Critical Impact of Peptidoglycan Precursor Amidation on the Activity of l,d-Transpeptidases from Enterococcus faecium and Mycobacterium tuberculosis

Article abstract of DOI:10.1002/chem.201706082

The bacterial cell wall peptidoglycan contains unusual l- and d-amino acids assembled as branched peptides. Insight into the biosynthesis of the polymer has been hampered by limited access to substrates and to suitable polymerization assays. Here we report the full synthesis of the peptide stem of peptidoglycan precursors from two pathogenic bacteria, Enterococcus faecium and Mycobacterium tuberculosis, and the development of a sensitive post-derivatization assay for their cross-linking by l,d-transpeptidases. Access to series of stem peptides showed that amidation of free carboxyl groups is essential for optimal enzyme activity, in particular the amidation of diaminopimelate (DAP) residues for the cross-linking activity of the l,d-transpeptidase Ldt_Mt2 from M. tuberculosis. Accordingly, construction of a conditional mutant established the essential role of AsnB indicating that this DAP amidotransferase is an attractive target for the development of anti-mycobacterial drugs.

Full text of DOI:10.1002/chem.201706082

A Journal of
Accepted Article
Title: Critical impact of peptidoglycan precursor amidation on the
activity of L,D-transpeptidases from Enterococcus faecium and
Mycobacterium tuberculosis
Authors: Flora Ngadjeua, Emmanuelle Braud, Saidbakhrom
Saidjalolov, Laura Iannazzo, Dirk Schnappinger, Sabine Ehrt,
Jean-Emmanuel Hugonnet, Dominique Mengin-Lecreulx,
Delphine Patin Patin, Mélanie Ethève-Quelquejeu, Matthieu
Fonvielle, and Michel Arthur
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201706082
Link to VoR: http://dx.doi.org/10.1002/chem.201706082
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10.1002/chem.201706082
Chemistry - A European Journal
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Critical impact of peptidoglycan precursor amidation on the
activity of L,D-transpeptidases from Enterococcus faecium and
Mycobacterium tuberculosis
Flora Ngadjeua#,[a], Emmanuelle Braud#,[b], Saidbakhrom Saidjalolov[b], Laura Iannazzo[b], Dirk
Schnappinger[c], Sabine Ehrt[c], Jean-Emmanuel Hugonnet[a], Dominique Mengin-Lecreulx[d], Delphine
Patin[d], Mélanie Ethève-Quelquejeu*,[b], Matthieu Fonvielle,*,[a] and Michel Arthur*,[a]
Abstract: The bacterial cell wall peptidoglycan contains unusual L
and D amino acids assembled in branched peptides. Insight into the
biosynthesis of the polymer has been hampered by limited access to
substrates and to suitable polymerization assays. Here we report the
full synthesis of the peptide stem of peptidoglycan precursors from
two pathogenic bacteria, Enterococcus faecium and Mycobacterium
tuberculosis, and the development of a sensitive post-derivatization
assay for their cross-linking by L,D-transpeptidases. Access to series
of stem peptides showed that amidation of free carboxyl groups is
essential for optimal enzyme activity, in particular the amidation of
diaminopimelate (DAP) residues for the cross-linking activity of the
L,D-transpeptidase LdtMt2 from M. tuberculosis. Accordingly,
construction of a conditional mutant established the essentiality of
AsnB indicating that this DAP amidotransferase is an attractive target
for the development of anti-mycobacterial drugs.
subunits containing the canonic GlcNAc-MurNAc motif. The most
frequent variations in the sequence of the pentapeptide stem
occur at the 3rd (e.g. L-Lys instead of diaminopimelic acid [DAP])
and at the 5th (e.g. D-Lac instead of D-Ala) positions (Figure 1b).
Modifications of the pentapeptide stem involve the addition of a
side-chain to the 3rd residue (e.g. D-isoAsn or Gly5) and the
amidation of the carboxyl groups (e.g. the α-carboxyl of D-Glu and
D-isoAsp or the ε-carboxyl of DAP). A last source of polymorphism
originates from the presence of 3→3 instead of 4→3 cross-links
in mycobacteria (e.g. Mycobacterium tuberculosis) and in β-
lactam-resistant mutants generated in vitro (e.g. Enterococcus
faecium) (Figure 1a). The 3→3 cross-links are formed by
transpeptidases of the L,D specificity, which cleave the L-Lys3-D-
Ala4 or DAP3-D-Ala4 bond of an acyl donor containing a
tetrapeptide stem and form L-Lys3→L-Lys3 or DAP3→DAP3 cross-
links.
Variability in the peptidoglycan structure has been known for
decades based on biochemical analyses of the cell wall.[3a] The
corresponding enzymes have been described more recently,
mostly because the complexity of their substrates has hampered
their characterization.[4] Consequently, the biological significance
of structural variability is poorly understood. It may involve various
selective advantages.[3b] Resistance to vancomycin is mediated
by replacement of D-Ala by D-Lac or D-Ser at the 5th position of
peptide stems since this prevents binding of the drug to the
precursors. Specific links in mature peptidoglycan are cleaved by
hydrolytic enzymes produced by eukaryote hosts, such as
lysozyme, which cleaves the MurNAc-GlcNAc β-1,4 bond, or by
competing bacteria, such as lysostaphin, which cleaves glycyl-
glycine bonds in the D-Ala4→(Gly5)-L-Lys3 cross-bridges of
Staphylococcus aureus. Variations in peptidoglycan structure are
therefore potential defense mechanisms against hydrolytic
enzymes.
Diversification of the structure of peptidoglycan precursors
associated with speciation is thought to lead to a parallel evolution
of the substrate specificity of the transpeptidases.[5] Genetic
evidence in favor of this hypothesis is limited since impaired
maturation of peptidoglycan precursors may have combined
effects on numerous peptidoglycan biosynthetic steps in addition
to transpeptidation. Scarce evidence has been provided by
biochemical studies due to limited access to purified enzymes and
substrates.[6] In this study, we have developed the chemical
synthesis of peptidoglycan precursor analogues and a post-
derivatization assay to directly assess the impact of amidation of
peptidoglycan precursors on the formation of cross-links by
purified L,D-transpeptidases from E. faecium and M. tuberculosis.
We show that defects in amidation strongly impair the efficacy of
these enzymes indicating that the amidotransférases[7] are
attractive targets to develop alternatives to transpeptidase
inhibition by β-lactam antibiotics in drug resistant bacteria.
Peptidoglycan is an essential and specific component of the
bacterial cell wall.[1] The main role of this giant (cell-sized)
macromolecule is to protect bacterial cells against the osmotic
pressure of the cytoplasm. The peptidoglycan subunit consists of
a disaccharide substituted by a pentapeptide stem (Figure 1),
which is polymerized by glycosyltransferases for the elongation of
the glycan chains (all glycosidic bonds are β-1,4) and by D,D-
transpeptidases for cross-linking the glycan chains to each
other.[2] The amide bond formed by the D,D-transpeptidases links
the carbonyl of D-Ala at the 4th position of an acyl donor stem to
the side-chain amino group at the 3rd position of an acyl acceptor
stem (4→3 cross-link). These enzymes are the targets of β-
lactam antibiotics such as penicillin.
The structure of peptidoglycan is generally conserved in
bacteria belonging to the same species, but highly diverse
between species, including members of the same genus.[3] The
polymorphisms include the N-deacetylation, O-acetylation, and N-
glycolylation of either or both GlcNAc and MurNAc. These
modifications are mostly, if not exclusively, due to maturation of
[a]
Dr. Flora Ngadjeua, Dr. Jean-Emmanuel Hugonnet, Dr. Matthieu
Fonvielle*, Dr. Michel Arthur*, INSERM UMRS 1138, Sorbonne
Universités, UPMC Univ Paris 06; Sorbonne Paris Cité, Université Paris
Descartes, Université Paris Diderot; Centre de Recherche des
Cordeliers, 75006 Paris, France. E-mail: michel.arthur@crc.jussieu.fr;
matthieu.fonvielle@crc.jussieu.fr
[b]
Dr. Emmanuelle Braud, Saidbakhrom Saidjalolov, Dr. Laura Iannazzo,
Dr. Mélanie Ethève-Quelquejeu*, Laboratoire de Chimie et de Biochimie
Pharmacologiques et Toxicologiques, Université Paris Descartes, UMR
8601, Paris, F-75005 France; CNRS UMR 8601, Paris, F-75006 France.
E-mail: melanie.etheve-quelquejeu@parisdescartes.fr
[c]
Dr. Dirk Schnappinger, Dr. Sabine Ehrt, Department of Microbiology and
Immunology, Weill Cornell Medical College, New York, NY 10021, USA.
Dr. Dominique Mengin-Lecreulx, Delphine Patin, Institute for Integrative
[d]
Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-
Saclay, 91198, Gif-sur-Yvette cedex, France.
[#] and [*] These authors contributed equally to this work.
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Peptidoglycan subunit (Disaccharide-pentapeptide)
Enterococcusfaecium Mycobacterium tuberculosis
GlcNAc-MurNGlyc
a)
b)
GlcNAc-MurNAc
L-Ala
L-Ala
1
2
1
2
D-isoGln
D-isoGln
DAPNH2
D-Ala
L-Lys
D-Ala
D-Ala
D-isoAsn
3
4
5
3
4
5
D-Ala
Subunit
strand
4→3
Figure 1. Peptidoglycan structure. a) Peptidoglycan is a giant mesh-like polymer that completely surrounds bacterial cells. b) Peptidoglycan is polymerized from
disaccharide-pentapeptide subunits, which are assembled in the cytoplasm. MurNAc, N-acetyl muramic acid; MurNGlyc, N-glycolyl muramic acid; GlcNAc, N-acetyl
glucosamine; D-isoGln, D-iso-glutamine; D-isoAsn, D-iso-asparagine; DAPNH2, diaminopimelic acid amidated at the ε position.
Peptidoglycan precursors contain unusual amino acids (D-
isoGln, D-Ala, D-isoAsn, amidated DAP), a non-peptide bond
linking the γ-carboxyl of D-isoGln to the α-amino group of L-Lys or
DAP, and a side-chain linked to the ε-amino group of L-Lys (Figure
1b). Synthesis of peptides containing these amino acids have
been previously reported using chemo-enzymatic methods,[8]
chemical synthesis in solution,[9] or on solid phase.[10] For this
study, we used the solid-support peptide synthesis strategy,
which required access to non-commercial Fmoc-protected amino
acids and the use of orthogonal protecting groups for the
synthesis of branched peptides (Scheme 1). Synthesis of the
Fmoc-protected DAP and amidated DAP has been performed
using a cross-metathesis reaction as a key step (Scheme 1a).[11]
Synthesis of the other protected amino acids is described in the
Supplementary Material. Orthogonal Fmoc and 1-(4,4-dimethyl-
A post-functionalization assay was developed to quantify the
substrates and the product of the enzymatic cross-linking reaction
catalyzed by Ldtfm from E. faecium, the prototypic enzyme of the
L,D-transpeptidase family (Figure 2a). We took advantage of the
presence of a single primary amine on these molecules to
specifically introduce a 4-fluoro-7-nitrobenzofurazan group by
nucleophilic aromatic substitution. This post-functionalization
reaction applied to the crude enzymatic reaction converted the
unreacted donor and acceptor substrates as well as the L,D-
transpeptidation product (dimer) into fluorescent peptides. The
post-functionalized peptides were separated by rpHPLC and
detected by fluorescence (Figure 2b). Since the fluorescence
yield may vary with the acetonitrile concentration required for
elution and with the molecular environment of the fluorophore
within the peptides, calibration curves were obtained for every
substrate and every product (Figure 2c). For this purpose, each
substrate and each product were individually post-functionalized
with 4-fluoro-7-nitrobenzofurazan and known amounts of each
fluorescent peptide were analyzed by rpHPLC. Access to
sensitive determination of the peptides in the 10- to 200-pmol
range provided a versatile method to determine the rate of the
cross-linking reaction catalyzed by Ldtfm.
2,6-dioxocyclohexylidene)-3-methylbutyl
(ivDde)
protecting
groups were used for sequential assembly of the peptide stem
and of the side-chain residue branched at the 3rd position,
respectively (Scheme 1b). Using this approach 9 peptides
mimicking peptidoglycan precursors have been synthesized and
fully characterized.
a)
b)
Scheme 1. a) Synthetic scheme for the protected DAP and amidated DAP building blocks. i) 5% Grubb’s II catalyst, CH2Cl2, RT, 12 h; ii) 3% PtO2, H2,
CH2Cl2/CH3OH/H2O (9/1/1), RT, 12 h; iii) 20% Grubb’s II catalyst, 1,2-dichloroethane, 70°C, 48 h; iv) PtO2, H2, CH3OH, 4 atm, RT, 18 h. b) Solid-phase-synthesis
of the peptidoglycan precursor analogues using orthogonal Fmoc and ivDde protecting groups.
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The reaction catalyzed by Ldtfm involves two peptidoglycan
precursors that act as an acyl donor and as an acyl acceptor
(Figure 2a). For the donor, we used a linear tetrapeptide that
cannot be used as an acyl acceptor since it does not harbor the
D-isoAsn residue branched to L-Lys. Conversely, the peptides
used as acyl acceptors cannot be used as acyl donors since they
do not harbor the essential C-terminal D-Ala residue (D-Ala4). To
specifically assess the impact of amidation in the acceptor
substrate, we tested a tetrapeptide donor containing a D-isoGln
residue (acyl donor 3A) and acceptors containing the four
combinations of amidation of the D-isoGlu and D-isoAsp α-
carboxyl groups (acyl acceptors 4A to 4D). The rate of formation
of peptidoglycan dimers by Ldtfm was the highest for the fully
amidated acceptor, corresponding to the amidation status found
in the peptidoglycan of the E. faecium host (Figures 2d and 2e).
The lack of amidation of D-isoGln had a moderate impact on the
transpeptidase activity of Ldtfm (36% residual activity). The impact
of the lack of amidation was greater for the side-chain D-isoAsn
residue (8.4% residual activity), whereas the combination of both
modifications almost completely abolished the activity of Ldtfm
(0.83% residual activity). No transpeptidation product was
observed for substrates fully lacking amidation both in the acyl
donor and acceptor substrates (data not shown). In conclusion,
amidation of the D-isoGlu and D-isoAsp α-carboxyl groups of the
acyl acceptor substrate was essential for optimal formation of
peptidoglycan cross-links by Ldtfm in vitro. It has previously been
shown that amidation of D-isoGlu is also required for effective
formation cross-links by purified PBPs from Streptococcus
pneumoniae.[12]
Our following objective was to evaluate the role of amidation
of the α- and ε-carboxyl groups of D-isoGlu and DAP on the
efficacy of formation of 3→3 cross-links by L,D-transpeptidases
from M. tuberculosis. We based our analysis on LdtMt2 as a
representative of the five L,D-transpeptidase paralogues
produced by this species. Substrates containing the four
combinations of amidation of the linear tetrapeptide stem of M.
tuberculosis were synthesized and independently tested in the
quantitative cross-linking assay (Figure 3). No cross-linked dimer
was observed with substrates lacking amidation of DAP, D-isoGlu,
or both. To assess the impact of the lack of amidation of
peptidoglycan precursors on the growth of M. tuberculosis, we
constructed mutants of strain H37Rv conditionally producing the
DAP amidotransferase AsnB (Rv2201). This analysis showed that
the amidation of the ε-carboxyl of DAP is required for growth of M.
tuberculosis H37Rv.
Donor
3A
Acceptor
4A to D
Dimer
3A/4A to D
a)
pH 8.0
65°C
Ldtfm
R = unreacted donor and acceptor
substrates and dimer product
Substrate
Product
3A/4C
A
A: Acceptor substrate
D: Donor substrate
P: Dimer product
b)
d)
c)
4A
4B
4C
4D
3A
A
3A/4D
3A/4B
3A/4A
D
P
A
P
D
0 min
15 min
120 min
D
16
14
12
10
8
Dimer
3A/4A
Peptideamount[pmol]
Peptideamount [pmol]
3A/4B
3A/4C
3A/4D
e)
Substrate
Donor
Product
Dimer
Cross-linking efficacy
Acceptor
4A R1=NH2 R2=NH2
4B R1=OH R2=NH2
4C R1=NH2 R2=OH
4D R1=OH R2=OH
Turnover x 106 (s-1)
%
100
36
6
3A
3A
3A
3A
(3A/4A)
(3A/4B)
(3A/4C)
(3A/4D)
3,100
1,100
260
±
±
±
±
1,000
500
150
7
4
2
8.4
0.83
0
0
30
60
90
120
26
Time [min]
Figure 2. Post-functionalization assay for the cross-linking activity of peptidoglycan transpeptidases. a) Reaction catalyzed by the L,D-transpeptidase Ldtfm. Inset,
post-functionalization reaction. b) Separation by rpHPLC of the post-functionalized substrates 3A and 4A and of the reaction product (dimer 3A/4A), which were
detected by fluorescence (λex = 470 nm; λem = 530 nm). c) Calibration curves for quantitative determination of the substrates (left panel) and reaction products (right
panel). d) Kinetics of dimer synthesis by Ldtfm. e) Impact of amidation of the acyl acceptor on the cross-linking efficacy of Ldtfm
.
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Donor
3B to E
Acceptor
3B to E
Dimer
3B/3B
3C/3C
3D/3D
3E/3E
TetOn-2 TetOn-6
a)
c)
1
2
3
1
2
3
100
10-1
10-2
10-3
10-4
LdtMt2
10-5
10-6
With anhydrotetracycline
TetOn-2 TetOn-6
1
2
3
1
2
3
b)
100
Substrate
Product
(Dimer)
(3B/3B)
(3C/3C)
(3D/3D)
(3E/3E)
Cross-linking efficacy
Turnover x 106 (s-1)
10-1
10-2
10-3
10-4
10-5
10-6
(Used as donor and acceptor)
%
3B
3C
3D
3E
R1=NH2 R2=NH2
R1=OH R2=NH2
R1=NH2 R2=OH
R1=OH R2=OH
1,300
±
100
100
< 2
< 2
< 2
< 26
< 26
< 26
Without anhydrotetracycline
Figure 3. Impact of impaired peptidoglycan precursor amidation on the activity of M. tuberculosis L,D-transpeptidase LdtMt2. a) Transpeptidation reaction catalyzed
by LdtMt2. b) In vitro cross-linking activity of LdtMt2. c) Impact of impaired amidation of DAP by AsnB (Rv2201) on the growth of M. tuberculosis H37Rv. Ten-fold
dilutions of cultures of mutants Rv2201-TetON-2 and Rv2201-TetON-6 were spotted on the indicated media showing that depletion of AsnB in the absence of
anhydrotetracycline prevents growth.
[1] A. J. Egan, R. M. Cleverley, K. Peters, R. J. Lewis, W.
Vollmer, FEBS J. 2017, 284, 851-867.
[2] E. Sauvage, M. Terrak, Antibiotics (Basel) 2016, 5, pii: E12.
In conclusion, we have developed routes of synthesis of the
peptide stems of peptidoglycan precursors that provide access to
the full structural diversity found in bacteria, including the
presence of DAP or L-Lys at the 3rd position, the presence or
absence of a side-chain, and the amidation of carboxyl groups.
We have also developed a sensitive assay for the detection of the
products of the cross-linking reaction. Based on these new tools,
functional analysis was focused on the impact of amidation of
carboxyl groups in the D-isoGlu and DAP residues of
peptidoglycan precursors on the activity of L,D-transpeptidases.
For the first time, we directly establish that the diversification of
the structure of peptidoglycan precursors is associated with a
parallel evolution of the substrate specificity of cross-linking
enzymes. Amidation of carboxyl groups was essential for the in
vitro activity of L,D-transpeptidases from E. faecium and M.
tuberculosis. Amidation of DAP was also essential for growth of
M. tuberculosis indicating that the amidotransferase AsnB is a
potential target for development of new drugs active on multi-drug
resistant bacilli.
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Acknowledgements
This work was supported by the project NAPCLI from the JPI
AMR program to MA (N° ANR-14-JAMR-0003-01).
Keywords: Amidation • Amidotransferase • Mycobacterium
tuberculosis • Peptidoglycan • Transpeptidase
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Layout 1:
COMMUNICATION
Sensitive fluorescent
cross-linking assay
Synthetic routes to the peptide stem of
peptidoglycan precursors and a sensitive
fluorescent cross-linking assay were
developed to assess the impact of
structural variability on peptidoglycan
polymerization. In the search for new
targets for anti-mycobacterial drug
development, this strategy was applied to
Flora Ngadjeua#, Emmanuelle Braud#,
Saidbakhrom Saidjalolov, Laura Iannazzo,
Dirk Schnappinger, Sabine Ehrt, Jean-
Emmanuel Hugonnet, Dominique Mengin-
Lecreulx, Delphine Patin, Mélanie Ethève-
Quelquejeu*, Matthieu Fonvielle,* and Michel
Arthur*
asnB
Off On
Impactof
amidation
Page No. – Page No.
the
evaluation
of
diaminopimelate
Critical
precursor amidation on the activity of L,D-
transpeptidases from Enterococcus
faecium and Mycobacterium tuberculosis
impact
of
peptidoglycan
amidation in Mycobacterium tuberculosis,
revealing the essential role of the AsnB
Synthesis of
peptidoglycan
fragments
amidotransferase
for
peptidoglycan
AsnB amidotransferase:new
targetin M.tuberculosis
transpeptidation both in vitro and in vivo.
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