Transpeptidase-mediated incorporation of d-amino acids into bacterial peptidoglycan

Article abstract of DOI:10.1021/ja2040656

The β-lactams are the most important class of antibiotics in clinical use. Their lethal targets are the transpeptidase domains of penicillin binding proteins (PBPs), which catalyze the cross-linking of bacterial peptidoglycan (PG) during cell wall synthesis. The transpeptidation reaction occurs in two steps, the first being formation of a covalent enzyme intermediate and the second involving attack of an amine on this intermediate. Here we use defined PG substrates to dissect the individual steps catalyzed by a purified E. coli transpeptidase. We demonstrate that this transpeptidase accepts a set of structurally diverse d-amino acid substrates and incorporates them into PG fragments. These results provide new information on donor and acceptor requirements as well as a mechanistic basis for previous observations that noncanonical d-amino acids can be introduced into the bacterial cell wall.

Full text of DOI:10.1021/ja2040656

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Transpeptidase-Mediated Incorporation of D-Amino Acids into
Bacterial Peptidoglycan
Tania J. Lupoli,† Hirokazu Tsukamoto,† Emma H. Doud,†,‡ Tsung-Shing Andrew Wang,†,‡
Suzanne Walker,*,‡ and Daniel Kahne*,†
†Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States
‡Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, United States
S Supporting Information
b
incorporate a variety of noncanonical D-amino acids into peptido-
ABSTRACT: The β-lactams are the most important class of
antibiotics in clinical use. Their lethal targets are the trans-
peptidase domains of penicillin binding proteins (PBPs),
which catalyze the cross-linking of bacterial peptidoglycan
(PG) during cell wall synthesis. The transpeptidation reaction
occurs in two steps, the first being formation of a covalent
enzyme intermediate and the second involving attack of an
amine on this intermediate. Here we use defined PG sub-
strates to dissect the individual steps catalyzed by a purified
E. coli transpeptidase. Wedemonstratethatthistranspeptidase
accepts a set of structurally diverse D-amino acid substrates
and incorporates them into PG fragments. These results
providenew informationondonorandacceptorrequirements
as well as a mechanistic basis for previous observations that
noncanonical D-amino acids can be introduced into the
bacterial cell wall.
glycan via transpeptidation. In addition to providing new tools to
monitor the transpeptidation reaction, these results emphasize
the range of enzymatic activities of TPs and suggest a role for
these proteins in biological signaling through modifications of the
cell wall.
To study the activation step of transpeptidation, we needed an
un-cross-linked PG polymer that would serve as a donor but not
an acceptor in the reaction (Step 1, Figure 1). We have shown that
such polymers can be obtained by PGT-mediated polymerization
of synthetic Lipid II (1, Figure 2a).7 Most Gram-negative bacteria,
including E. coli, make PG with cross-links to meso-diaminopi-
melic acid (m-DAP) as shown in Figure 1 (R = COOH).5
However, a lysine-containing (R = H) precursor (1), which is
found in most Gram-positive bacteria, is simpler to obtain. To
study donor activation, we needed to ensure that cross-linking
does not occur. Therefore, PG precursor Lipid II (1)7a,8 was
acylated on the reactive ɛ-amine of the lysine side chain to produce
Lipid II analog 2.7e Radiolabeled acetic anhydride was used for the
acylation to enable detection of future peptide modifications.
Un-cross-linked PG (3, PG) was then produced by polymerizing
[14C]Ac-Lipid II using the E. coli PBP1A PGT domain.9 Follow-
ing heat inactivation of the PGT, 3 was incubated with or without
E. coli PBP1A* (Figure 2b), which contains an inactive PGT
domain and an active TP domain (see Supporting Information
(SI), Figures S1À2).3d The reaction was quenched after 20 min,
and a previously characterized amidase was added to cleave the
peptides from the MurNAc residues of the glycan polymers
(Figure 2a, dashed red line).6 As shown in Figure 2c, reactions
containing PBP1A* produce two products upon amidase cleavage:
the unmodified pentapeptide and a tetrapeptide that lacks the
terminal D-Ala (lane 4) as confirmed by mass spectrometry
analysis (Figure S3). The tetrapeptide is not observed when
penicillin G, a general PBP inhibitor, is included in the reaction
(lane 6), nor is it observed when the TP is absent from the reaction
(lane 2). Furthermore, it was not observed if only Ac-Lipid II (2)
was incubated with PBP1A* (Figure S4), which is not capable of
producing glycan polymers. We conclude that the formation of
tetrapeptide results from TP-dependent activation of pentapep-
tide followed by hydrolysis, a reaction expected to occur in the
absence of an acceptor substrate (Step 2a, Figure 1). Similar results
were obtained for other TPs from both Gram-negative and
-positive organisms (Figure S5). These results show that
eptidoglycan (PG) is an essential polymer that surrounds
P
bacterial cells. It is composed of linear glycan chains coupled
through peptide cross-links. The glycan strands are synthesized by
peptidoglycan glycosyltransferases (PGTs). The peptide cross-
links are formed by enzymes called transpeptidases (TPs),1 which
are the lethal targets of the most widely used class of antibiotics:
the β-lactams.2 The interactions of β-lactams with TPs are far
better characterized than transpeptidation reactions involving
natural substrates.3 This is mainly due to difficulties in obtaining
suitable substrates to study the TPs. TPs catalyze a two-step
reaction that begins with the activation (Step 1, Figure 1) of a
donor peptide through formation of an acyl-enzyme intermediate
with concomitant release of a terminal D-Alaresidue. This activated
donor caneither be hydrolyzedby water(Step 2a)4 or react with an
amino group on an acceptor peptide attached to an adjacent glycan
strand (Step 2b). While the amino acid residue containing the
reactive amine varies depending on the bacterial species,5 the
overall mechanism is conserved.
Here we report a method to dissect the individual steps of
transpeptidation using substrates that act only as either donors or
acceptors. By examining donor peptides following postreaction
cleavage from the glycan strand,6 we have established that the donor
must be a polymeric fragment of PG. In contrast, the acceptor
can be a single amino acid as long as the amine nucleophile is
found in a D-stereocenter like that in the native substrate. Since
single amino acids serve as acceptors, it is possible to efficiently
Received: May 3, 2011
Published: June 17, 2011
r
2011 American Chemical Society
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Figure 1. Stepwise mechanism of peptidoglycan (PG) transpeptidases
(TPs). TPs activate PG donor strands (Step 1) resulting in release of
terminal D-Ala and formation of an acyl-enzyme intermediate. Either
hydrolysis of this activated donor (Step 2a) or peptide cross-linking to an
acceptor strand (Step 2b) can then occur. For most Gram-negative bacteria,
including E. coli, R = COOH (meso-diaminopimelic acid, m-DAP), while
R=H(L-Lys) for most Gram-positive bacteria5 (MurNAc:N-acetylmuramic
acid; GlcNAc: N-acetylglucosamine).
Figure 2. TPs activate and hydrolyze PG donor strands. (a) Chemical
structure of Lipid II analogs and nascent (un-cross-linked) PG where
n ≈ 30. Amidases cleave peptides from MurNAc residues (dashed red
line) of PG.6 (b) Schematic depicting detection method for modifica-
tions to the PG peptide. PG activated by PBP1A* results in release of
terminal D-Ala followed by hydrolysis of the acyl-enzyme intermediate,
which is indicated by a mixture of tetrapeptides and unreacted penta-
peptides upon amidase cleavage. (c) SDS-PAGE analysis showing that
E. coli PBP1A* incubated with PG followed by amidase treatment
produces pentapeptides and tetrapeptides (lane 4), while PBP1A*
inhibited by penicillin G (penG) produces only pentapeptides (lane
6) as seen in the reaction lacking PBP1A* (lane 2) (PBP1A* contains an
inactivating mutation, E86Q, in the PGT domain; see SI).
polymerized N-acyl Lipid II (3) serves as a donor substrate for
TPs from various organisms.
The ability of activated PG donors to undergo transpeptida-
tion was investigated by adding amino acids as potential accep-
tors to the PBP reaction (Figure 3a, top). For the initial studies,
amino acids were chosen for their similarity to natural peptide
acceptors, which contain reactive amines on m-DAP or lysine
residues (Figure 1).5 E. coli PBP1A, which contains an active PGT
and an active TP domain, was incubated with [14C]Ac-Lipid II
(2) and L-Lys, D-Lys, or m-DAP. The reactions were treated with
amidase and analyzed by SDS-PAGE. m-DAP and D-Lys were
found to be substrates, but L-Lys was not (Figure S6aÀb). m-DAP
contains two stereocenters, one L and one D, and in PG the
L-stereocenter is incorporated into the peptide backbone while
the cross-linking side chain contains the D-stereocenter. Since
D-Lys, but not L-Lys, reacts, we concluded that reaction with D-Lys
occurs on the R-amino group rather than the ɛ side chain. These
results suggest that the enzyme recognizes R-amino acids but
discriminates against the L-configuration.10 Consistent with this
conclusion, both Gly and D-Ala were found to be good substrates
but L-Ala did not react (Figure S6c).
We further examined incorporation of D-Ala into activated
PG polymers to verify the position of labeling. Lipid II (1) was
incubated with [14C]-D-Ala and PBP1A, and the resulting radi-
olabeled PG polymer ([14C]-PG) was treated with amidase. The
reaction was analyzed by paper chromatography to separate PG
polymers from low molecular weight cleavage products (Figure 3a,
bottom and SI).6,7a,7b,11 Amidase treatment resulted in release of
the radiolabel from the polymer (Figure 3b).6 Labeled PG was also
incubated with PBP5 in the presence and absence of penicillin G.
PBP5 is a known carboxypeptidase that hydrolyzes the terminal
D-Ala residue from the pentapeptide side chain of PG (Figure S7).12
Paper chromatography analysis showed that PBP5 causes release
of the radiolabel from [14C]-PG only in the absence of penicillin G,
which inhibits its activity. These data confirm that PBP1A intro-
duces[14C]-D-Alaintothefifth position of the peptide side chain of
PG. The ability to efficiently makelabeled PG via transpeptidation
should enable further mechanistic studies of the transpeptidase
reaction.
The ability of transpeptidases to accept both D-Ala and D-Lys
suggested that other amino acids would be tolerated. Notably,
there have been several reports describing the incorporation of
noncanonical D-amino acids into the existing PG of both Gram-
negative and -positive bacteria.13 The incorporation of these D-
amino acids affects bacterial cell shape,13f as well as biofilm
formation,13g and is proposed to increase the sensitivity of cells
to β-lactams and serve as a means of biological regulation.13h The
mechanism(s) for introduction of D-amino acids into the cell wall
has not been established. Since our results indicate that
D-amino acids can be introduced into PG via transpeptidation,
we evaluated the ability of PBP1A to incorporate several non-
canonical amino acids, including phenylalanine, tyrosine, and
tryptophan, into PG. SDS-PAGE analysis of reactions containing
these amino acids revealed new bands of higher molecular weight
than the substrate pentapeptide in all lanes containing D- (lanes 4,
6, and 8), but not L- (lanes 3, 5, and 7) isomers (Figure 4a).
Liquid chromatographyÀmass spectrometry (LC-MS) anal-
ysis confirmed that D-Phe, D-Tyr, and D-Trp replace one of the
alanine residues in these PG pentapeptides (Figure 4b, Table
S1).14 MS/MS fragmentation of the D-Tyr peptide (4) confirmed
that the added amino acid was in the fifth position (Figure 4c). Hence,
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Figure 3. PG fragments synthesized by E. coli PBP1A are labeled with
excess D-Ala via transpeptidation. (a) Top: Chemical reaction depicting
transpeptidation of an acyl-enzyme intermediate (donor) and D-amino
acid (acceptor) to produce a pentapeptide side chain in PG. Bottom:
Schematic of assay to analyze TP product using D-Ala as an acceptor. PG
is treated with hydrolases (PBP5 or amidase) followed by paper
chromatography to separate unreacted PG (low) from cleaved D-Ala
or peptide (high). (c) Assay results: PBP1A (400 nM) was incubated
with unlabeled Lipid II (1, 40 μM) and [14C]-D-Ala (1 mM) to produce
[14C]-PG. The [14C]-label is removed by treatment with PBP5 or
amidase (4 μM each), but not PBP5 inactivated by penG (5 kU/mL).
Background was not subtracted. Error bars indicate the standard
deviation of triplicate experiments.
Figure 4. E. coli PBP1A incorporates D-, but not L-, noncanonical amino
acids into the termini of PG peptides via transpeptidation. (a) SDS-
PAGE analysis of PBP1A (400 nM) reactions with aromatic amino acids
(1 mM) and labeled Ac-Lipid II (2, 4 μM). Amidase treatment of PG
products reveals a new peptide band in the D-amino acid reactions (lanes
4, 6, and 8) but not the corresponding L-isomer reactions (lanes 3, 5,
and 7) or with added penG (1 kU/mL, lane 1). (b) Overlaid LC-MS
extraction traces of amidase-treated PG produced by incubation of
D-Phe, D-Tyr, or D-Trp (each 1 mM) with Lipid II (1, 40 μM) and PBP1A
(400 nM). Peaks represent mass values of pentapeptides containing
aromatic amino acids in place of terminal D-Ala ([M + H]+ calculated,
observed m/z values for each, respectively: 565.2980, 565.2984;
581.2930, 581.2933; 604.3089, 604.3085). (c) MS/MS fragmentation
of the D-Tyr reaction trace at t = 18.7 min confirms the sequence of the D-
Tyr pentapeptide (4) indicated by the chemical structure (inset with
calculated m/z values).
even D-amino acids with large aromatic substituents function as
acceptors. We found that D-Met, D-Thr, D-Asp, D-Gln, and D-Pro,
but not the corresponding L-isomers, also function as substrates
for transpeptidation (Figures S6dÀe, S8; Table S1). Hence, all
classes of amino acids can be incorporated into the nascent
(un-cross-linked) PG polymer by transpeptidation.
We have reported the development of methods to probe the
transpeptidation step of PG synthesis. We can observe substrate
activation and hydrolysis by postreaction detection of the tetra-
peptide product. Using this method we have established that
activation requires a polymeric substrate since Lipid II does not
react. Although E. coli PBP1A normally utilizes substrates contain-
ing m-DAP in the peptide side chain, activation does not depend on
the presence of this amino acid since polymer-containing N-acyl
lysine (3) can be used as a donor. By monitoring the formation of
new pentapeptides, we have also shown that single amino acids
undergo transpeptidation; however, only amines in R-amino acids
react and there is a stringent selection against the L-configuration.
Since transpeptidation using amino acid acceptors is the reverse of
activation and D-Ala is released during activation,15 the preference
for the D-configuration is unsurprising, but the tolerance for a wide
range of side chains was unexpected. This finding provides a
possible mechanism to explain the observation that noncanonical
D-amino acids are incorporated in vivo into the PG of bacteria;13
there is good agreement between promiscuity seen for D-amino
acid incorporation in vivo to what we have observed in vitro.13bÀ-
e,16
The susceptibility of nascent PG containing noncanonical D-amino acids to
subsequent hydrolysis or cross-linkingby PG biosynthetic enzymes
is unknown but can now be studied in vitro since our results have
provided a facile method to obtain the required substrates. The
method also allows for incorporation of radiolabeled D-Ala into
substrates for future PBP rate studies.17
Our results explain the observation that L-Lys-D-Ala-D-Ala
analogs, commonly used substrates to study PBP-catalyzed
PG hydrolysis,10a,12b do not undergo transpeptidation with E. coli
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PBPs.3b,18 This tripeptide cannot serve as a TP donor since
polymeric PG substrates are required. Furthermore, it cannot serve
as a TP acceptor because the lysine side-chain amine does not
contain an R-D-amino acid.19 In closing, we note that the ability to
reconstitute TP activity using defined substrates and purified
proteins will enable detailed studies into the cross-linking step of
the transpeptidation reaction. Understanding the chemistry of these
enzymes will provide insight into the various roles that TPs play in
building up a new cell wall, editing the existing cell wall, and perhaps
transmitting cellular signals through these chemical modifications.
(7) (a) Ye, X.-Y.; Lo, M.-C.; Brunner, L.; Walker, D.; Kahne, D.;
Walker, S. J. Am. Chem. Soc. 2001, 123, 3155–3156. (b) Chen, L.;
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Supporting Information. Experimental procedures, syn-
b
thesis of substrates and compound analysis, protein construct
descriptions, protein cloning and purification protocols, Q-TOF
LC-MS and SDS-PAGE analysis of peptides. This material is
available free of charge via the Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
suzanne_walker@hms.harvard.edu; kahne@chemistry.harvard.edu
(11) Anderson, J. S.; Matsuhashi, M.; Haskin, M. A.; Strominger, J. L.
Proc. Natl. Acad. Sci. U.S.A. 1965, 53, 881–889.
’ ACKNOWLEDGMENT
This research was supported by the National Institutes of Health
(R01 GM076710; R01 GM066174). All LC-MS data were
acquired on an Agilent 6520 Q-TOF spectrometer supported by
theTaplinFunds forDiscoveryProgram(P.I.:S.W.). Wethankthe
Bernhardt Lab (Harvard Medical School) for PBP.
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undergo cross-linking with E. coli PBP1A..
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