Effect of the inserted active-site-covering lid loop on the catalytic activity of a mutant: B. subtilis γ-glutamyltransferase (GGT)

Article abstract of DOI:10.1039/c9ra05941e

Γ-Glutamylpeptides are compounds derived from the acylation of an amino acid or a short peptide by the γ-carboxyl carbon of the side chain of glutamic acid. Due to their altered chemico-physical and organoleptic properties, they may be interesting substitutes or precursors of parent compounds used in pharmaceutical, dietetic and cosmetic formulations. Some of them are naturally occurring flavor enhancers or are endowed with biological activities. Enzymatic approaches to the synthesis of γ-glutamyl derivatives based on the use of γ-glutamyltransferases (GGTs, EC 2.3.2.2) have been proposed, which should be able to alleviate the problems connected with the troublesome and low-yielding extraction from natural sources or the non-economical chemical synthesis, which requires protection/deprotection steps. With the aim of overcoming the current limitations in the use of GGTs as biocatalysts, a mutant GGT was investigated. The mutant GGT was obtained by inserting the active-site-covering lid loop of the E. coli GGT onto the structure of B. subtilis GGT. With respect to the wild-type enzyme, the mutant showed a more demanding substrate specificity and a low hydrolase activity. These results represent an attempt to correlate the structural features of a GGT to its different activities. However, the ability of the mutant enzyme to catalyze the subsequent addition of several γ-glutamyl units, inherited by the parent B. subtilis GGT, still represents a limitation to its full application as a biocatalyst for preparative purposes.

Full text of DOI:10.1039/c9ra05941e

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Effect of the inserted active-site-covering lid loop
on the catalytic activity of a mutant B. subtilis g-
glutamyltransferase (GGT)†
Cite this: RSC Adv., 2019, 9, 34699
a
b
c
ad
Michela Massone, Cinzia Calvio,
Marco Rabuffetti, Giovanna Speranza
a
and Carlo F. Morelli
*
g-Glutamylpeptides are compounds derived from the acylation of an amino acid or a short peptide by the
g-carboxyl carbon of the side chain of glutamic acid. Due to their altered chemico-physical and
organoleptic properties, they may be interesting substitutes or precursors of parent compounds used in
pharmaceutical, dietetic and cosmetic formulations. Some of them are naturally occurring flavor
enhancers or are endowed with biological activities. Enzymatic approaches to the synthesis of g-
glutamyl derivatives based on the use of g-glutamyltransferases (GGTs, EC 2.3.2.2) have been proposed,
which should be able to alleviate the problems connected with the troublesome and low-yielding
extraction from natural sources or the non-economical chemical synthesis, which requires protection/
deprotection steps. With the aim of overcoming the current limitations in the use of GGTs as
biocatalysts, a mutant GGT was investigated. The mutant GGT was obtained by inserting the active-site-
covering lid loop of the E. coli GGT onto the structure of B. subtilis GGT. With respect to the wild-type
enzyme, the mutant showed a more demanding substrate specificity and a low hydrolase activity. These
results represent an attempt to correlate the structural features of a GGT to its different activities.
However, the ability of the mutant enzyme to catalyze the subsequent addition of several g-glutamyl
units, inherited by the parent B. subtilis GGT, still represents a limitation to its full application as
a biocatalyst for preparative purposes.
Received 31st July 2019
Accepted 11th October 2019
DOI: 10.1039/c9ra05941e
rsc.li/rsc-advances
2
its biological activity as Se-methyl-selenocysteine. Also, the
bitter taste of branched-chain and aromatic amino acids 3–5 is
1
. Introduction
3
g-Glutamyl compounds (Fig. 1) are dened as being derived by severely reduced upon g-glutamylation.
the acylation of an amino acid or a short peptide through the g-
g-Glutamyl derivatives are also widespread in nature.
carboxyl carbon of a glutamic acid moiety. The prototypical g- Glutathione 1 represents the most abundant thiol in cells.
glutamyl derivative is glutathione (g-glutamyl-cysteinyl-glycine, Several g-glutamyl compounds, including glutathione itself, are
4,5
GSH) 1. With respect to parent compounds, g-glutamyl deriva- taste-active compounds with kokumi properties. Kokumi is
tives exhibit different physico-chemical and organoleptic a Japanese term that refers to gustative sensations related to
properties. For example, since the g-glutamyl moiety intro- mouthfulness, roundness and continuity. Kokumi compounds
6
duced has polar functional groups, the solubility of g-glutamyl are naturally occurring avor enhancers tasteless by them-
compounds in water may be enhanced compared to that of the selves but able to elicit their activity in conjunction with other
1
underivatized amino acids. g-Glutamyl-Se-methyl-seleno- taste-active components in food through the interaction with
7,8
cysteine 2 represents a convenient source of the micronutrient a Calcium sensing receptor (CaSR). g-Glutamyl derivatives
selenium, free of the unpleasant smell but equally effective in with kokumi properties have been isolated from several foods,
9
–12
e.g. compounds 3–8 among others,
and they are character-
13
a
istic constituents of edible vegetables (3 and 4), especially
Department of Chemistry, Universit a` degli Studi di Milano, Via Golgi, 19, 20133
those commonly used as seasoning ingredients in worldwide
Department of Biology and Biotechnology, Universit a` degli Studi di Pavia, Via Ferrata, cuisine, such as garlic (9) and onion (10). In addition, some
Milano, Italy. E-mail: carlo.morelli@unimi.it
b
14
15
9
, 27100 Pavia, Italy
of such g-glutamyl derivatives (8 and 10) show interesting bio-
c
16,17
Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente, Universit a` degli
logical activities,
nutraceuticals.
which lead to considering them as
Studi di Milano, Via Mangiagalli, 25, 20133 Milano, Italy
d
Istituto di Scienze e Tecnologie Molecolari (INSTM), CNR, c/o Department of
Despite their relatively simple chemical structure, a practical
transformation into g-glutamyl derivatives is not a trivial task.
The enzymatic approach has been envisaged as a more
Chemistry, Via Golgi, 19, 20133 Milano, Italy
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c9ra05941e
1
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convenient preparation method with respect to both the labo-
rious and low-yielding extractions from natural sources and the recently proposed,
Despite the use of glutaminases (EC 3.5.1.2) has also been
17,19–21
most of the enzymatic synthesis of g-
synthesis through the classical peptide chemistry, plagued by glutamyl compounds relies on the use of a g-glutamyltransfer-
2,22–42
the need of protection–deprotection steps.
ase (GGT, EC 2.3.2.2) as the biocatalyst.
GGT is a heterodimeric enzyme arising from a post-
Enzymatic synthesis of g-glutamyl derivatives can be tackled
by the use of synthetases such as g-glutamylmethylamide translational cleavage of a single polypeptide chain into
1
8
43–45
synthetase (EC 6.3.4.12), which however requires an ATP- a small and a large subunit.
It is mainly involved in gluta-
46
supplying and recycling system.
thione metabolism and is highly conserved from bacteria to
47
plants and animals. It cleaves glutathione, or other g-glutamyl
donors, transiently binding the g-glutamyl moiety in a g-
glutamyl-enzyme intermediate through an ester bond formed
by the hydroxyl group of the catalytically active threonine
residue, located at the N-terminal end of the small enzyme
48,49
subunit.
The g-glutamyl residue is then either transferred to
a water molecule in a hydrolysis reaction, or to an amino acid or
a short peptide in a transpeptidation reaction, leading to a new
50
g-glutamyl derivative (Scheme 1). The extent of the hydrolase
and transpeptidase activities exerted by GGTs is usually pH-
dependent, with basic pHs promoting the transpeptidation
reaction over the hydrolysis. The optimum pH of the reaction
a
medium for transpeptidation depends on the pK of the amino
group of the acceptor substrate, which should be in the
unprotonated form in order to be competent as a nucleo-
51,52
phile.
Although the transpeptidation-to-hydrolysis ratio for
mammalian GGT is usually higher and better controlled
53
through pH adjustment, bacterial GGTs are preferred as bio-
catalysts for preparative purposes. They are easily produced and
41,54–58
puried,
avoiding the risk of viral contamination associ-
ated with an enzyme isolated from an animal organ. In addi-
tion, glutamine can be used as a less expensive g-glutamyl-
donor substrate with respect to glutathione, required by GGTs
35,54,56
from higher organisms.
The GGT from E. coli (EcGGT) has been extensively used for
29
preparative purposes. The synthesis of g-glutamylvaline 3, g-
glutamylphenylalanine 5 (ref. 3) and g-glutamylglutamine 7
(ref. 32) has been employed to obtain tasteless precursors of the
bitter parent amino acids. Biologically active compounds such
27
as g-glutamyltaurine 11, g-D-glutamyl-L-tryptophan 12 (ref. 30)
34
and b-N-(g-L-glutamyl)phenylhydrazine 13, or prodrugs such
as g-glutamyl-L-DOPA 14 (ref. 23) have also been prepared using
EcGGT as biocatalyst. A common byproduct of EcGGT-catalyzed
reactions is g-glutamylglutamine 7, arising from a so-called
autotranspeptidation reaction, i.e. a reaction in which the
same compound acts as both the donor and the acceptor
substrate.
In addition to EcGGT, some GGTs from Bacillus spp. have
been proposed as biocatalysts. An evident structural feature that
characterizes the GGT of B. subtilis (BsGGT) is the lack of
a sequence, called lid loop, that covers the g-glutamyl binding
59
site in most of the known GGTs.
The synthesis of the amino acid theanine 15 was accom-
3
3,36,38
plished using GGTs from different strains of B. subtilis.
g-
39
Glutamyl-S-allylcysteine 9, g-glutamylphenylalanine 5 (ref. 40)
and g-glutamylleucine 4 (ref. 42) were obtained through reac-
tions catalyzed by the GGT from B. licheniformis, while GGT
from B. atrophaeus was used for the enzymatic synthesis of g-D-
41
Fig. 1 Examples of g-glutamyl derivatives.
glutamyl-L-tryptophan 12.
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Scheme 1 g-glutamyltransferase-catalyzed reactions.
Although attractive for their simplicity and for the lack of counterpart. LL-BsGGT then could have potentialities as a bio-
required protection/deprotection steps, in general the enzy- catalyst for the enzymatic synthesis of g-glutamyl derivatives
matic approaches to the synthesis of g-glutamyl derivatives with kokumi properties.
suffer from rather low yields and formation of byproducts from
autotranspeptidation (g-glutamylglutamine) and hydrolysis
reactions (glutamic acid).
2
. Experimental
2.1 General
We recently obtained a mutant GGT by inserting the lid loop
of EcGGT onto the structure of BsGGT and we called this L-Glutamine, L-glutamic acid, L-serine, glycilglycine (GlyGly), L-
enzyme LL-BsGGT (from Lid Loop B. subtilis GGT) (Fig. 2). glutamic acid 5-(p-nitroanilide) (GPNA) and 1-uoro-2,4-
Through the use of synthetic oligo-g-glutamylglutamines as dinitrobenzene (Sanger's reagent) were from Sigma Aldrich
model donor compounds in hydrolysis reactions, we concluded (Darmstadt, Germany); L-alanylglycine, L-2-aminobutrylglycine
that the presence of the lid loop is able to affect donor substrate (Abu-Gly) and L-leucylglycyne were from Bachem (Bubendorf,
60
selection on the basis of molecular size. In this work, the Switzerland). All reagents were used as received without further
promising features of LL-BsGGT have been elucidated, purication.
demonstrating that the recombinant enzyme shows enhanced
L-valylglycine and g-glutamylvalylglicine were prepared as
tranpeptidase activity with respect to its wt (wild-type) reported in ESI.† S-Allylcysteine and g-glutamyl-S-allylcysteine
52
were prepared as previously described.
HPLC-grade solvents were from Aldrich.
Analytical TLC was performed on silica gel F254 pre-coated
aluminum sheets (0.2 mm layer) (Merck, Darmstadt, Ger-
many). Elution solvents are reported for each specic product's
preparation. Detection: UV lamp (l 254 nm), 4.5% w/v CeSO4/
(
NH ) Mo O $4H O solution or 5% w/v ninhydrin solution in
4 6 7 24 2
ꢀ
ethanol, followed by heating at 150 C ca.
HPLC analyses were carried out with a Jasco instrument
equipped with UV/Vis detector, using a 250 ꢁ 4.6 mm Gemini
RP C18 column (Phenomenex, Torrance, CA, USA). Eluent A was
0.1% triuoroacetic acid; eluent B was a 80 : 20 mixture of
acetonitrile and eluent A. The compounds, derivatized with
Sanger's reagent, were analyzed with the following gradient: 0–
Fig. 2 Excerpt of the sequence alignment of E. coli GGT (upper line),
B. subtilis GGT (middle line) and the mutant LL-BsGGT (lower line). The
catalytically active N-terminal Thr residue of the small subunit is
marked with *. The sequence of the lid loop starts after residue 460
10 min, eluent A : eluent B 80 : 20 isocratic elution; 10–15 min,
linear gradient to eluent A : eluent B 70 : 30; 15–25 min, linear
gradient to eluent A : eluent B 40 : 40; 25–35 min, linear
(
overall numbering).
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gradient to eluent A : eluent B 40 : 60; 35–40 min, isocratic 2.5 pH-activity prole of LL-BsGGT
elution eluent A : eluent B 40 : 60; 40–60 min, column riequili-
Measurements were carried out in the same conditions of
enzyme activity assays, in the presence (“transpeptidation”) and
in absence (“hydrolysis”) of added GlyGly (100 mM), using
buffers at different pH values from 7 to 10. In particular, pHs
bration through linear gradient to eluent A eluent B 80 : 20.
Flow rate was 1 mL min and detection was at 356 nm.
ꢂ1
Ion exchange chromatography was performed with Dowex 1
ꢁ
8 resin 200–400 mesh (Aldrich, Darmstadt, Germany) in the
7.0, 7.5, 8.0, 8.5 were controlled by using 0.1 M Tris HCl buffers,
acetate form. Resin was equilibrated before use with 5 volumes
of 0.5 M acetic acid.
while pHs 9.0, 9.5, 10.0 through 0.1 M sodium carbonate/
sodium hydrogencarbonate buffers. Experiments were per-
formed in duplicate.
1
13
H-NMR and C-NMR spectra were acquired at 400.13 MHz
and 100.61 MHz, respectively, on a Bruker Advance 400 spec-
trometer (Bruker, Karlsruhe, Germany) interfaced with a work-
station running Windows operating system and equipped with
2.6 Substrate specicity
1
3
a TOPSPIN soware package. C signal multiplicities were Measurements were carried out in the same conditions of
based on attached proton test experiments (APT) and attribu- enzyme activity assays, by replacing GlyGly with the proper
tions were based on HSQC (Hetero Single Quantum Correlation) acceptor (see Table 1) at 100 mM concentration. Experiments
and HMBC (Hetero Multiple Bond Correlation) experiments. were performed in triplicate, using the wt BsGGT and the
Chemical shis are given in ppm (d) and are referenced to mutant enzyme LL-BsGGT aer normalization of their activity
solvent signal (dH D O 4.79 ppm) or to TSP (3-(trimethylsilyl) in the absence of added acceptor.
2
propionic-2,2,3,3-d4 acid sodium salt) as external standard
(d
0.00 ppm). Spectra analyses were carried out with inmr
Me
2.7 Pre-column derivatization procedure
Reader soware (http://www.inmr.net).
Standard solutions for calibration curves or aliquots of reaction
mixtures to be analyzed (20 mL) were rst diluted 1 : 20 with
water. 100 mL of the diluted solution was transferred into a pyrex
tube equipped with a perforated screw cap tted with a forcible
sealing septum. 50 mL of 5 mM L-serine in water was added as
the internal standard, followed by 350 mL borate buffer at pH
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 photo-
diode array detector and interfaced with the ESI Thermo Fin-
nigan LCQ Advantage spectrometer using column and elution
parameters reported previously.
8.5. The mixture was shaken and 500 mL of 10 mM 1-uoro-2,4-
dinitrobenzene (Sanger's reagent) solution in acetone was
ꢀ
added. The sealed tube was heated at 70 C for 45 minutes in
the dark. Then a needle was introduced into the septum and the
tube was le in the heated water bath for further 10 minutes, to
evaporate most of acetone. The tube was cooled under running
water and 300 mL of the resulting mixture was diluted 1 : 2 with
2.2 The mutant lid-loop B. subtilis GGT (LL-BsGGT)
The mutant enzyme LL-BsGGT was obtained during our studies
6
0
about the role of the lid-loop in GGTs.
.3 Enzyme activity assay
0.1% TFA solution, affording the sample for HPLC analysis.
2
2.8 LL-BsGGT-catalyzed reactions
ꢂ1
Enzyme activities were measured for BsGGT (0.639 mg mL
)
2.8.1 Assessment of reaction conditions. Reaction condi-
ꢂ1
and LL-BsGGT (0.524 mg mL ) prior to every experiments,
tions (effect and enzyme concentration and pH) were prelimi-
nary investigated as described in 2.3 (enzyme activity assay) and
in 2.5 (pH-activity prole of LL-BsGGT), respectively, using
ValGly or S-allylcysteine as acceptor substrates (100 mM)
instead of GlyGly.
Effect of temperature was investigated by carrying out reac-
tions at analytical level at different temperatures, as described
in 2.8.2.
55
following the procedure reported for B. subtilis (natto), with
some modications.
Reactions (2.0 mL) were carried out in plastic cuvettes con-
taining 1 mM GPNA and 100 mM GlyGly in Tris buffer at pH 8.5.
Reactions were initiated by adding 20 mL of diluted enzyme
solution. The release of p-nitroaniline was monitored at 410 nm
ꢀ
for 3 minutes at 21–23 C, recording data every 10 s. One
enzyme unit was dened as the amount of enzyme that liberates
2.8.2 Analytical level. General procedure. A 1 mL-mixture of
ꢂ1
ꢂ1
1
mmol mL min of p-nitroaniline in the presence of glycyl-
glutamine and the acceptor substrate (valylglycine or S-allyl-
cysteine), each at 100 mM concentration in 0.1 M Tris$HCl
buffer at the right pH (8.5 for ValGly, 9.5 for S-allylcysteine) and
glycine. p-Nitroaniline concentrations were estimated through
a calibration curve.
ꢂ1
LL-BsGGT (0.6, 1.2 or 4.0 U mL ) was stirred in a thermostatic
2.4 Assessment of hydrolase and transpeptidase activities
ꢀ
water bath at a xed temperature (either 25 or 40 C). At xed
Hydrolase and transpeptidase activities were evaluated with the time points, 20 mL aliquots were withdrawn, derivatized as
same procedure used for enzyme activity assays, in the presence described previously and analyzed by HPLC.
and in the absence of GlyGly as the acceptor, respectively.
2.8.3 Effect of a further addition of the donor substrate. A 1
Experiments were performed in duplicate using unstandardized mL-mixture of glutamine (100 mM), valylglycine (100 mM) and
ꢂ1
solutions of the three enzymes (BsGGT, EcGGT, LL-BsGGT).
LL-BsGGT (4.0 U mL ) in 0.1 M Tris$HCl buffer at pH 8.5 was
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ꢀ
a
stirred in a thermostatic water bath at 25 C. Aer 45 min Table 1 Acceptor substrate specificity of LL-BsGGT
reaction time, a further equivalent of glutamine (14 mg) was
added. Reaction was monitored at xed time points as
Entry
Acceptor
Relative activity
described in 2.8.2.
1
2
3
4
5
6
7
8
9
Gly-Gly
None
L-Ala
L-Cys
L-His
L-Ile
L-Leu
L-Met
D-Met
L-Phe
L-Ser
L-Val
S-Methyl-L-Cys
S-Ethyl-L-Cys
S-Allyl-L-Cys
Ala-Gly
Abu-Gly
Val-Gly
Leu-Gly
100
2.8.4 Preparative level. General procedure. A 5 mL-mixture
3.91 ꢃ 1.01 A
of glutamine and the acceptor substrate (valylglycine or S-
allylcysteine), each at 100 mM concentration in 0.1 M Tris$HCl
buffer at the right pH (8.5 for valylglycine, 9.5 for S-allylcysteine)
2.27 ꢃ 0.18
0.82 ꢃ 1.01
19.41 ꢃ 2.84 C
7.45 ꢃ 2.83 B
8.98 ꢃ 0.08 B
21.95 ꢃ 2.96 C
8.17 ꢃ 0.35 B
8.59 ꢃ 0.45 B
4.03 ꢃ 0.69 A
4.54 ꢃ 1.55 A
16.43 ꢃ 0.83 C
19.16 ꢃ 0.55 C
46.31 ꢃ 5.15
65.43 ꢃ 3.33
38.23 ꢃ 2.32
10.32 ꢃ 1.65 B
4.44 ꢃ 0.57 A
ꢂ1
and LL-BsGGT (0.6 or 4.0 U mL ) was stirred in a thermostatic
ꢀ
water bath at 25 C for the appropriate time (5 hours for valyl-
glycine and 6 hours for S-allylcysteine). Reactions were stopped
by inactivating the enzyme at 95 C for 10 min. The pH of the
mixture was adjusted to 9.5 with 1 M NaOH and the solution
ꢀ
10
1
1
1
1
2
3
was loaded onto a column packed with Dowex 1 ꢁ 8 ion
exchange resin in the acetate form. The column was eluted with 14
water (4 column volumes) and then with a stepwise gradient of 15
1
1
1
1
6
7
8
9
acetic acid solution (0.5, 1.0, 1.5, and 2.0 M, three column
volumes each), collecting eluate in fractions. On the basis of
TLC analysis (silica gel, n-BuOH/water/AcOH 3 : 1 : 1; staining
reagent: ninhydrin), fractions were combined and freeze-dried.
a
Experiments were carried out at pH 8.5 in Tris HCl buffer using 1 mM
2.8.5 g-Glutamyl-L-valylglycine. Obtained in 35% isolated
g-glutamyl-p-nitroanilide as the donor substrate in the presence of 100
mM acceptors in order to favor transpeptidation over hydrolysis and
autotranspeptidation reactions. Liberation of p-nitroaniline was
monitored continuously at 410 nm and initial velocities were derived
from the slopes of the resulting curves in their initial, linear range.
ꢂ1
yield aer 5 hours reaction time using 0.6 U mL LL-BsGGT.
1
H-NMR (400 MHz; D
2
O): d 4.17 (d, J ¼ 6.8 Hz, 1H, Ha Val),
4
.00 (d, J ¼ 17.8 Hz, 1H, Ha1 Gly), 3.97 (d, J ¼ 17.8 Hz, 1H, Ha2
Gly), 3.83 (t, J ¼ 6.4 Hz, 1H, Ha Glu), 2.61–2.48 (m, 2H, Hg Glu), Results are expressed in relation to the activity measured using GlyGly
as the acceptor, taken as 100. Values below that recorded in the
absence of the acceptor substrate are assumed as
transpeptidase activity. Values labelled with the same letter are not
2
.19–2.09 (m, 3H, Hb Glu and Hb Val), 0.97 (d, J ¼ 6.8 Hz, 3H,
a lack of
Me1 Val), 0.98 (d, J ¼ 6.8 Hz, 3H, Me2 Val).
1
3
C NMR (101 MHz; D O): d 174.9 (COg Glu), 174.1 (CO Val), statistically different at 95% condence level (Tukey test, p > 0.05).
2
1
4
73.49 (CO Gly), 173.40 (COa Glu), 59.6 (Ca Val), 53.8 (Ca Glu),
1.3 (Ca Gly), 31.1 (Cg Glu), 29.9 (Cb Val), 26.1 (Cb Glu), 18.3
(
Me-1 Val), 17.3 (Me-2 Val).
bacterial GGTs due to the presence of an added acceptor is
ꢂ
52,54,61
ESI-MS, negative mode: m/z 302.56 [M ꢂ H] ; 324.55 [M ꢂ 2H normally modest,
and the mutant LL-BsGGT differs from
ꢂ
+
Na]
the parent wt BsGGT only for the presence of the inserted lid-
2
.8.6 g-Glutamyl-S-allyl-L-cysteine. Obtained in 21% iso- loop, thus underlining the importance of this structural
lated yield aer 6 hours reaction time using 0.6 U mL LL- feature in modulating enzyme functions.
ꢂ1
BsGGT.
The activity of LL-BsGGT was then compared with that of
O): d 5.84 (ddt, J ¼ 17.1, 9.9, 7.2 Hz, the wt counterpart aer normalization of the two enzymes
H, vinyl CH), 5.23–5.17 (m, 2H, vinyl CH
), 4.56 (dd, J ¼ 8.2, through reactions carried out in the absence of the acceptor
.8 Hz, 1H, Ha Cys), 3.90 (t, J ¼ 6.3 Hz, 1H, Ha Glu), 3.23 (d, J ¼ substrate. In repeated experiments the activity of the wt enzyme
.2 Hz, 2H, allyl CH
), 3.05 (dd, J ¼ 14.1, 4.8 Hz, 1H, Hb1 Cys),
.89 (dd, J ¼ 14.1, 8.2 Hz, 1H, Hb2 Cys), 2.61–2.49 (m, 2H, Hg
1
H-NMR (400 MHz; D
2
1
4
7
2
2
2
Glu), 2.27–2.13 (m, 2H, Hb Glu).
1
3
C NMR: see ref. 67.
ꢂ
ESI-MS, negative mode: m/z 289.15 [M ꢂ H] ; 311.16 [M ꢂ 2H
ꢂ
+
Na] .
3. Results and discussion
3.1 Transpeptidase activity of LL-BsGGT
The transpeptidase activity of the mutant LL-BsGGT was
preliminarily compared to those of the wt enzymes EcGGT and
BsGGT. In the standard activity assay, based on the use of the Fig. 3 Comparison of the activities of BsGGT, EcGGT and LL-BsGGT
in the absence (grey bars) and in the presence (black bars) of the added
acceptor GlyGly. Measurements were carried out at pH 8.5 in Tris HCl
chromogenic g-glutamyl-p-nitroanilide as the donor substrate
and glycylglycine as the standard acceptor, the activity of LL-
buffer, by continuously monitoring the release of p-nitroaniline at
BsGGT was much higher (ca. 23 folds) in the presence of
410 nm and deriving the initial reaction rates from the slope of the
added GlyGly than in its absence (Fig. 3). The result is quite
resulting curves in their linear range. Results are averages of three
remarkable, in that the increase in the enzymatic activity of experiments; error bars refer to standard deviation.
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was 2.5–4.6-fold higher in the presence of GlyGly than in its
The mutant LL-BsGGT generally gave better results with
absence, while the mutant LL-BsGGT showed an unusual 25–30- respect to the wt enzyme, although the improvement was
fold increase in activity in the presence of GlyGly. Overall, the modest for most of the tested acceptors. The mutant enzyme
mutant enzyme appeared to be ca. 5–7-fold more effective in showed low activities when apolar aromatic and branched-
catalyzing the transpeptidation reaction than the wt. In the chain amino acids were used as acceptor substrates (Table 1,
absence of the acceptor substrate, the measurement of specic entries 6, 7, 10 and 12), with a ca. 1.5-fold higher activity with
activities of the two enzymes gave quite similar results, namely respect to the wt counterpart (Fig. 5A). A modest improvement
ꢂ1
ꢂ1
1
122 and 1304 mmol min mg for the wt and the mutant was observed also for methionine (Table 1, entry 8), which is
enzyme, respectively, while in the presence of GlyGly the activ- among the best acceptors for BsGGT. Conversely, the S-
ꢂ1
ꢂ1
ities were 5198 and 29 686 mmol min mg , respectively.
.2 pH dependence
substituted cysteines showed the higher activities (Table 1,
entries 13–15), with a 2.5–4.1 increase in comparison with
the wt enzyme (Fig. 5A).
3
It therefore appears that LL-BsGGT showed an astonishingly
high activity only towards GlyGly as the acceptor. The result is
intriguing, as the active sites of the wt and mutant enzymes are
essentially the same, the main difference being the presence of
the lid-loop covering the g-glutamyl binding site on the mutant.
As the lid-loop has a gate function, allowing the selection of the
donor substrate on the basis of steric requirements, the mutant
LL-BsGGT may have become more selective also in the recogni-
tion of the acceptor substrate. In order to test this hypothesis,
aminoacyl derivatives of glycine with increasing steric demand
The pH-activity prole of LL-BsGGT in the absence of the
acceptor substrate seems to assume a at bell shape, while in
the presence of GlyGly the activity is favored by basic pH values
(Fig. 4) according to the assumption that pH affects not only the
intrinsic enzymatic activity, but also the proportion of the
acceptor substrate present in its non-protonated, nucleophilic
60
51,52
form.
activity cannot be univocally determined, as it depends on the
pK of the nucleophilic amino group of the acceptor substrate.
As a consequence, the optimum pH for transpeptidase
a
(
16–20, Fig. 6) were assayed (Table 1, entries 1 and 16–19). While
the transpeptidase activity of wt BsGGT towards compounds 16–
0 varied comparatively little (Fig. 5B), the mutant enzyme
3
.3 Transpeptidase activity of LL-BsGGT towards selected
acceptor substrates
2
The relative activities towards different acceptor substrates
measured for LL-BsGGT in relation to that of GlyGly are re-
ported in Table 1. Fig. 5A shows the activities measured for
selected acceptors in comparison with the activities displayed
by the wt counterpart, aer normalization of enzyme activities
as reported above.
seemed much more selective in recognizing the acceptor
substrate on the basis of the steric bulkiness. Indeed, the activ-
ities measured in LL-BsGGT-catalyzed reactions decreased as the
steric demand of the amino acid side chains increased.
Curiously enough, in several independent experiments the
activities showed always an inverse linear correlation with the
number of carbon atoms (0 for GlyGly, 3 for Val) on the acceptor
side chains (Fig. 5B). The activity of the mutant enzyme towards
LeuGly 20 was only slightly higher than that in the absence of
acceptor substrate (Table 1, entry 19). The relatively high
activities observed in reactions involving the sulfur-containing
amino acids (Table 1, entries 8 and 13–15) could then be
related to the minor steric requirements of their straight side-
chains with respect to the more demanding ones of branched-
chain and aromatic amino acids.
56
In addition to available data, also S-substituted cysteines S-
methyl- and S-allylcysteine proved to be good acceptors for
the wt enzyme. On the contrary, the transpeptidation of alanine
and cysteine were negligible, and comparable to the mock
reactions in the absence of the acceptor substrate. A possible
inhibitory activity of alanine towards BsGGT has been previ-
52
ously hypothesized.
Unfortunately, while the g-glutamyl binding site of several
GGTs has been investigated in detail through X-ray crystallog-
59,62–66
raphy,
the acceptor binding site remains, on the contrary,
elusive. It is therefore difficult to correlate the hydrolase and
transpeptidase activities of GGTs towards different acceptor
substrates on structural basis. Computational studies are ongoing
in our laboratories in order to gain insights into this topic.
3.4. Synthetic application of mutant LL-BsGGT
The possible use of the mutant LL-BsGGT as biocatalyst for
the synthesis of g-glutamyl derivatives of applicative interest
Fig. 4 pH-activity profile for LL-BsGGT in the absence (grey bars) and was tested towards both a dipeptide and an amino acid. The
in the presence (black bars) of GlyGly as the acceptor substrate.
Measurements at pH 7.0–8.5 were carried out in Tris HCl buffer;
measurements at pH 9.0–10 were carried out in sodium hydrogen
carbonate/sodium carbonate buffer. Bars labelled with the same letter
acceptor dipeptide was valylglycine 19 and the amino acid
was S-allyl cysteine. The choice of the two acceptors was
dictated by our interest in the search of an enzyme-catalyzed
reaction able to afford taste-active compounds with kokumi
are not statistically different at 95% confidence level (Tukey test, p >
.05).
7
,35
0
properties.
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Fig. 5 Comparison of the activities of BsuGGT and LL-BsuGGT towards selected acceptors. The activities of the two enzymes were prior
normalized in the reaction without added acceptor. (A) Activity towards simple amino acids. (B) Activity towards some representative amino-
acylglycines with increasing steric demand near the nucleophilic nitrogen atom (see Fig. 6).
3.4.1 Enzymatic synthesis of g-glutamylvalylglycine. The
dipeptide valylglycine 19 was prepared through the classical
methods of peptide synthesis in solution (see ESI†).
It is to note that, although the activity of LL-BsGGT towards
Val-Gly as acceptor compound seemed to be rather low (Table 1,
entry 18), it was still 1.5 fold higher than that of the wt enzyme
(Fig. 5A). Enzymatic activity in the presence of Val-Gly varied
little with pH between 7 and 10, with higher yields obtained at
ꢀ
pH 8.5. The reaction was rst tested at 25 C in the presence of
equimolar amounts of glutamine as the donor substrate and
Val-Gly at 100 mM and was monitored by HPLC upon pre-
column derivatization with 1-uoro-2,4-dinitrobenzene (Sang-
er's reagent) (Fig. 7A). The concentration of the donor substrate
glutamine dropped rapidly in addition to the desired g-Glu-
60
ValGly 21, also g-GluGln 7 and g-Glu
Notably, the concentration of g-GluGln 7 rose quickly at the
beginning of the reaction, indicating strong auto-
2
Gln were identied.
a
transpeptidase activity of the enzyme, and then decreased
slowly, while the concentration of the transpeptidation product
21 reached a plateau at 4–5 hours, and then remained fairly
constant up to 24 hours. The concentration of the hydrolysis
product glutamic acid 23 rose slowly in the initial stages, and
then increased when the concentrations of glutamine and g-
GluGln 7 fell below a lower threshold. HPLC-MS analysis of the
non-derivatized reaction mixture (Fig. 8) showed the presence of
numerous poly-g-glutamylated compounds, deriving from both
the donor glutamine and the acceptor ValGly (e.g. 22 in Fig. 7).
This behavior was not unexpected, as the enzyme can accept as
substrates the g-glutamyl derivatives formed in the early stages
Fig. 6 Acceptor aminoacyl-glycines with increasing steric demand
near the nucleophilic amino group.
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Fig. 7 Time course of the LL-BsGGT-catalyzed synthesis of g-GluValGly. (A) Reaction carried out using equimolar amounts of donor and
acceptor substrates. (B) Reaction carried out by supplementing one equivalent of the donor substrate after 45 min reaction time (see text for
details).
of the reaction. Thus, g-GluGln 7 is formed as long as Gln is
The reaction was repeated at a preparative level in a volume
present in sufficient amount and behaves as a donor substrate of 5 mL and aer 5 hours reaction time the product was ob-
when the concentration of glutamine drops. Also the trans- tained in 35% isolated yield aer ion exchange column chro-
peptidation product g-GluValGly is recognized as a donor matography. This amount is in good agreement with the
substrate, but the presence of ValGly ensures its continuous re- concentration of the product in the reaction mixture as deter-
formation, maintaining its concentration fairly stable. This mined by HPLC, thus conrming the reliability of the analytical
balance is broken by the irreversible hydrolysis reaction leading method used for reaction monitoring.
to glutamic acid, which is not recognized by GGTs as a donor
The ability of the synthetized g-glutamyl derivatives to also
substrate. However, the hydrolysis reaction is slow in compar- act as acceptor substrates affording poly-g-glutamylated
ison with transpeptidation, so explaining the slow decline of the compounds, represents an undesirable trait of the reaction.
product from six to 24 hours, paralleled by the equally slow This functional feature of the mutant enzyme is very likely an
increase of glutamic acid concentration. Maximum concentra- inheritance from the parent wt BsGGT. Indeed, it has been
tion of the desired g-GluValGly was 38 mM and was reached shown that BsGGT is able to accept polymeric substrates, being
52,60,68
between four and six hours.
actually a poly-g-glutamic acid (g-PGA) hydrolase.
g-PGA is
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by the relative concentration of the possible acceptor species
present in the reaction mixture. When the concentration of the

rst transpeptidation product, g-GluValGly, nearly equaled the
concentration of ValGly, it started to compete to a signicant
extent with the latter as the acceptor substrate. This might
explain the steady concentration of the rst product from
a certain time point on, usually 4–6 h, and the appearance of
several peaks attributable to poly-glutamylated species in the
HPLC-MS spectra, the concentration of which decreased with
the increase in number of bound g-glutamyl residues.
3.4.2 Enzymatic synthesis of g-glutamyl-S-allylcysteine.
The reaction in which S-allylcysteine was used as the acceptor
substrate showed a similar trend. The reaction was carried out
ꢀ
at an analytical level at pH 9.5 and 25 C, and HPLC monitoring
revealed that a maximum product concentration of ca. 22 mM
was obtained between 5 and 6 hours. At a preparative level, the
reaction allowed the isolation of the product 9 in 21% yield,
Fig. 8 HPLC-MS chromatogram of an underivatized sample from the
LL-BsGGT-catalyzed reaction between Gln as the donor and ValGly as
the acceptor at the end of the reaction. Detection was in negative conrming also in this case the good correlation with the
mode. Recorded m/z values are reported and are attributable as concentrations estimated by HPLC. Surprisingly enough, the
follow: m/z 173: ValGly; m/z 274: g-GluGln; m/z 275: g-GluGlu; m/z
yield of g-glutamyl-S-allylcysteine 9 was lower than that of g-
4
03: g-Glu
g-Glu
18: g-Glu
2
Gln; m/z 404: g-Glu
ValGly; m/z 560: g-Glu
ValGly.
2
Glu; m/z 302: g-GluValGly; m/z 431:
ValGly; m/z 689: g-Glu ValGly; m/z
3 4
glutamylvalylglycine obtained in the previous reaction, despite
the activity of the enzyme was higher in the presence of the
former than in the presence of the latter (Table 1 and Fig. 5). A
possible explanation could rely on the higher activity of the
enzyme not only towards S-allylcysteine, but also towards the g-
glutamyl derivatives thereof. The product did not accumulate in
the reaction mixture because it was used as acceptor in further
g-glutamyl-transfer reactions. Unfortunately, except the peak
attributable to g-glutamyl-g-glutamyl-S-allylcysteine, which was
easily assigned, it was not possible to identify and quantify
other peaks attributable to poly-g-glutamylated-S-allylcysteine
derivatives, as those would be expected in a crowded area of the
chromatogram. It is also conceivable that poly-g-glutamylated
species could compete as acceptor substrates being structurally
similar to g-PGA, the physiological substrate of the wt enzyme,
i.e. the glutamate moiety of the molecule, and not the S-allyl-
cysteine portion, was recognized by the enzyme.
2
8
5
a natural polymer composed by very long chains of L- and D-
glutamic acid residues in different proportions, linked together
through amide bonds formed by the g-carboxyl group of a glu-
tamic acid residue and the amino group of the next residue in
the chain. It is produced by several microorganisms and
notably by some Bacillus spp.
For the principle of microscopic reversibility, BsGGT should
then be able to catalyze the formation of oligomers under
appropriate conditions.
Attempts to improve the conversion of the substrates into the
desired product by raising the concentration of the enzyme in
69
70
ꢀ
the reaction medium or the temperature up to 40 C affected
only the reaction time, but did not result in a substantial
improvement in product formation or changes in products
distribution. The observation that the concentration of g-Glu-
ValGly remained quite stable in the reaction mixture aer the
disappearance of the donor substrates prompted us to try a fed-
batch procedure, restoring the concentration of the donor
glutamine immediately before the concentration of the trans-
peptidation product reached the plateau (Fig. 7B). A higher
amount of enzyme was used in these experiments, so as to
shorten the reaction time. Aer the addition of the second
In summary, our results conrm that the mutant enzyme
inherited some functional features from the wt BsGGT. BsGGT
is indeed considered a hydrolase with adventitious trans-
peptidase activity and relaxed acceptor substrate speci-
56,67,71

city.
In addition, present results suggest that the
transpeptidase activity of the mutant enzyme depends in part
on the concentration of the possible nucleophilic species in the
reaction mixture.
equivalent of glutamine, the concentration of g-GluGln 7 4. Conclusions
showed an initial steep increase; when its concentration started
In our ongoing search on the possible exploitation of bacterial
to decrease, the concentration of g-Glu-g-GluValGly grew more
signicantly than that of g-GluValGly (Fig. 7B). At the same
time, several peaks of decreasing intensities, attributable to
poly-g-glutamyl glutamine derivatives (at least 5 g-glutamyl
residues linked to a single glutamine moiety), eluted before g-
GGTs as biocatalysts, the transpeptidase activity of a mutant
GGT, obtained by inserting the lid-loop from E. coli GGT on the
structure of B. subtilis GGT, naturally lacking it, has been
investigated.
Our results suggest that the lid-loop is able to shield the g-
glutamyl enzyme intermediate from bulk solvent, preventing
hydrolysis and thus favoring the transpeptidation reaction. In
addition, it contributes to substrate selection on the basis of
Glu Gln. Also, poly-g-glutamylated derivatives of the acceptor
valylglycine were identied. This result suggests that the
transpeptidase activity of the enzyme is affected at least in part
2
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steric requirements near the nucleophilic amino group. On the
other hand, being the active site of the mutant enzyme essen-
tially identical to that of the wt, the mutant enzyme catalyzes the
formation of poly-g-glutamylated species, thus reecting the
ability of the parent B. subtilis GGT to accept polymeric
substrates. The substrate specicity appears to be relaxed and
the relative concentration of the possible nucleophiles in the
reaction mixture gain importance in determining products
distribution.
3 H. Suzuki, Y. Kajimoto and H. Kumagai, J. Agric. Food Chem.,
2002, 50, 313–318.
4 Y. Ueda, M. Yonemitsu, T. Tsubuku, M. Sakaguchi and
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5 K. X. Tang, C. J. Zhao and M. G. Ganzle, J. Agric. Food Chem.,
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6 T. Miyaki, H. Kawasaki, M. Kuroda, N. Miyamura and
T. Kouda, Flavour, 2015, 4, 17.
The mutant enzyme does appear promising as a biocatalyst
for preparative purposes in case poly-glutamylated species are
the desired products, however these studies allow a deeper
understanding of GGTs behavior. Another interesting aspect
emerged from our investigation: as previously reported, there
does not appear to be a straightforward relationship between
the enzymatic activity towards a given acceptor substrate, as
7 T. Ohsu, Y. Amino, H. Nagasaki, T. Yamanaka, S. Takeshita,
T. Hatanaka, Y. Maruyama, N. Miyamura and Y. Eto, J. Biol.
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52
9 S. Toelstede, A. Dunkel and T. Hofmann, J. Agric. Food Chem.,
2009, 57, 1440–1448.
measured through the standard colorimetric assay, and the 10 S. Toelstede and T. Hofmann, J. Agric. Food Chem., 2009, 57,
ability of the same acceptor to afford satisfactory results in 3738–3748.
reactions carried out at a preparative level. Indeed, the actual 11 H. Hillmann, J. Behr, M. A. Ehrmann, R. F. Vogel and
event monitored by the colorimetric assay is the release of p- T. Hofmann, J. Agric. Food Chem., 2016, 64, 1784–1793.
nitroaniline due to the formation of the g-glutamyl-enzyme 12 C. J. Zhao and M. G. Ganzle, J. Agric. Food Chem., 2016, 64,
intermediate. Then the g-glutamyl-enzyme intermediate must 7561–7568.
be resolved and, using a large excess of acceptor substrate, the 13 A. Dunkel, J. Koester and T. Hofmann, J. Agric. Food Chem.,
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pathway. However, such a large excess of acceptor substrate 14 Y. Ueda, M. Sakaguchi, K. Hirayama, R. Miyajima and
cannot be replicated at a preparative level, so other reaction A. Kimizuka, Agric. Biol. Chem., 1990, 54, 163–169.
pathways, such as hydrolysis, autotranspeptidation and the 15 Y. Ueda, T. Tsubuku and R. Miyajima, Biosci., Biotechnol.,
formation of poly-glutamylated species, become evident. Biochem., 1994, 58, 108–110.
Indeed, the assay based on the use of g-glutamyl-p-nitroanilide 16 H. A. Wetli, R. Brenneisen, I. Tschudi, M. Langos, P. Bigler,
was assessed and validated for applications with mammalian
GGTs and it is thus conceivable that the same assay is not able
T. Sprang, S. Schuerch and R. C. M u¨ hlbauer, J. Agric. Food
Chem., 2005, 53, 3408–3414.
72
to account satisfactorily for the different behavior exerted by 17 Y. Juan, S. Dongxiao, C. Chun, D. Keming and Z. Mouming,
bacterial GGTs in standard reaction conditions.
Int. J. Food Sci. Technol., 2018, 53, 1166–1175.
1
1
2
8 S. Liu, Y. Li and J. Zhu, Process Biochem., 2016, 51, 1458–
1463.
Conflicts of interest
9 J. Yang, D. Sun-Waterhouse, J. Xie, L. Wang, H.-z. Chen,
C. Cui and M. Zhao, Food Chem., 2018, 247, 89–97.
0 J. Yang, D. Sun-Waterhouse, C. Cui, K. Dong and W. Wang, J.
Agric. Food Chem., 2017, 65, 8696–8703.
Authors have no conicts of interest to declare.
Acknowledgements
21 T. Itoh, Y. Hoshikawa, S.-i. Matsuura, J. Mizuguchi,
H. Arafune, T.-a. Hanaoka, F. Mizukami, A. Hayashi,
H. Nishihara and T. Kyotani, Biochem. Eng. J., 2012, 68,
This work was founded by Fondazione Cariplo, Bando per le
Biotecnologie Industriali e la Bioeconomia 2016, project
207–214.
“Added-value products through biocatalysis: tailored g-gluta-
2
2
2
2
2
2 R. Nakayam, H. Kumagai, S. Akashi, S. Hiroyoshi and
T. Tochikura, Agric. Biol. Chem., 1985, 49, 1041–1046.
3 H. Kumagai, T. Echigo, H. Suzuki and T. Tochikura, Agric.
Biol. Chem., 1988, 52, 1741–1745.
4 H. Kumagai, T. Echigo, H. Suzuki and T. Tochikura, Agric.
Biol. Chem., 1989, 53, 1429–1430.
5 H. Kumagai, H. Suzuki, M. Shimizu and T. Tochikura, J.
Biotechnol., 1989, 9, 129–138.
6 H. Kumagai, T. Echigo, H. Suzuki and T. Tochikura, Lett.
Appl. Microbiol., 1989, 8, 143–146.
myltranspeptidases – TailGluTran”, grant no. 2016-0741; and by
the Italian Ministry of Education, University and Research
(MIUR): Departments of Excellence, Program 2018–2022 to the
Dept. of Biology and Biotechnology, University of Pavia. MM
was assigned a scholarship within the TailGluTran Project
(
Borsa per la formazione dei giovani pi u` promettenti).
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