Synthetic tripeptides as alternate substrates of murein peptide ligase (Mpl)

Article abstract of DOI:10.1016/j.biochi.2012.12.011

Murein peptide ligase (Mpl) is an enzyme found in Gram-negative bacteria. It catalyses the addition of tripeptide l-Ala-γ-d-Glu-meso-diaminopimelate to nucleotide precursor UDP-N-acetylmuramic acid during the recycling of peptidoglycan. Although not essential, this enzyme represents an interesting target for antibacterial compounds through the synthesis of alternate substrates whose incorporation into peptidoglycan might be deleterious for the bacterial cell. Therefore, we have synthesised 10 tripeptides l-Ala-γ-d-Glu-Xaa in which Xaa represents amino acids different from diaminopimelic acid. Tripeptide with Xaa = ε-d-Lys proved to be an excellent substrate of Escherichia coli Mpl in vitro. Tripeptides with Xaa = p-amino- or p-nitro-l-phenylalanine were poor substrates, while tripeptides with Xaa = d- or l-2-aminopimelate, dl-2-aminoheptanoic acid, l-Glu, l-norleucine, l-norvaline, l-2-aminobutyric acid or l-Ala were not substrates at all. Although a good Mpl substrate, the d-Lys-containing tripeptide was devoid of antibacterial activity against E. coli, presumably owing to poor uptake.

Full text of DOI:10.1016/j.biochi.2012.12.011

Biochimie 95 (2013) 1120e1126
Contents lists available at SciVerse ScienceDirect
Biochimie
journal homepage: www.elsevier.com/locate/biochi
Research paper
Synthetic tripeptides as alternate substrates of murein peptide
ligase (Mpl)
,
b
,
,
ꢀ c
Mireille Hervé a , Andreja Kovac 1, Cécile Cardoso a, Delphine Patin a b, Boris Brus c,
Hélène Barreteau a b, Dominique Mengin-Lecreulx a b, Stanislav Gobec c, Didier Blanot a
,
,
,b,
*
a Univ Paris-Sud, Laboratoire des Enveloppes Bactériennes et Antibiotiques, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619,
F-91405 Orsay, France
b Centre National de la Recherche Scientifique, F-91405 Orsay, France
c
ꢀ
ꢀ
Fakulteta za Farmacijo, Askerceva 7, Univerza v Ljubljani, 1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history:
Murein peptide ligase (Mpl) is an enzyme found in Gram-negative bacteria. It catalyses the addition
of tripeptide -Ala- -Glu-meso-diaminopimelate to nucleotide precursor UDP-N-acetylmuramic acid
during the recycling of peptidoglycan. Although not essential, this enzyme represents an interesting
target for antibacterial compounds through the synthesis of alternate substrates whose incorporation
into peptidoglycan might be deleterious for the bacterial cell. Therefore, we have synthesised 10
Received 24 October 2012
Accepted 14 December 2012
Available online 25 December 2012
L
g-D
Keywords:
Escherichia coli
Mpl
Murein peptide ligase
Peptidoglycan recycling
Tripeptide substrates
tripeptides
acid. Tripeptide with Xaa ¼ ε-
Tripeptides with Xaa ¼ p-amino- or p-nitro-
with Xaa ¼ - or -2-aminopimelate, DL-2-aminoheptanoic acid,
aminobutyric acid or -Ala were not substrates at all. Although a good Mpl substrate, the
L
-Ala-
g
-
D
-Glu-Xaa in which Xaa represents amino acids different from diaminopimelic
-Lys proved to be an excellent substrate of Escherichia coli Mpl in vitro.
-phenylalanine were poor substrates, while tripeptides
-Glu, -norleucine, -norvaline, -2-
-Lys-
D
L
D
L
L
L
L
L
L
D
containing tripeptide was devoid of antibacterial activity against E. coli, presumably owing to poor
uptake.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
the cytoplasm, in the membrane and on the outer side of the
membrane (periplasm in Gram-negative bacteria) (Fig. 1) [2e4].
Peptidoglycan (murein) is a major component of the cell wall of
most eubacteria. It is composed of long glycan chains cross-linked
by short peptides [1]. Its main function is to preserve cell integ-
rity by withstanding the internal osmotic pressure. Its biosynthesis
is a complex process occurring in different cell compartments: in
Due to the essential role of the macromolecule, the enzymes
involved in this biosynthetic process represent important targets
for new antibacterial compounds.
The cytoplasmic steps include inter alia a series of four enzymes, the
Mur ligases, responsible for the assembly of the peptide stem of
peptidoglycan [2]. These enzymes catalyse the successive addition of
Ala (MurC), -Glu (MurD), meso-diaminopimelic acid (A2pm) or -lysine
(MurE), and -Ala- -Ala (MurF) to UDP-MurNAc, leading to the
synthesis of Park’s nucleotide, UDP-MurNAc-pentapeptide (Fig. 1).
Nevertheless, besides this de novo synthesis, there exists in Gram-
L-
D
L
Abbreviations: Abu, 2-aminobutyric acid; AcOH, acetic acid; Apm,
2-aminopimelic acid; A2pm, 2,6-diaminopimelic acid; Bn, benzyl; Boc, t-butylox-
ycarbonyl; DCC, N,N0-dicyclohexylcarbodiimide; DCM, dichloromethane; DMF,
dimethylformamide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; Hep,
heptyline (2-aminoheptanoic acid); HOBt, hydroxybenzotriazole; HOSu,
N-hydroxysuccinimide; HPLC, high-performance liquid chromatography; IPTG,
D
D
negative bacteria another process called recycling [5,6]: tripeptide
L-
Ala- -Glu-meso-A2pm, which results from the action of hydrolase
g-D
isopropyl b-D-thiogalactopyranoside; MALDI-TOF, matrix-assisted laser desorption/
and permease activities, is directly added to UDP-MurNAc, generating
UDP-MurNAc-tripeptidewhich can re-enter the main synthetic process
through MurF (Fig.1). The demonstration that a defect inpeptidoglycan
recycling in Escherichia coli brings about bacteriolysis suggests that this
process is a promising target for novel antibacterial agents [7].
Murein peptide ligase (Mpl) is the enzyme which catalyses the
addition of the tripeptide to UDP-MurNAc. Mpl has been identified
in and purified from E. coli [8,9], and the tridimensional structure of
ionisation time-of-flight; Me, methyl; Mpl, murein peptide ligase; MppA, murein
peptide permease A; MurNAc, N-acetylmuramoyl; Nle, norleucine; NMM,
N-methylmorpholine; Nva, norvaline; TFA, trifluoroacetic acid; TLC, thin-layer
chromatography; Z, benzyloxycarbonyl.
* Corresponding author. Laboratoire des Enveloppes Bactériennes et Anti-
biotiques, IBBMC, UMR 8619, CNRS, Bâtiment 430, Université Paris-Sud, F-91405
Orsay, France. Tel.: þ33 1 69 15 81 65; fax: þ 33 1 69 85 37 15.
E-mail address: didier.blanot@u-psud.fr (D. Blanot).
1
ꢀ
Present address: LEK Pharmaceuticals, Verovskova 57, 1000 Ljubljana, Slovenia.
0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.biochi.2012.12.011

M. Hervé et al. / Biochimie 95 (2013) 1120e1126
1121
2.3. Assay for Mpl activity
The assay mixture (50
m
L) contained 100 mM TriseHCl buffer
(pH 8.4), 5 mM ATP, 15 mM MgCl2, 0.4 mM UDP-[14C]MurNAc
(500 Bq), purified Mpl enzyme (20 ng of protein), and peptide 1, 2
or 12 (variable concentrations). Mixtures were incubated at 37 ꢀC
for 30 min and reactions were stopped by the addition of 10 mL of
acetic acid, followed by lyophilisation. The residue was dissolved in
50 mM ammonium formate, pH 3.8 (buffer A) and injected onto
a Nucleosil 100C18
5
m
m column (4.6 ꢁ 150 mm; Grace Davison
Discovery Sciences) using buffer A at 0.6 mL minꢂ1 as the mobile
phase. Detection was performed with a radioactive flow detector
(model LB506-C1, Berthold) using the Quicksafe Flow 2 scintillator
(Zinsser Analytic) at 0.6 mL minꢂ1. Quantification was carried out
with the Winflow software (Berthold).
For the determination of the kinetic constants, the same assay
was used with various concentrations of one substrate and fixed
concentrations of the others. In all cases, the substrate consump-
tion was <20%, the linearity being ensured within this interval even
at the lowest substrate concentration. The data were fitted to the
equation v ¼ VmaxS/(Km þ S) or v ¼ VmaxS/(Km þ S þ S2/Ki) using the
MDFitt software developed by M. Desmadril (IBBMC, Orsay,
France).
Identical assay conditions were employed when other peptides
were tested as substrates. However, in order to make sure that the
putative product is eluted from the column, isocratic elution with
buffer A (10 min) was followed by a gradient of methanol in buffer
A (from 0 to 40% over 35 min).
Fig. 1. Metabolism of peptidoglycan. Mature peptidoglycan is produced by de novo
synthesis (right hand side). These reactions occur in the cytoplasm (synthesis of UDP-
MurNAc-pentapeptide from UDP-GlcNAc), at the level of the cytoplasmic membrane
(enzymes MraY and MurG), and in the periplasm (polymerisation reactions). During
bacterial growth, peptidoglycan is partly degraded by numerous hydrolases. The
degradation fragments are re-imported by permeases (AmpG, MppA) and further
degraded by cytoplasmic enzymes (NagZ, AmpD, LdcA). The final degradation product,
2.4. Microbiology
tripeptide L-Ala-g-D-Glu-meso-A2pm, is re-introduced into the synthetic pathway by
enzyme Mpl (left hand side), a process named recycling.
The E. coli BW25113 strain [14] was used to test the antibacterial
activity of tripeptide 12. Cells were grown in 2YT rich medium or
M63 minimal medium [15]. When required, media were supple-
mented with ampicillin (25 mg mLꢂ1) and IPTG (0.001e1 mM).
the orthologue from Psychrobacter arcticus has been solved [10].
Although the mpl gene is not essential for bacterial growth [8], Mpl
is interesting from a medicinal chemistry standpoint for two
reasons. First, alternate Mpl peptide substrates might be incorpo-
rated into peptidoglycan and display an antibacterial effect through
inhibition of subsequent steps, in particular transpeptidases. The
2.5. Docking simulation
The docking procedure is described in the Supplementary
material.
fact that tetrapeptide
L-Ala-g-D-Glu-meso-A2pm-D-Ala, or tri- and
tetrapeptide containing
moderate substrates of E. coli Mpl [9] indicates that the enzyme is
L
-Lys in lieu of meso-A2pm, were good or
3. Results
permissive enough to accept other compounds than the regular
3.1. Tripeptide design and synthesis
L-Ala-g-D-Glu-meso-A2pm tripeptide. Second, such alternate
substrates might increase the susceptibility to existing antibiotics:
indeed, it has been shown that the deletion of the mpl gene in
Starting from the structures of known tripeptide substrates of
Mpl,
(Fig. 2), we decided to synthesise peptides of core structure
-Glu-Xaa. Amino acids at position 3 (Xaa) were selected so that
L
-Ala-
g
-
D
-Glu-meso-A2pm (1) and
L
-Ala-
g
-
D
-Glu-
L-Lys (2)
Acinetobacter baylyi brings about a hypersensitivity to
b-lactam
L
-Ala-
antibiotics [11]. In this paper, we wish to report on the synthesis
and biological evaluation of tripeptides as alternate substrates of
Mpl from E. coli.
g-D
they lacked a proximal (i.e., on the main chain) or distal (i.e., on
the lateral chain) function, thereby possibly hampering subse-
quent biosynthetic reactions (MurF or transpeptidases). These
amino acids were 2-aminopimelic acid (Apm) (tripeptide 3) and
its lower homologue glutamic acid (tripeptide 4); heptyline (Hep)
and its lower homologues norleucine (Nle), norvaline (Nva),
2-aminobutyric acid (Abu) and alanine (tripeptides 5, 6, 7, 8 and 9,
respectively); p-aminophenylalanine (tripeptide 10) and its
synthetic precursor, p-nitrophenylalanine (tripeptide 11); and
2. Materials and methods
2.1. Chemicals
UDP-MurNAc and UDP-[14C]MurNAc were obtained according
to published procedures [12,13]. The Mpl enzyme from E. coli was
produced as the C-terminal His-tagged form according to Hervé
et al. [9].
D
-lysine acylated on its ε-amino function (tripeptide 12) (Fig. 2).
Apm in compound 3 represents meso-A2pm lacking the distal
NH2 group. Hep in compound 5 is an isostere of lysine with the
distal NH2 group replaced by methyl. p-Aminophenylalanine in
compound 10 is also an isostere of lysine, but the distal NH2 group
is present while the aliphatic side-chain is replaced by a phenyl
ring. Finally, in tripeptide 12, the distal functions (amino and
2.2. Peptide synthesis
The peptides were synthesised as described in the
Supplementary material.

1122
M. Hervé et al. / Biochimie 95 (2013) 1120e1126
Fig. 2. Formulae of the tripeptides synthesised.
carboxyl) of meso-A2pm are maintained while the proximal
carboxyl group is absent.
acidolysis, and resulting derivatives 18 and 19 were coupled with
Boc- -Ala 20. Finally, the protecting groups of tripeptide derivatives
L
The peptides were synthesised by the classical methods of
21 and 22 were removed by trifluoroacetic acid treatment followed
by catalytic hydrogenolysis.
peptide synthesis. Amino acids Xaa were of the
except Apm and Hep, for which only the racemic compound is
commercially available, and of course -lysine. A first series of
compounds (4, 9) were synthesised according to the scheme of
Fig. 3. Boc- -Glu-OBn 15 was coupled with benzyl-protected amino
L configuration,
A second series of tripeptides (3, 5e8, 12) was synthesised
D
according to the scheme of Fig. 4. Z-
DCC/HOSu and coupled with -Glu-OBn 24, yielding partially
protected dipeptide derivative 25. The -carboxyl group of 25
was activated and coupled with benzyl-protected amino acids
26e30, or Z- -Lys-OBn 31. Finally, the protecting groups of
L-Ala 23 was activated with
D
D
g
acids 13 and 14 by the EDC/HOBt method, yielding protected
dipeptide 16 or 17, respectively. The Boc group was eliminated by
D
Fig. 3. Synthesis of tripeptides 4 and 9. Reagents and conditions: (a) EDC, HOBt, NMM, DMF; (b) TFA, DCM; (c) 1. TFA, DCM; 2. H2, Pd/C, MeOH.

M. Hervé et al. / Biochimie 95 (2013) 1120e1126
1123
Fig. 4. Synthesis of tripeptides 3, 5e8 and 12. Reagents and conditions: (a) 1. 23, DCC, HOSu, DCM; 2. 24, NMM; (b) EDC, HOBt, NMM, DMF; (c) H2, Pd/C, glacial AcOH.
tripeptide derivatives 32e37 were removed by catalytic
hydrogenolysis.
A similar scheme (Fig. 5) was used for tripeptides 11 and 10, but
owing to the presence of the p-nitrophenyl group, carboxyl groups
With tripeptide 10 or 11 at 1 mM, a radiolabelled product
appeared only upon prolonged incubation (16 h) with a large
amount of enzyme (2 g): in these conditions, ca. 50% (11) or 10%
(10) conversion with respect to UDP-MurNAc occurred. In order to
verify the structure of these new products, the reactions
were performed with non-labelled UDP-MurNAc and the products
were purified by HPLC. MALDI-TOF mass spectrometry was in
m
were protected as methyl esters. Dipeptide derivative Boc-
Glu-OMe 39 was synthesised from Boc- -Ala 20 and -Glu-OMe 38
as described above. Coupling to p-nitro- -phenylalanine methyl
L-Ala-D-
L
D
L
ester 40 yielded tripeptide derivative 41. The protecting groups
were removed by alkaline hydrolysis followed by acidolysis, giving
agreement with the structures UDP-MurNAc-L-Ala-g-D-Glu-p-
amino-
L
-phenylalanine (m/z ratio of the [M ꢂ H]ꢂ ion, 1040.31;
p-nitro-L-phenylalanine-containing tripeptide 11. A small amount
calculated molecular mass of the nucleotide product, 1041.26) and
of 11 was submitted to catalytic hydrogenation to obtain p-amino-
UDP-MurNAc-L-Ala-g-D-Glu-p-nitro-L-phenylalanine (m/z ratio of
L-phenylalanine-containing tripeptide 10.
the [M ꢂ H]ꢂ ion, 1070.32; calculated molecular mass of the
The tripeptides were purified by HPLC. They were characterised
nucleotide product, 1071.24).
by TLC, analytical HPLC, and MALDI-TOF mass spectrometry using
carbon nanotubes [16]. This method has recently been shown to
give good spectra for low molecular weight compounds without
interference of intrinsic matrix ions. Tripeptides appeared mainly
as cationised species in the positive mode, and as the molecular ion
in the negative mode.
No radiolabelled product could be detected with tripeptides 3a,
3b, 4e9 at 1 mM after 16 h with 2 mg Mpl.
3.3. Attempts to incorporate tripeptide 12 into the peptidoglycan
via Mpl
Tripeptides 3 and 5 are mixtures of LDL and LDD diastereoisomers.
While diastereoisomers of 5 were eluted on the semi-preparative
column as a largely unresolved peak, those of 3 could be sepa-
rated. The stereochemistry of resulting compounds 3a and 3b was
determined by chiral chromatography of acid hydrolysates to be LDD
and LDL, respectively.
Since tripeptide 12 is an excellent substrate of Mpl, attempts
were made to test a possible antibacterial effect on E. coli cells.
Indeed, its in vivo incorporation would produce a nucleotide which
could not be a substrate for the subsequent biosynthetic enzyme,
MurF, owing to the absence of proximal carboxyl group. In this
purpose, the BW25113 strain was plated on rich 2YT medium or
glucose minimal medium, and different concentrations of tripep-
tide 12 were spotted. However, even the addition of large amounts
of peptide (up to 5 mg mLꢂ1) had no effect on cell growth. The
BW25113 strain was transformed either with the pMLD131 [9] or
pMLD1285 [18] plasmid, allowing overexpression of the mpl or
mppA gene, respectively, upon IPTG induction. The aim of these
experiments was to stimulate the recycling process (Mpl over-
production) or to increase tripeptide 12 uptake (MppA over-
production, see Section 4). But even in these conditions, no
deleterious effect of the tripeptide was observed.
3.2. Tripeptides as Mpl substrates
Tripeptides were tested for their ability to be added to radio-
active UDP-MurNAc by purified Mpl. When classical conditions of
incubation were used (30 min incubation time, 20 ng enzyme),
tripeptide 12 gave rise to the appearance of a radiolabelled product
which had the same retention time as authentic UDP-MurNAc-
Ala- -Glu-ε- -Lys [17]. The kinetic parameters of 12 were deter-
mined and found to be similar to those of peptide 1 (Table 1).
L-
g-
D
D

1124
M. Hervé et al. / Biochimie 95 (2013) 1120e1126
Fig. 5. Synthesis of tripeptides 10 and 11. Reagents and conditions: (a) 20, DCC, HOSu, DCM; 2. 38, NMM; (b) EDC, HOBt, NMM, DMF; (c) 1. NaOH, dioxane; 2. TFA, DCM; (d) H2, Pd/C,
10% AcOH.
4. Discussion
which is in the lateral chain) (Fig. 6) [19]. Mpl recognises the amino
and carboxyl groups in the distal site of the natural substrate 1.
The Mpl enzyme intervenes in peptidoglycan recycling by cata-
The carbon atom bearing these groups should be of the D configu-
lysing the addition of meso-A2pm-containing tripeptide 1 to UDP-
ration. The absence of carboxyl group in 2 brings about a decrease
of specificity constant (700-fold); nevertheless, 2 remains a fairly
good substrate. By contrast, the absence of amino group in 3b or 5
prevents the recognition between enzyme and peptide (Fig. 6). This
is a fortiori the case when an additional modification, namely
shortening of the lateral chain, occurs (4, 6e9).
The case of peptides 10 and 11 is particular. Peptide 10 is a poor
substrate of the enzyme. Interestingly, it contains an amino group
(borne by a phenyl ring) at the C-terminal position. This would
suggest that an amino group on the distal site is essential for
a peptide to be a substrate. However, peptide 11 with a nitro group
at the same place is also a substrate, albeit even less efficient than
10. Moreover, Olrichs et al. have shown that peptide 2 labelled at
the lysine residue with N-7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) is
substrate of Mpl in vitro [20]. As for 10 and 11, a large amount of
enzyme had to be added. Together, these results indicate that
a primary amine at the distal site of position 3 is not absolutely
necessary.
MurNAc. We had previously shown that it can also utilise
containing tripeptide 2 in vitro [9]. The most remarkable result of
the present study is that -Lys-containing tripeptide 12 constitutes
L-Lys-
D
another substrate for Mpl. The examination of the kinetic parame-
ters (Table 1) shows that tripeptide 12 is as good a substrate as 1, the
specificity constant kcat/Km being of the same order of magnitude.
A more disappointing result is that no other peptide (apart from
10 and 11, see below) is a substrate of the enzyme. Nevertheless, we
can draw a few conclusions concerning the interactions of Mpl and
its tripeptide substrate. As mentioned in Section 3.1, the residue at
position 3 possesses two recognition sites: the proximal site (the
one which is in the main peptide chain) and the distal site (the one
Table 1
Kinetic parameters of Mpl for tripeptide substrates.
Compound
Km (mM)
kcat (minꢂ1
)
kcat/Km
(minꢂ1 mMꢂ1
)
Tripeptide 12 is very interesting. Its
D
-lysine residue is linked
/ ε bond. Such
1a
0.10 ꢃ 0.02
4.1 ꢃ 0.4
290 ꢃ 70
16 ꢃ 1
2900
3.9
2400
“upside down” to the -glutamyl residue with a
D
g
2a
a structure has been found in the peptidoglycan of Thermotoga
maritima [17,21]. The distal site of 12 is constituted by amino and
12b
0.15 ꢃ 0.04
360 ꢃ 40
a
Ref. [9].
carboxyl functions borne by a
D carbon, i.e. identical to that of
b
The concentration range of tripeptide 12 was 0.1e1.2 mM. Owing to inhibition
by excess tripeptide, data were fitted to the equation v ¼ VmaxS/(Km þ S þ S2/Ki); the
Ki value determined was 0.70 ꢃ 0.20 mM.
peptide 1 (Fig. 6). This is the reason why the enzyme recognises
peptide 12 quite well. It should be noted that the modification of

M. Hervé et al. / Biochimie 95 (2013) 1120e1126
1125
Fig. 6. Proximal and distal sites of amino acids found at position 3 of tripeptides. The specificity constant of those tripeptides which are substrates for Mpl is shown in the lower part
of the figure. R, L-Ala-D-Glu.
the proximal site (suppression of the carboxyl group and trans-
formation of an carbon to an achiral one) has no influence on the
interaction between the peptide and the enzyme. This is in keeping
with the fact that tetra- and pentapeptides, in which the carboxyl
function of the proximal site is substituted, are as good substrates
as the tripeptide [9].
In spite of being an excellent substrate for Mpl, tripeptide 12 was
devoid of antibacterial activity against E. coli. Such a disappointing
result had already been encountered with tripeptide 2, a less effi-
cient Mpl substrate [9]. An explanation could be the poor uptake of
12 by E. coli cells. Elements in favour of this hypothesis can be found
upon examination of the murein peptide permease A (MppA). This
protein is responsible for the internalisation of tripeptide 1 during
the recycling process of peptidoglycan [18]. Maqbool et al. [22] have
solved the crystal structure of MppA in complex with 1 (PDB entry:
3O9P). They have found that the tripeptide forms multiple inter-
actions with the active site of the protein, in particular through
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L
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confirms that the peptide has lost an important interaction with
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Appendix A. Supplementary material
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.biochi.2012.12.011.

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