine
therefore becomes the preferred reaction path up to
2 h of reaction time.
The reaction was repeated at at a 1-mmol prepara-
tive level with 10 mL of starting solution. After 1.5 h
of reaction time, the enzyme was inactivated, and the
products were isolated with ion exchange chromatog-
raphy. c-Glutamylmethionine was obtained in a yield
of 45%. From the reaction mixture, unreacted methio-
nine (~ 40%) and low amounts of glutamic acid and
c-glutamylglutamine (< 10% each) were also isolated
and identified with NMR and MS.
Conclusions
Our results offer an unprecedented overview of the
activities exerted by B. subtilis c-GT from the view-
point of the qualitative and quantitative determination
of the products formed in the enzyme-catalyzed reac-
tions under various experimental conditions.
The constant concentration of the transpeptidation
product even after the disappearance of the donor sub-
strate glutamine can be explained, in our opinion, by
the early formation of c-glutamylglutamine and other
poly-glutamylated species, which became able to func-
tion as c-glutamyl donors in later reaction stages. In
these circumstances, a complex equilibrium arises, in
The common thread emerging in our investigation
was the strong influence of pH in modulating enzyme
activity. When the pH was increased beyond the pKa
value of the nucleophilic amino group, glutamine,
which is commonly considered to be a poor acceptor
in c-GT-catalyzed transpeptidation reactions, under-
went a rapid autotranspeptidation reaction, affording
not only c-glutamylglutamine, but also oligomeric spe-
cies, in which up to four c-glutamyl residues were
linked to a single glutamine molecule. This behavior
was also seen with other known acceptor peptides or
amino acids. Poly-glutamylation products were indeed
found when glycylglycine, methionine and phenylala-
nine were used, provided that the pH of the reaction
mixtures was basic enough to ensure the presence of a
deprotonated, nucleophilic amino group in a reason-
able concentration.
This finding, together with the observation that
D-glutamine is also recognized both as a donor and as
an acceptor substrate, leads us to assume the involve-
ment of B. subtilis c-GT in the homeostasis of extra-
cellular c-PGA, supporting the hypothesis that the
enzyme acts in vivo as an exo-hydrolase towards
c-PGA [49]. The liberated glutamic acid is then used
by the organism as a source of carbon and nitrogen.
In this context, some structural features of the enzyme
appear to gain significance. The lack of the lid loop can
Fig. 6. Time
course
of
the
B. subtilis
c-GT-catalyzed
transpeptidation reaction of glutamine (donor) and methionine
(acceptor) at pH 10 and 22 °C to give c-glutamylmethionine. The
formation of glutamic acid was also monitored. Reaction
conditions: equimolar amounts of glutamine and methionine
(100 mM) in sodium carbonate/hydrogencarbonate buffer, pH 10.0,
22 °C, B. subtilis c-GT 4.36 mUÁmLÀ1
.
FEBS Journal 281 (2014) 232–245 ª 2013 FEBS
239

B. subtilis c-glutamyltransferase activities
C. Calvio et al.
be related to the molecular sizes of the physiological
substrates. c-GTs that accept only discrete, low molecu-
lar mass c-glutamyl derivatives (e.g. glutathione) as sub-
strates possess the lid loop, whereas, in enzymes
involved in the homeostasis or remodeling of large poly-
mers composed of a huge number of c-glutamyl resi-
dues, the lid loop seems to be absent, as in some strains
of B. subtilis c-GT, e.g. natto, or capD from B. anthra-
cis, even if these enzymes have different physiological
roles. The significance of the lid loop, although not com-
pletely understood, could therefore be related to sub-
strate selection [56], although, at the current state of
knowledge, its involvement in the modulation of the
hydrolase/transpeptidase activity of the enzyme cannot
be ruled out (Calvio C, Morelli CF, unpublished).
Although the involvement of c-GT in the biosynthesis
of poly-c-glutamic acid has been proposed [28], its cata-
lytic properties do not support this view. Glutamic acid
is indeed unable to act as a c-glutamyl donor, and the
autotranspeptidase activity of B. subtilis c-GT towards
glutamine becomes appreciable at high substrate
concentrations and rather high pH values (~ 10). The
discovery of a multienzymatic system able to synthetize
c-PGA [57,58] therefore points towards a hydrolytic
function of B. subtilis c-GT in the homeostasis of
c-PGA.
Because of the transpeptidase activity, B. subtilis c-
GT could be used as a biocatalyst for the enzymatic
synthesis of c-glutamyl derivatives, avoiding the pro-
tection/deprotection steps required with chemical
approaches. The glutaminase activity allows glutamine
to be used as the donor compound. A possible draw-
back is the formation of poly-c-glutamylglutamine
through the autotranspeptidation reaction. In a preli-
minary, unoptimized experiment, we obtained the
desired product in moderate yield, but adjustment of
reaction conditions (temperature, time, and reactant
molar ratio) could lead to improved results.
Analytical TLC was performed on Silica gel 60 F254 pre-
coated aluminum sheets (Merck, Darmstadt, Germany).
Plates were 9 cm high; mixtures were spotted 1.5 cm from
the inferior edge, and plates were eluted with n-BuOH/
AcOH/water (4 : 1 : 1) to a line drawn 7 cm from the start.
Components were detected by inspection under a UV lamp
(254 nm) in the case of chromophoric substances, and by
spraying a 1% ninhydrin solution in ethanol followed by
heating at ~ 150 °C.
Ion exchange chromatography was carried out with
Dowex 1 9 8 resin 200–400 mesh (Aldrich) in the acetate
form. The resin was regenerated with standard methods,
and equilibrated by passing five volumes of 0.5 M acetic
acid through it prior to use.
Analytical HPLC was performed on a Waters 600 (Milli-
pore) instrument equipped with a Hewlett-Packard diode
array detector Series 1050 (Palo Alto, CA, USA) at
436 nm, with a 250 9 4.60-mm Gemini RP C18 column
(5 lm) (Phenomenex, Torrance, CA, USA). Eluents: 0.1%
aqueous solution of trifluoroacetic acid (solvent A) and
acetonitrile/solvent A (80 : 20) (solvent B). Elution gradi-
ent: 0–5-min isocratic elution with solvent A/solvent B
(80 : 20); 5–35-min linear gradient up to solvent A/
solvent B of 20 : 80; 35–45-min isocratic -elution with
solvent A/solvent B (20 : 80). Flow rate: 1.5 mLÁminÀ1
.
1H-NMR and 13C-NMR spectra were acquired at
400.13 MHz and 100.61 MHz, respectively, in D2O or in
dimethylsulfoxide d6 on a Bruker Advance 400 spectrometer
(Bruker, Karlsruhe, Germany) interfaced with a workstation
running a Windows operating system and equipped with a
TOPSPIN software package. 13C-signal multiplicities were
based on attached proton test experiments. Chemical shifts
are given in p.p.m. (d), and are referenced to 3-(trimethylsi-
lyl)propionic-2,2,3,3-d4 acid sodium salt (dMe 0.00 p.p.m.)
as an external standard, or to dimethylsulfoxide signal as an
internal standard (dH dimethylsulfoxide 2.50 p.p.m.; dC
dimethylsulfoxide 39.52 p.p.m.). Spectral analyses were car-
ried out with INMR READER software (www.inmr.net, last
access September, 2013) on an Apple computer.
ESI-MS spectra were recorded on a Thermo Finnigan
LCQ Advantage spectrometer (Hemel Hempstead, UK).
UV measurements were carried out with a Jasco V-360
Spectrophotometer (Jasco International, Tokyo, Japan).
HPLC-MS was performed with a Thermo Finnigan
Surveyor LC pump equipped with a Thermo Finnigan
Surveyor photodiode array detector and interfaced with the
ESI ThermoFinnigan LCQ Advantage spectrometer.
The search for suitable reaction conditions and for a
readily available donor compound other than gluta-
mine, in order to minimize autotranspeptidation
side reactions, is currently being pursued in our
laboratories.
Experimental procedures
General
Enzyme production and purification
L-Glutamine, D-glutamine, L-methionine, L-phenylalanine,
L-serine, L-alanine, L-glutamic acid, N-phtaloyl-L-glutamic
acid, phtalic anhydride, GPNA, DbsCl and anhydrous dim-
ethylformamide were from Aldrich, and were used as
received.
For enzyme production and purification and for the synth-
esis of reference compounds, see Doc. S1.
Enzyme aliquots (60 lL) were diluted to 500 lL with
appropriate buffer prior to use.
240
FEBS Journal 281 (2014) 232–245 ª 2013 FEBS

C. Calvio et al.
B. subtilis c-glutamyltransferase activities
heating was continued for a further 5 min, in order to
evaporate most of the acetone. The vial was cooled with
current water, and 500 lL of the resulting orange–red solu-
tion was diluted with 500 lL of a 0.1% aqueous solution
of trifluoroacetic acid and used for HPLC analysis. Deriva-
tized samples are stable for months, and can be stored at
–20 °C.
Enzyme activity assay
One unit of B. subtilis c-GT was defined as the amount of
enzyme that liberates 1 lmole of p-nitroaniline per minute
from GPNA in the presence of the acceptor glycylglycine.
To
a solution of GPNA (0.25 mM) and glycylglycine
(2.0 mM) in Tris buffer at pH 8.5 and 22 °C, B. subtilis
c-GT was added (10 lL; final volume of 2.0 mL), and the
release of p-nitroaniline was monitored for 4 min at
410 nm by means of a UV spectrophotometer. The amount
of released p-nitroaniline was determined by use of a cali-
bration curve obtained with freshly recrystallized, pure
p-nitroaniline. Experiments were carried out in triplicate.
Time course of the c-GT-catalyzed reaction of
glutamine at three pH values
Calibration curves were constructed for glutamine, glutamic
acid and c-glutamylglutamine with increasing concentra-
tions of authentic materials in a sodium carbonate/hydrog-
encarbonate buffer at pH 8.5. Aliquots of 100 lL of each
solution were derivatized in the presence of 100 lL of
50 mM L-leucine as the internal standard, as described
above, and the obtained samples were analyzed with
HPLC.
pH-activity profile
The pH-activity profile was determined at 22 °C with solu-
tions at different pH values from 6.0 to 11.0 (final volume
of 2.0 mL), containing 0.25 mM GPNA alone (hydrolysis),
and in the presence of 2.0 mM glycylglycine (transpeptida-
tion). Reactions were initiated by the addition of 10 lL of
enzyme solution (21.9 mU). Initial velocities were measured
by monitoring the release of p-nitroaniline at 410 nm for
2 min, with the absorbance being read every 10 s. The
amount of released p-nitroaniline was calculated from a
calibration curve obtained with freshly recrystallized, pure
p-nitroaniline. Experiments were carried out in duplicate.
To three solutions of glutamine (L-glutamine or D-gluta-
mine, 1 mL, 100 mM) at three different pH values (7.4,
phosphate buffer; 8.2 and 9.8, sodium carbonate/hydrogen-
carbonate buffers) in a 2-mL Eppendorf tube, B. subtilis
c-GT (50 lL, 0.109 U) was added. The Eppendorf tube
was sealed and shaken briefly by means of a vortex appara-
tus. Reactions were carried out at 22 °C for 24 h, and, at
fixed time intervals (0, 1, 2, 3, 5, 7 and 24 h), 100-lL aliqu-
ots were withdrawn from each mixture, derivatized as
described, and analyzed for glutamine, glutamic acid and
c-glutamylglutamine content by use of the corresponding
calibration curves.
Precolumn derivatization procedure for HPLC
analysis
The method of Keillor [59] was used, with some modifica-
tions.
A 10 mM solution was prepared by dissolving
Time course of the transpeptidation reaction
between glutamine donor and glycylglycine
acceptor
32.4 mg of DbsCl in 7–8 mL of dry acetone. The solution
was filtered through a Millipore membrane into a 10-mL
volumetric flask, and the volume was adjusted with dry
acetone. The solution was transferred into a dark-glass,
screw-capped bottle, and stored at 4 °C until use. In the
precolumn derivatization procedure, the DbsCl was used in
at least two-fold excess with respect to the material to be
derivatized, taking into account that the initial reaction
mixtures were 100 mM in both the donor glutamine and the
acceptor. Reaction times were carefully observed. To a 100-
lL aliquot of the mixture to be derivatized, 100 lL of a
50 mM solution of the internal standard L-leucine dissolved
in a 100 mM hydrogencarbonate buffer at pH 8.2 was
added. The final volume was adjusted to 1.0 mL by adding
800 lL of buffer. From this solution, a 100-lL aliquot was
withdrawn and diluted with 400 lL of buffer in a glass vial
equipped with a screw cap with a rubber septum. To the
500 lL of aqueous solution, 500 lL of DbsCl in acetone
was added; the vial was then tightly sealed and placed in a
preheated water bath at 70 °C for 10 min, during which
time complete dissolution was achieved. After this time, a
needle was introduced through the rubber septum, and
Calibration curves were constructed for glutamine, glutamic
acid, glycylglycine, and c-glutamylglycylglycine, with
increasing concentrations of authentic materials in
a
sodium carbonate/hydrogencarbonate buffer at pH 8.5.
Aliquots of 100 lL of each solution were derivatized in the
presence of 100 lL of 50 mM L-leucine as the internal stan-
dard, as described above, and the obtained samples were
analyzed with HPLC.
The transpeptidation reaction was carried out with 1 mL
of a solution of glutamine (100 mM) and glycylglycine
(100 mM) in sodium carbonate/hydrogencarbonate buffer at
pH 8.5 in a 2-mL Eppendorf tube. The reaction was initi-
ated by the addition of B. subtilis c-GT (50 lL, 0.109 U).
After addition of the enzyme, the Eppendorf tube was
sealed and shaken briefly with a vortex apparatus. The
reaction was allowed to proceed at 22 °C for 24 h. At time
intervals (0, 1, 2, 3, 5, 7 and 24 h), 100 lL of the reaction
mixture was withdrawn, derivatized as described previously,
FEBS Journal 281 (2014) 232–245 ª 2013 FEBS
241

B. subtilis c-glutamyltransferase activities
C. Calvio et al.
and analyzed for glutamine, glutamic acid, glycylglycine
and c-glutamylglycylglycine content by use of the corre-
sponding calibration curves.
NaOH, and the solution was charged onto a column
packed with Dowex 1 9 8 200–400 mesh ion exchange
resin in the acetate form. The column was eluted with
water and with a scalar gradient of AcOH solutions
(0.5, 1.0, 1.5 and 2.0 M, three column volumes each).
Fractions were collected on the basis of TLC analysis,
and lyophilized. Four fractions were obtained: unreact-
ed methionine (76 mg), eluted with 0.5 M AcOH;
glutamic acid (12 mg), eluted with 1.5 M AcOH;
c-glutamylmethionine (117 mg, 42% yield, eluted
between 1.5 and 2.0 M AcOH; and c-glutamylgluta-
mine (23 mg), eluted with 2.0 M AcOH.
pH dependence of the transpeptidation reactions
of selected acceptor amino acids
Eight stock solutions (50 mL each) of glutamine (100 mM)
and the candidate amino acid (100 mM) were prepared with
sodium hydrogencarbonate or sodium carbonate/hydrogen-
carbonate solutions (0.1 M in different proportions, pH 7.5,
8.0, 8.5, 9.0, 9.5, 10.0, 10.5, and 11.0). The pH of each
solution was carefully adjusted with 1 M or 0.1 M NaOH.
One milliliter of each stock solution was then transferred
into an Eppendorf tube, and the reaction was initiated by
addition of the enzyme (50 lL, 0.109 U; total final volume
of 1.05 mL). After 1 h of reaction at 20 °C, 100 lL of each
reaction mixture was withdrawn, derivatized with DbsCl in
the presence of the internal standard as described, and ana-
lyzed with HPLC. The amounts of the transpeptidation
products in the reaction mixtures were estimated on the
basis of response factors calculated with respect to the
internal standard and determined by the use of synthetic,
pure samples. Experiments were carried out in triplicate.
Acknowledgements
This work was made possible by financial support
from Fondazione Alma Mater Ticinensis. We thank
A. M. Albertini for valuable discussions. We also wish
to thank A. Daghetti for performing HPLC-ESI-MS
experiments.
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Supporting information
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Additional supporting information may be found in
the online version of this article at the publisher’s web
site:
Doc. S1. Supplementary experimental procedures.
Fig. S1. His-tagged B. subtilis c-GT purification.
Fig. S2 HPLC-MS of sample at pH 8.2 after 2 h of
reaction time.
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Enzyme Microb Technol 30, 883–888.
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c-glutamyl transpeptidase: c-glutamyl peptide
formation. Life Sci 33, 1757–1762.
Fig. S3. HPLC-MS of sample at pH 9.8 after 2 h of
reaction time.
49 Kimura K, Tran LP, Uchida I & Itoh Y (2004)
Characterization of Bacillus subtilis c-
Fig. S4. HPLC-MS of sample at pH 9.8 after 2 h of
reaction time (D-glutamine).
Fig. S5. Time course of the reactions.
glutamyltransferase and its involvement in the
degradation of capsule poly-c-glutamate. Microbiology
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244
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B. subtilis c-glutamyltransferase activities
Fig. S6. HPLC-MS of sample from the reaction of
25 mM glutamine after 1 h of reaction time.
Fig. S7. HPLC-MS of sample from the reaction of
50 mM glutamine after 2 h of reaction time.
Fig. S8. HPLC-MS of sample at 2 h of reaction time.
Fig. S9. c-GT-catalyzed reaction between glutamine
and glycylglycine at pH 10.5.
and phenylalanine at pH 8.0.
Fig. S13. c-GT-catalyzed reaction between glutamine
and phenylalanine at pH 10.5.
Fig. S14. c-GT-catalyzed reaction between glutamine
and serine at pH 8.5.
Fig. S15. c-GT-catalyzed reaction between glutamine
and serine at pH 10.5.
Fig. S10. c-GT-catalyzed reaction between glutamine
and methionine at pH 8.0.
Fig. S16. c-GT-catalyzed reaction between glutamine
and alanine at pH 8.5.
Fig. S11. c-GT-catalyzed reaction between glutamine
and methionine at pH 10.5.
Fig. S17. c-GT-catalyzed reaction between glutamine
and alanine at pH 10.5.
Fig. S12 c-GT-catalyzed reaction between glutamine
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