The mechanism of action of ramoplanin and enduracidin
Xiao Fang,adKittichoat Tiyanont,bdYi Zhang,aJutta Wanner,cDale Bogercand Suzanne Walker*d
Received 28th October 2005, Accepted 16th November 2005
First published as an Advance Article on the web 29th November 2005
The lipoglycodepsipeptide antibiotic ramoplanin is proposed to inhibit bacterial cell wall
biosynthesis by binding to intermediates along the pathway to mature peptidoglycan, which
interferes with further enzymatic processing. Two sequential enzymatic steps can be blocked by
ramoplanin, but there is no definitive information about whether one step is inhibited
preferentially. Here we use inhibition kinetics and binding assays to assess whether ramoplanin
and the related compound enduracidin have an intrinsic preference for one step over the other.
Both ramoplanin and enduracidin preferentially inhibit the transglycosylation step of
peptidoglycan biosynthesis compared with the MurG step. The basis for stronger inhibition is a
greater affinity for the transglycosylase substrate Lipid II over the MurG substrate Lipid I. These
results provide compelling evidence that ramoplanin’s and enduracidin’s primary cellular target is
the transglycosylation step of peptidoglycan biosynthesis.
Peptidoglycan is a crosslinked carbohydrate polymer that
surrounds bacterial cells and prevents them from rupturing
under high internal osmotic pressures. Because peptidoglycan
is essential for survival and has no eukaryotic counterpart,
peptidoglycan biosynthesis (Fig. 1) is the target of a large
number of clinically used antibiotics, including the b-lactams,
cephalosporins, and glycopeptide antibiotics.1The emergence
of resistance to all these classes of antibiotics represents a
significant threat to public health and has stimulated efforts to
develop structurally novel antibacterial agents that inhibit the
peptidoglycan biosynthetic pathway. One molecule that has
received considerable attention in recent years is ramoplanin
(Fig. 2, 1), a lipoglycodepsipeptide antibiotic discovered in the
1980s in a screen for peptidoglycan synthesis inhibitors.2,3
Ramoplanin has good activity against a wide range of Gram-
positive organisms and is regarded as a promising candidate
for the treatment of many Gram-positive infections.4It is
currently in late stage clinical trials for two different
indications.5,6Due to hydrolytic instability and other issues,
however, neither of these indications involves systemic
administration of ramoplanin, and the full potential of this
compound has yet to be realized.4A better understanding of
the mechanism of action of ramoplanin may enable the
development of derivatives to treat systemic infections.
A mechanism of action for ramoplanin was first proposed in
1990 by Somner and Reynolds, who showed, using a cell-free,
particulate membrane assay, that the antibiotic blocks the
MurG-catalyzed conversion of Lipid I to Lipid II (Fig. 1) on
the biosynthetic pathway to peptidoglycan.7Although no
direct evidence for an interaction with Lipid I was presented,
these authors suggested that ramoplanin kills bacterial cells by
binding to this substrate, rendering it inaccessible to MurG.7A
decade later, also using a cell-free, particulate membrane
system, we showed that ramoplanin inhibits the transgly-
cosylase-catalyzed coupling of Lipid II molecules to form the
carbohydrate chains of peptidoglycan.8We established that
ramoplanin binds to synthetic Lipid II analogues, and so we
proposed that ramoplanin acts primarily by binding to Lipid II
and inhibiting the transglycosylation step of peptidoglycan
Our hypothesis that the primary mechanism of action of
ramoplanin involves binding to Lipid II and inhibition of
transglycosylation rather than binding to Lipid I and inhibition
of MurGrestedlargely onthe factthat Lipid IIistranslocated to
the external surface of the bacterial membrane as soon as it is
produced whereas Lipid I remains on the internal surface of the
membrane.10Ramoplanin is a large and highly water-soluble
molecule, and in the absence of a dedicated transport mecha-
nism, it seemed improbable that it could diffuse readily through
bacterial membranes to reach an intracellular target. In fact,
Somner and Reynolds made this point in their mechanistic
papers on ramoplanin, but when they did their studies it was not
known that Lipid I and MurG are intracellular.7,10
Comparative information on how well ramoplanin and
various analogues inhibit MurG and the bacterial trans-
glycosylases could provide more insight into the mechanism of
action of the molecule. We have developed synthetic routes to
Lipid I and Lipid II substrates and have developed assays to
study E. coli MurG and E. coli PBP1b, the major bacterial
transglycosylase in this organism.9,11,12These tools enable us
to carry out the studies required to assess the importance of the
different proposed targets of ramoplanin and structurally
related compounds. Below we report a comparative analysis of
aDepartment of Chemistry and Chemical Biology, Harvard University,
12 Oxford St., Cambridge, MA 02128, USA.
E-mail: email@example.com.; Tel: 617-432-5498
bDepartment of Chemistry, Princeton University, Princeton, NJ 08544,
cDepartment of Chemistry and The Skaggs Institute for Chemical
Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
dDepartment of Microbiology and Molecular Genetics, Harvard Medical
School, Harvard University, Boston, MA 02115, USA.
PAPERwww.rsc.org/molecularbiosystems | Molecular BioSystems
This journal is ? The Royal Society of Chemistry 2006 Mol. BioSyst., 2006, 2, 69–76 | 69
the inhibition kinetics of ramoplanin, the ramoplanin aglycon
(Fig. 2, 3), and the related antibiotic enduracidin (Fig. 2, 2)
with respect to MurG and PBP1b. We also present quantita-
tive information on the binding affinities of ramoplanin for
Lipid I and Lipid II. These studies support the hypothesis that
the primary mechanism of action of ramoplanin involves
binding to Lipid II and inhibiting the transglycosylation step
of peptidoglycan biosynthesis. They also indicate that endur-
acidin operates by the same mechanism as ramoplanin.
Ramoplanin was a gift from Oscient Pharmaceuticals.
Enduracidin was purchased as the hydrochloride salt from
Sigma-Aldrich (approx. 75% pure) and was further purified by
HPLC (see below for conditions). The ramoplanin aglycon was
prepared as previously reported.13Alkaline phosphatase was
purchased from Roche. UDP-[14C*]-GlcNAc (specific activity =
288 mCi mmol21) was purchased from Perkin-Elmer Life
Sciences, Inc. 6-(fluorescein-5-carboxamido)-hexanoic acid, suc-
A solution of 5 mg enduracidin in 1.5 mL DMF was purified
by reverse phase HPLC (VyDAC protein & peptide C18
column, Solvent A: H2O/0.1% trifluoroacetic acid; Solvent B:
CH3CN/0.1% trifluoacetic acid) using a linear gradient from
0% B to 100% B over 145 min.
Fig. 1 Peptidoglycan biosynthesis.
Fig. 2Structure of ramoplanin (1), enduracidin (2) and the ramoplanin aglycon (3).
70 | Mol. BioSyst., 2006, 2, 69–76 This journal is ? The Royal Society of Chemistry 2006
Kinetic assay for enduracidin and ramoplanin aglycon in MurG
described.14The reactions contained 0.01 mg mL21MurG
(260 nM), reaction buffer (50 mM HEPES, pH 7.9), 5 mM
MgCl2, 35mM UDP-[14C*]-GlcNAc, 10% methanol, varied
concentrations of tetraprenyl Lipid I15and inhibitor (see Fig. 4
for concentrations) in a total volume of 10mL in eppendorf
tubes. After 2 min, the reactions were quenched with 1% SDS.
The product was separated using paper chromatography
(3 MM Whatman chromatography paper, 5 : 3 isobutyric
acid : 1N NH4OH), and quantified by scintillation counting.
Kaleidagraph (Version 3.6.4, Synergy Software) was used to fit
the data to an equation for substrate depletion.9
was overexpressedand purifiedas previously
Kinetic assays for enduracidin and ramoplanin aglycon in
E.coli PBP1b was expressed and purified as previously
described.16Assays were done in Eppendorf tubes containing
transglycosylase buffer (50 mM HEPES at pH 7.5, 10 mM
CaCl2, 1000 U mL21penicillin G, 0.2 mM decyl PEG and 11%
DMSO) and varying concentrations of [14C*]-GlcNAc-labeled
heptaprenyl Lipid II9(specific activity = 288 mCi mmol21),
6 mM of 2 or 3. The reactions were initiated by adding 1 mL
PBP1b (from a solution freshly prepared by diluting a 50%
glycerol stock of PBP1b 5-fold into 5 mM Tris buffer, pH 8.0,
containing 8 mM decyl PEG) to the reaction mixtures for a
final volume of 10 mL and an enzyme concentration of 30 nM.
The reactions were stopped after 8 minutes by adding 10 mL of
ice cold 10 mM Tris (pH 8.0) containing 0.2% Triton X-100.
The products and starting material were separated using paper
chromatography (3MM Whatman chromatography paper, 5:3
isobutyric acid:1 N NH4OH), and quantified by scintillation
counting. Kaleidagraph (Version 3.6.4, Synergy Software) was
used to fit the data to an equation for substrate depletion.9
Synthesis of fluorescein-labeled Lipid I analog (4)
One mg (1 mmol) of citronellyl Lipid I (synthesized as
previously described14) was dissolved in 120 mL saturated
sodium bicarbonate solution. A solution of 2 mg (5 mmol)
ester in 120 mL dioxane was added, and the reaction was
stirred for 1 h at room temperature. Another milligram
(2.5 mmol) of 6-(fluorescein-5-carboxamido)-hexanoic acid,
succinimidyl ester was added to the reaction mixture and
stirred for an additional hour. The reaction mixture was then
concentrated under vacuum, and purified by reverse phase
HPLC using a linear gradient of H2O/CH3CN containing 0.1%
ammonium bicarbonate. The product was eluted at approxi-
mately 75% H2O/CH3CN. The yield was 28%. Product 4 was
analyzed by ESI-MS2(calculated: 1533.5, found 1532.5).
Synthesis of fluorescein-labeled UDP-GlcNAc analog (5)
The fluoresceinated UDP-GlcNAc analog (5) was prepared by
diluting UDP-glucosamine (11.4 mmoles) in a 1 : 1 mixture of
aqueous NaHCO3 (0.4 M) and dioxane. 6-(fluorescein-5-
carboxamido) hexanoic acid, succinimidyl ester (20 mg,
34 mmoles) was added as a solution in dioxane. The reaction
was run overnight at room temperature. To obtain the desired
product 5, the reaction mixture was purified by reverse phase
HPLC (Phenomenex Luna 5m C18(2), 100A 250 6 21.2 mm;
solvent A: H2O/0.1% ammonium bicarbonate; solvent B:
CH3CN/0.1% ammonium bicarbonate). The following gradi-
ent was used for the purification: t = 0, %B = 0; t = 130, %B =
50; t = 145, %B = 100.1H NMR (400 MHz, D2O): d 8.08 (s,
1H, fluorescein), 7.87 (d, J = 6.4 Hz, 1H, fluorescein), 7.65 (d,
J = 7.8 Hz, 1H, H6-uracyl), 7.19 (d, J = 6.0 Hz, 1H,
fluorescein), 6.94 (m, 2H, fluorescein), 6.55 (m, br, 4H,
fluorescein), 5.61 (d, J = 7.8 Hz, 1H, H5-uracyl), 5.56 (d, J =
4.8 Hz, 1H, H1-ribose), 5.33 (d, br, J = 4.0 Hz, 1H, H1-hexose),
4.16–3.18 (m, 13H), 2.22 (t, J = 7.2 Hz, 2H, He), 1.60–1.42 (m,
br, 4H, Hband Hd), 1.32–1.22 (m, 2H, Hc).
Synthesis of fluorescein-labeled Lipid II analog (6)
0.5 mL of the fluoresceinated UDP-GlcNAc analog (5, 10 mM)
and 0.5 mL of tetraprenyl C20 lipid I15(10 mM) were incubated
with 5 mL of 10 mg mL21MurG, 31.5 mL of alkaline
phosphatase (3.2 U mL21), 7.5 mL MeOH, and 5 mL reaction
buffer (50 mM HEPES, pH 7.9 and 5 mM MgCl2) for 1 h. Ten
of the above reactions were combined and purified using
reverse phase HPLC at a liner gradient from 0% B to 100% B
over 45 min (solvent A: H2O/0.1% trifluoroacetic acid; solvent
B: CH3CN/0.1% trifluoroacetic acid). Product 6 was analyzed
by ESI-MS2(calculated: 1827.7, found 1826.7).
Fig. 3Structures of fluorescein-labeled peptidoglycan precursors.
This journal is ? The Royal Society of Chemistry 2006Mol. BioSyst., 2006, 2, 69–76 | 71
Fluorescence binding assays of ramoplanin against 4 and 6
Fluorescent measurements were done using an Aminco
Bowman Series 2 Luminescence Spectrometer (lex= 492 nm,
lem= 525 nm). Methanol stock solutions of 4 or 6 were diluted
in buffer (25 mM potassium phosphate, 75 mM NaCl, pH 7.0)
to yield a final concentration of 100 nM in 1 mL cuvettes.
Aliquots of ramoplanin in DMSO were added and the
mixtures were allowed to equilibrate 10 min to 1 h until the
fluorescence intensity stopped changing. The titration data was
fit using Prism (version 4.0a, GraphPad Software, Inc.) to the
following equation describing binding of a ramoplanin dimer
to 4 or 6 (i.e., R2F > R2+ F):
I = I0+ [([R2] + [F] + Kd) 2 (([R2] + [F] + Kd)22
Where I denotes fluorescence intensity (lex= 492 nm, lem=
525 nm); I0denotes the fluorescence intensity in the absence of
ramoplanin (lex = 492 nm, lem = 525 nm); I3 denotes
fluorescence intensity at the end of ramoplanin titration (lex=
492 nm, lem= 525 nm); [R2] represents the total concentration
of ramoplanin dimer; and [F] represents the total concentra-
tion of 4 or 6.
Displacement assay of Lipid I and Lipid II against 4
Citronellyl Lipid I and Lipid II were prepared as previously
reported.8,14,15Fluorescent measurements were done using an
Aminco Bowman Series 2 Luminescence Spectrometer (lex=
492 nm, lem= 525 nm). A methanol stock of 4 was diluted in
buffer (25 mM potassium phosphate, 75 mM NaCl, pH 7.0) to
a final concentration of 100 nM and the fluorescence intensity
was monitored. Aliquots of a DMSO stock solution of 1 were
added to 4 to a final concentration of 600 nM. Then aliquots
of the unlabeled citronellyl Lipid I or Lipid II DMSO stock
were added to the mixture, which was allowed to equilibrate
until the fluorescence intensity stopped changing. The Lipid II
and presence of 1,2 and 3. (A) MurG reaction in the absence ($) and presence (m and .) of 1 (2 mM and 5 mM, respectively); (B) Transglycosylase
reaction in the absence ($) and presence (r) of 1 (6 mM); (C) MurG reaction in the absence ($) and presence (#) of 2 (6 mM); (D)
Transglycosylase reaction in the absence ($) and presence (#) of 2 (6 mM); (E) MurG reaction in the absence ($) and presence (&) of 3 (5 mM);
(F) Transglycosylase reaction in the absence ($) and presence (r) of 3 (6 mM).
Representative velocity versus substrate concentration curves for MurG (A, C, E) and transglycosylase (B, D, F) reactions in the absence
72 | Mol. BioSyst., 2006, 2, 69–76This journal is ? The Royal Society of Chemistry 2006
titration was stopped when the fluorescence intensity returned
to the level observed before 1 was added (7 6 1025M). At the
same concentration, the fluorescence intensity was observed to
increase only by 0.2 fluorescence units, corresponding to 10%
displacement of 4 from the complex. Because of the limited
amount of citronellyl Lipid I, the titration was not continued.
Previous results on the inhibition kinetics of ramoplanin with
respect to MurG and PBP1b
Preliminary results on the inhibition of MurG and PBP1b by
ramoplanin revealed significant differences in the mode of
inhibition of these enzymes (Fig. 4A and 4B).9,17Notably, the
curves for inhibition of MurG indicated a non-competitive
mode of inhibition whereas those for PBP1b were sigmoidal,
which is consistent with a mechanism involving binding to
Lipid II.9,17The sigmoidal shape of the inhibition curves
reflects the fact that the reaction rate is negligible at low
concentrations because the substrate is bound to ramoplanin
and unavailable for reaction; however, as soon as the substrate
concentration exceeds a critical value, the rate jumps
dramatically and inhibition is largely overcome. From the
concentration at which the reaction rate was observed to jump
(always one half the ramoplanin concentration) and structural
studies, we concluded that ramoplanin binds to Lipid II as a
dimer.9,18A Job titration confirmed this stoichiometry.19In
contrast, the inhibition curves for MurG suggested a mode of
inhibition involving a direct interaction between ramoplanin
itself and the enzyme.17Surprisingly, the inhibition was found
not to depend on binding to Lipid I.17Since ramoplanin had
been shown to bind to Lipid I analogues in vitro,17we were
perplexed by these results. Therefore, we decided to investigate
the behavior of a structurally related antibiotic, enduracidin, in
the hope that this molecule would shed some more light on the
behavior of ramoplanin.
Inhibition kinetics of enduracidin with respect to MurG and
Enduracidin (Fig. 2, 2) is a lipodepsipeptide that was isolated
in the 1960s and found to be active against a wide range of
Staphylococcus aureus (MRSA).22Enduracidin was proposed
to inhibit peptidoglycan biosynthesis because it causes the
in bacteria;23however, the site of inhibition was never
Like ramoplanin, enduracidin contains 17 amino acid
residues, 16 of which form a macrocycle joined by a lactone
linkage from the b-hydroxyl group on residue 2 (Thr2) to the
carboxyl group of residue 17 (Hpg17). Enduracidin is identical
to ramoplanin at residues 3 through 8, and at residues Gly14,
Ala16, and Hpg17. In addition, it has the same chirality at all 17
amino acids and bears the same charge (+2) at physiological
pH. Differences between enduracidin and ramoplanin occur at
residue 9 (Phe in ramoplanin and Cit in enduracidin), at
residue Hpg11(which contains a dimannosyl moiety in
ramoplanin but is unmodified in enduracidin), at residue 13
(Dpg in ramoplanin and Hpg in enduracidin) and at residues
10 and 15 (which are ornithine and leucine in ramoplanin but
are both enduracididines in enduracidin).
A comparison of the solution structures of ramoplanin and
enduracidin by NMR shows that they have quite similar
backbone structures, as expected given the identical number
and chirality of the residues in the macrocycles.24,25The
backbone structures feature two antiparallel b-sheets formed
by residues 2–7 and 10–14, respectively.24,25The side-chains of
residues 3, 9 and 17, although located far apart in primary
sequence, show strong NOEs, indicating that they are in close
proximity and revealing a cup-shaped curvature in the
molecule.24,25Although minor structural differences may exist
between ramoplanin and enduracidin,24,25they are sufficiently
similar that one might reasonably attribute any differences in
behavior to specific differences in individual side chain
The curves for inhibition of MurG and PBP1b by
enduracidin are shown in Fig. 4C and 4D. The curve for
inhibition of PBP1b is sigmoidal and inhibition is overcome at
high concentrations of Lipid II, indicating that enduracidin
inhibits this enzyme by binding to Lipid II (Fig. 4D).
Furthermore, the reaction rate was negligible until the
substrate concentration exceeded half the total enduracidin
concentration, implying a 2 : 1 enduracidin : Lipid II binding.
The kinetics shows that enduracidin is virtually identical to
ramoplanin with respect to the mode of inhibition of this
bacterial transglycosylase. However, the manner in which
enduracidin inhibits MurG is different from ramoplanin.
Whereas ramoplanin displays noncompetitive inhibition,
enduracidin produces a sigmoidal inhibition curve that is
consistent with substrate binding (Fig. 4A and 4C).
Based on the differences in the curves for the inhibition of
MurG, we surmised that ramoplanin must contain some
features, which enduracidin lacks, that explain its ability to
interact directly with MurG. Although ramoplanin and
enduracidin differ by several residues, the single largest
structural difference between these two molecules is the
presence of a dimannosyl moiety on residue Hpg11of
ramoplanin. This dimannosyl group on ramoplanin is not
required for biological activity.26–28To investigate whether it
plays a role in MurG inhibition, we removed this glycosyl
group by treatment with anhydrous HF to obtain the
aglycon.13The aglycon was tested for inhibition of both
MurG and PBP1b, and the results are described in the
Inhibition kinetics of ramoplanin aglycon with respect to MurG
Kinetic curves for MurG and PBP1b in the presence of the
ramoplanin aglycon are shown in Fig. 4E and 4F. The curve
for inhibition of PBP1b is sigmoidal, as it is for ramoplanin
itself. The curve for MurG inhibition is also sigmoidal, in
contrast to the behavior of ramoplanin. Therefore, the
ramoplanin aglycon, like enduracidin, inhibits E. coli MurG
by binding to Lipid I. We have concluded that the dimannosyl
unit on ramoplanin entirely accounts for the noncompetitive
inhibition pattern observed for the parent compound. This
This journal is ? The Royal Society of Chemistry 2006 Mol. BioSyst., 2006, 2, 69–76 | 73
dimannosyl unit evidently promotes an interaction with E. coli
MurG that is independent of Lipid I binding and that obscures
the effects of binding to Lipid I.17It remains to be established
whether MurG homologues from other organisms also interact
with ramoplanin directly, or whether this phenomenon is
specific to the E. coli enzyme. However, even if ramoplanin
directly interacts with other MurG homologues via the
dimannosyl unit, the interaction is not likely to be biologically
relevant because our studies with E. coli MurG suggest that it
is not of sufficiently high affinity to compete with the much
tighter binding observed to Lipid II.17
Quantification of Lipid I and Lipid II binding using inhibition
Inhibition kinetics reveals a considerable amount about the
interactions between a substrate-binding antibiotic and the
target substrate. For example, the kinetics of transglycosylase
inhibition has revealed that ramoplanin, the ramoplanin
aglycon, and enduracidin all bind to Lipid II with a
stoichiometry of 2 : 1. Furthermore, the observation that
there is virtually no reaction at low substrate concentrations in
the presence of low micromolar concentrations of antibiotic
implies that all of the substrate is sequestered at these
concentrations, which indicates in turn that the Kd’s for
binding are in the submicromolar range for all three
compounds. By fitting the inhibition curves to an equation
for inhibition via substrate sequestration, it is possible to
estimate Kd’s for Lipid II binding of approximately 10 nM for
the three compounds.
Although we could estimate the Kdfor Lipid II binding to
ramoplanin via analysis of the curves for transglycosylase
inhibition, we could not estimate the Kdfor ramoplanin and
Lipid I from the curves for MurG inhibition because the
inhibition pattern was not consistent with substrate binding.
However, we found that the ramoplanin aglycon inhibits MurG
bybinding to Lipid I. Because the dimannosyl unit does not play
any role in the binding of ramoplanin to Lipid II (as determined
by a comparison of ramoplanin and the ramoplanin aglycon
inhibition patterns for PBP1b), it seems safe to assume that it
does not play a role in binding to Lipid I. Therefore, we thought
it would be reasonable to use the kinetic data obtained for
inhibition of MurG by the ramoplanin aglycon to estimate the
Kdfor ramoplanin itself. Visual inspection of the kinetic curves
for MurG and PBP1b inhibition by the ramoplanin aglycon
reveals weaker binding to Lipid I, and fitting these data to the
same equation used for PBP1b inhibition yields an estimated Kd
for Lipid I binding that is approximately five-fold higher than
the Kdfor Lipid II binding (see Experimental). Nevertheless,
because the kinetic experiments were done at high concentra-
tions relative to the estimated binding constants, and thus are
not as accurate as one might wish, we sought an alternative
method that allows the direct measurement of binding constants
at lower concentrations.
Quantification of Lipid I and Lipid II binding using fluorescent
Measuring the binding of ramoplanin to Lipid I and Lipid II
analogues using standard biophysical methods has proven to
be a considerable challenge because ramoplanin : ligand
complexes self-associate to form fibrils.8,29Thus, prior to this
work, the only quantitative data on binding reported for
ramoplanin came from the laboratory of McCafferty and
coworkers, who studied the binding of ramoplanin to
analogues of Lipid I using NMR.29In order to prevent
aggregation of the ramoplanin complexes, these authors
performed their experiments in DMSO–water mixtures.30
They reported a Kdof 180 ¡ 20 mM for UDP–MurNAc–
pentapeptide (Fig. 1) and estimated the Kdfor ramoplanin and
Lipid I to be less than 200 mM.30Because the inhibition
kinetics for MurG and transglycosylase shows that the Kd’s of
ramoplanin for both Lipid I and Lipid II are at least two
orders of magnitude lower than previously reported estimates
(i.e. , 1 mM), we sought a more appropriate method than
NMR to measure the dissociation constants. Fluorescence-
based methods are suitable for measuring binding constants in
the nanomolar range. Because studies in our laboratory have
suggested that the peptide chain on Lipid I plays a negligible
role in binding to ramoplanin,31we prepared an analogue of
Lipid I containing a fluorescent label on the e-amino group of
the lysine in the pentapeptide chain (Fig. 3, 4). To evaluate
the utilityof this compound
constants, we added increasing amounts of ramoplanin to a
100 nM solution of 4 and monitored the fluorescent emission
of the sample at 525 nm following excitation at 492 nm. A
decrease in fluorescence intensity was observed as ramoplanin
was added. The change in fluorescence was observed to
plateau by the time 2 mM of ramoplanin was in solution. Based
on two sets of data, the dissociation constant for binding of the
ramoplanin dimer to Lipid I was found to be 170 ¡ 30 nM
We attempted to prepare the corresponding fluorescently
labeled Lipid II analogue using MurG to form the b-(1,4)
linkage to GlcNAc. Although we have previously shown that
Lipid I analogues containing substituents on the lysine side
chain are substrates for MurG,14this particular compound did
not turn over efficiently. However, we were able to prepare a
Lipid II analogue (Fig. 3, 6) with a fluorophore on the C2-
N-acyl position of the GlcNAc sugar using the corresponding
fluorescently labeled UDP-GlcNAc donor 5. When ramopla-
nin was added to a 100 nM solution of compound 6, a
dramatic decrease in fluorescence was observed, plateauing
around 400 nM ramoplanin. A dissociation constant of 3 ¡
2 nM for binding of the ramoplanin dimer to Lipid II was
established from two sets of data (Fig. 5B).
Since the fluorophore on Lipid I analogue 4 and Lipid II
analogues 6 was installed at different sites, we wanted to make
certain that the preferential binding of ramoplanin to Lipid II
was not related to the position of the fluorophore. Therefore,
we sought to compare the concentrations of unlabeled Lipid I
and Lipid II analogues required to displace the fluorescent
probe 4 from ramoplanin. The displacement experiments were
complicated by the fact that ramoplanin complexes self-
associate; nevertheless, 1025M concentrations of Lipid II
displaced compound 4 fully; at the same concentrations of
Lipid I, only 10% of 4 was displaced. The titration of Lipid I
was not carried to completion due to compound limitations.
The results, however, were consistent with all previous data
74 | Mol. BioSyst., 2006, 2, 69–76This journal is ? The Royal Society of Chemistry 2006
indicating that Lipid II binds better to ramoplanin by a
difference of one magnitude.
The mechanism of action of ramoplanin has been the subject
of extensive examination over the past fifteen years, with two
possible cellular targets identified for the antibiotic.3,7–9The
first proposed target to be identified was MurG, the GlcNAc
transferase that converts Lipid I to Lipid II on the pathway to
peptidoglycan.7The experiments pointing to MurG as a target
were carefully performed but were not conclusive because they
were conducted at a time when there were limited assays to
identify the step at which peptidoglycan synthesis inhibitors
take effect.7Particulate membranes containing all of the
enzymes involved in the membrane-linked steps of peptidogly-
can biosynthesis were used, and the assays involved following
the conversion of UDP–MurNAc–pentapeptide first to Lipid I
and then to Lipid II and peptidoglycan polymer. Reynolds and
coworkers showed that ramoplanin prevents the formation of
Lipid II but were unable to investigate whether ramoplanin
also inhibits subsequent steps.7Ten years after Reynolds
and coworkers proposed the MurG step as the target of
ramoplanin, we adapted the particulate membrane assay to
report on the transglycosylation step of peptidoglycan
synthesis.8We showed that ramoplanin also blocks this step.8
The identification of two possible cellular targets for
ramoplanin raised the question of whether one target is
more responsible for the antibiotic effects than the other. We
argued that the primary mechanism of action of ramoplanin
most likely involves binding to Lipid II/inhibition of transgly-
cosylation rather than binding to Lipid I/inhibition of MurG
because Lipid I is intracellular while ramoplanin does not
have the physical properties characteristic of molecules
that penetrate cell membranes (MW = 2554, aqueous
solubility >100 mg mL21).10
While these arguments are reasonable, we had no direct,
quantitative evidence that ramoplanin binds preferentially to
Lipid II over Lipid I, or that it inhibits transglycosylases
more strongly than it inhibits MurG. Furthermore, as we
and others have discovered, the structural and energetic
aspects of ramoplanin–substrate complexation have proven
remarkably difficult to dissect.8,29,30As soon as Lipid I and
Lipid II are added to ramoplanin, the complexes self-
assemble to form fibrils.8,29,30This self-assembly process
precludes efforts to characterize and quantify the binding
interactions by most techniques. McCafferty and coworkers
have been able, by using organic solvent–water mixtures
and weakly binding ligands, to obtain some NMR data on 1 : 1
complexes of ramoplanin with substrate analogues,29but
it is not clear that these data provide insight into the 2 : 1
complexes that are involved in enzyme inhibition.
The inhibition kinetics described here for the ramoplanin
aglycon and enduracidin show that both of these molecules
bind with submicromolar affinity to Lipid I, more than 100-
fold below the upper limit for the Kdestablished by McCafferty
and coworkers using NMR. Therefore, we used fluorescent
derivatives of Lipid I and Lipid II to evaluate binding to
ramoplanin. Our studies show that there is a difference of
about one order of magnitude in the Kds of ramoplanin
for Lipid I and Lipid II, with Lipid II favored. The Kds
estimated from the inhibition kinetics are consistent with
aglycon binds Lipid II preferentially over Lipid I by a factor
of approximately five. Preferential binding to Lipid II,
combined with its greater accessibility on the outside
surface of the bacterial cell membrane, strongly supports the
hypothesis that the primary means by which ramoplanin
kills bacterial cells involves binding to Lipid II and
inhibiting transglycosylases. Because the inhibition kinetics
shows that enduracidin binds better to Lipid II than to
Lipid I, we propose that the primary mechanism of action of
this molecule is the same as that of ramoplanin: it inhibits
bacterial transglycosylases by binding to Lipid II. It is
presumed that both ramoplanin and enduracidin also bind to
the reducing end of the growing glycan chain, which
contains the same disaccharide diphosphate moiety as
Lipid II (see Fig. 1).
It is worth noting that the many residue differences between
ramoplanin and enduracidin have no obvious effect on Lipid
II binding. It has been suggested that the residues in these
molecules that are most critical for interaction with peptido-
glycan intermediates are located at positions 3–8, which
are identical in ramoplanin and enduracidin.29Therefore, it
has been suggested that residues 3–8 comprise the ‘‘minimal
concentration. (B) Fluorescence intensity of compound 6 as a function of ramoplanin concentration.
Fluorescent binding assays of ramoplanin with 4 and 6. (A) Fluorescence intensity of compound 4 as a function of ramoplanin
This journal is ? The Royal Society of Chemistry 2006Mol. BioSyst., 2006, 2, 69–76 | 75
other than the similarities in residues 3–8 may explain the
observation that ramoplanin and enduracidin behave similarly
with respect to ligand binding. For example, conformationally
defined backbone interactions requiring the full structure may
play the primary role in the recognition of Lipid I and Lipid
II.32Breukink and coworkers have reported that the lantibiotic
nisin, which also binds to Lipid II, uses primarily backbone
contacts in complexation, and they have identified a ‘‘pyro-
phosphate cage’’ as a key binding element.33Structural
studies of enduracidin and ramoplanin show that the back-
bones of these two molecules are very similar.24,25The
backbone conformations are determined by the number and
chirality of the amino acids in the macrocycles rather than by
the side chain identities, and it is quite possible that most of
the side chains are individually unimportant for binding.
Alanine scanning experiments on the ramoplanin structure
should shed more light on the importance of each particular
amino acid side chain in substrate binding and biological
activity, and changes in chirality can provide insight into the
importance of the backbone conformation in binding.
Having established a convergent synthetic route to the
ramoplanin aglycon analogues that enables such a detailed
examination of the importance of each residue,28,32,34–36and
with suitable assays in place to probe Lipid II vs. Lipid I
binding, transglycosylase vs. MurG inhibition, and biological
activity, we should be able to determine the essential
requirements for binding and biological activity, and we may
be able to identify analogues with more desirable properties to
use as antibiotics.
We compared the inhibitory behavior of ramoplanin and
two structurally related compounds, enduracidin and the
ramoplanin aglycon, with respect to two enzymes involved in
the late steps of peptidoglycan biosynthesis. We have shown
that the ramoplanin aglycon and enduracidin inhibit these two
enzymes, MurG and the bacterial transglycosylase PBP1b, by
binding to their substrates, Lipid I and Lipid II, respectively.
The inhibition kinetics shows that both compounds bind more
tightly to Lipid II than to Lipid I. We measured the Kds of
ramoplanin for fluorescent analogues of Lipid I and Lipid II
and found that Lipid II binds about ten-fold more tightly than
Lipid I. Based on our studies, we conclude that the primary
mechanism of action of both ramoplanin and enduracidin
involves inhibition of the transglycosylation step of peptido-
This work is supported by NIH grants AI50855 (to SW) and
CA41101 (to DLB). We thank Oscient Pharmaceuticals for a
generous gift of ramoplanin.
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