(Received for publication, July 5, 1995; and in revised form, January 17, 1996)
From the
Enzymes of the membrane cycle of reactions in bacterial
peptidoglycan biosynthesis remain as unexploited potential targets for
antibacterial agents. The first of these enzymes,
phospho-N-acetylmuramyl-pentapeptide-translocase (EC
2.7.8.13), has been overexpressed in Escherichia coli and
solubilized from particulate fractions. The work of W. A. Weppner and
F. C. Neuhaus ((1977) J. Biol. Chem. 252, 2296-2303) has
been extended to establish a usable routine fluorescence-based
continuous assay for solubilized preparations. This assay has been used
in the characterization of the natural product, mureidomycin A as a
potent slow binding inhibitor of the enzyme with K and K
* of 36 nM and 2
nM, respectively.
Enzymes responsible for the biosynthesis of the peptidoglycan
component of the bacterial cell wall are well precedented targets for
antibiotics(1) . The worldwide emergence of bacterial strains
resistant to current antibiotics necessitates the development of new
antimicrobial agents(2, 3) .
Phospho-N-acetylmuramyl-pentapeptide-translocase (also
translocase I) catalyzes the first step in the membrane cycle of
peptidoglycan biosynthesis, namely the transfer of
phospho-N-acetylmuramyl-L-Ala--D-Glu-m-DAP(
)-D-Ala-D-Ala
from uridine 5`-monophosphate (UMP) to a membrane-bound lipid carrier,
undecaprenyl phosphate (see Fig. 1)(4, 5) .
This enzyme is encoded by the mraY gene in Escherichia
coli. This gene has been cloned and sequenced; examination of the
inferred amino acid sequence indicates that the encoded enzyme is an
integral membrane protein whose molecular mass is 39.5 kDa(6) .
The lipid-linked product of MraY is further elaborated by attachment of
an N-acetylglucosamine unit and the precursor is somehow
flipped across the membrane and incorporated into peptidoglycan. This
and other such lipid-linked cycles have been reviewed(7) . No
commercial antibiotics in current use are directed against translocase
I. This enzyme represents a target for novel antibacterial agents which
is as yet unexploited.
Figure 1: Transfer of phospho-N-acetylmuramyl-pentapeptide to undecaprenyl phosphate catalyzed by translocase I.
Until recently, the only known inhibitors of
this step of peptidoglycan biosynthesis were tunicamycin, which is
known also to inhibit mammalian glycoprotein biosynthesis and other
lipid-linked glycosyl transfer reactions(8) , and amphomycin,
which chelates undecaprenyl-P in the presence of
Ca(9, 10) . In recent years two new
classes of natural products have been characterized as potent and
specific inhibitors of this step in peptidoglycan biosynthesis, the
mureidomycins and the
liposidomycins(11, 12, 13, 14) .
Both classes of compound share a uridine nucleoside moiety found in the
substrate UDPMurNAc-pentapeptide, but beyond this there is little
obvious similarity to the substrates of translocase I (see Fig. 2). The presence of the common uridine moiety suggests a
similar mode of action for this class of molecules.
Figure 2: Chemical structures of translocase I substrates and inhibitors.
The
mureidomycins are a class of novel peptidylnucleoside antibiotics
isolated from Streptomyces flavidovirens SANK 60486 which show
selective antipseudomonal activity (minimal inhibitory concentration
values of 0.1-3.13 µg/ml), while not being toxic in
mice(15) . Mureidomycin A has been demonstrated to inhibit
translocase I activity in particulate preparations from Pseudomonas
aeruginosa using a radiochemical assay (IC, 0.05
µg/ml), but not to significantly inhibit formation of lipid-linked N-acetylglucosamine for teichoic acid synthesis in Bacillus subtilis (IC
>100 µg/ml) or
dolichol-linked precursors for glycoprotein biosynthesis in a mammalian
system(11) . The antibacterial potency of mureidomycin A and
its novel structure prompted us to begin an investigation into the
molecular mechanism of action of this antibiotic. Here we report the
characterization of mureidomycin A as a slow-binding inhibitor of
solubilized translocase I from Escherichia coli using a
convenient fluorescence enhancement continuous assay.
Under optimized conditions, preparations of JM109 (pBROC525) solubilized with 1% Triton X-100 at 4 mg of protein/ml gave specific activities of 1-2 nmol/min/mg. Attempts to purify the solubilized enzyme by a wide range of conventional or affinity chromatographic methods gave >95% loss of enzyme activity. No loss of activity was observed upon gel filtration on Sephadex G-75, suggesting that a ``factor'' is being lost during adsorptive chromatography. Experiments are in progress to identify this factor.
In this assay system dansylated substrate is
converted into lipid-linked product which resides within detergent
micelles rather than the bulk aqueous compartment, resulting in a
measurable change in fluorescence emission. Conversion of dansylated
substrate to lipid-linked product resulted in a blue shift of the
fluorescence emission maximum (exciting at 340 nm) from 565 nm to 535
nm, and a fluorescence enhancement at equilibrium of approximately
two-fold. In our hands this assay method is of similar sensitivity to
the previous radiochemical assay since the amount of enzyme required to
generate 20 pmol of C-lipid-linked butanol-extractable
product in a 3-min assay gave a change in fluorescence of 3.24 units in
a 3-min assay.
Fluorescence enhancement was found to be directly proportional to protein concentration, and linear at low conversions for up to 30 min (data not shown). At higher enzyme concentrations nonlinearity was observed upon approach to chemical equilibrium. Activity is expressed as increase in units of fluorescence emission per minute at 30 °C. Since the quantum yield of the fluorophore under the conditions employed is not known, this unit is arbitrary.
Using the fluorescence enhancement
assay the K for dansyl-UDP-MurNAc-pentapeptide
(dPP) has been measured at 19 ± 3 µM and the
apparent K
for dodecaprenyl-P was 13 ± 3
µM with a V
of 4.7 units/min (see Fig. 3). For heptaprenyl-P the apparent K
was measured at 19 ± 5 µM (data not shown)
with a corresponding V
of 1.7 units/min.
The relative K
and V
values indicate that dodecaprenyl-P is a more efficient substrate
for translocase I than heptaprenyl-P, indicating that the enzyme is
selective for the larger substrate which is closer in chain length to
the natural substrate, undecaprenyl-P. Consequently, dodecaprenyl-P was
used in subsequent kinetic studies.
Figure 3:
Lineweaver-Burk determinations of K values. Panel A, K
determination for dPP. Panel
B, K
determination for dodecaprenyl
phosphate (DP) (see footnote 2). Initial rate measurements
were made using the fluorescence enhancement assay as described in the
experimental section, first varying DP concentration with dPP present
at 105 µM, then varying dPP concentration with DP present
at 40 µM. Phosphatidylglycerol was constant at 100
µg/ml. K
values were determined from
the slope using the reciprocal forms of the initial forward rate
equation for a Ping Pong Bi Bi
system(32) .
Dependence of activity in the
fluorescence enhancement assay upon magnesium ions and detergent
concentration was examined (see Fig. 4). The results reveal a
requirement for >10 mM Mg, with maximal
activity at 40 mM Mg
. Activity was
stimulated 2-fold by the inclusion of 50 mM KCl in the assay.
This is consistent with the results obtained with the S. aureus enzyme(25) . Assays with other divalent metal ions
revealed that Mg
could be replaced only by
Mn
. The optimal concentration for Mn
was 1 mM, but activity was 2.5-fold less than with the
optimal concentration of Mg
. No activity was observed
using Ni
, Ca
and
Zn
. Optimal detergent concentration in the assay was
found at 0.25% Triton X-100.
Figure 4:
Effect of Triton X-100 (panel A)
and MgCl (panel B) in the fluorescence enhancement
assay. Assays were carried out as under ``Experimental
Procedures'' except that the concentration of of MgCl
or Triton X-100 was varied
accordingly.
Activity was stimulated 5-10-fold by inclusion of 100 µg/ml phosphatidyl-glycerol in both radiochemical and fluorescence enhancement assays, but not by phosphatidylethanolamine at the same concentration. Specific activation by phosphatidylglycerol is precedented by the case of UDP-GlcNAc:dolichyl phosphate GlcNAc-1-phosphate transferase from pig aorta, a eukaryotic enzyme which catalyzes a similar phosphosugar transfer reaction(26) . Activation by phosphatidylglycerol and other phospholipids has also previously been observed with a gel-filtered preparation of the S. aureus translocase I activity in 1% Triton X-100, using a radiochemical exchange assay(27) . It is not possible to say whether this activation is due to maintenance of the structural integrity of the protein in detergent solution or perhaps more efficient solubilization of the lipid substrate.
Figure 5:
Inhibition of translocase I by
mureidomycin A. Progress curves in the presence of increasing
concentrations of mureidomycin A. Units of fluorescence are arbitrary.
Initial competitive inhibition is followed by time-dependent onset of
slow binding inhibition characterized by a steady-state final rate.
Nonlinearity in the absence of inhibitor is due to product inhibition
and approach to equilibrium. Diluted enzyme remains linear over an
increase of 2.5 fluorescence units.
The observed time-dependent inhibition is consistent either with irreversible or slow binding inhibition. In order to distinguish between irreversible and slow binding inhibition, assays containing 100 nM mureidomycin A were allowed to proceed for varying lengths of time, and intrinsic enzyme activity measured by addition of 0.4 mM UMP and subsequent decrease in fluorescence intensity due to the reverse reaction. The observed rate of reverse reaction upon addition of UMP was independent of incubation time over 5-50 min, indicating that no irreversible enzyme inactivation is taking place. No loss of potency of the inhibitor was observed upon prolonged storage in aqueous medium, ruling out the possibility that the inhibitor is being degraded during the time course of the assay.
In the absence of
irreversible inhibition, the biphasic time course observed can be
explained only by slow binding inhibition. Preincubation experiments in
the presence or absence of substrates showed that inhibition is not
substrate-dependent, indicating that mureidomycin A interacts with the
free enzyme. K and V
parameters were measured for fluorescent substrate and
dodecaprenyl-P in the presence 0, 200, 300, and 400 nM mureidomycin A.
K
values
increased with inhibitor concentration whilst V
values were unchanged showing that mureidomycin A is competitive
with respect to both substrates (data not shown). In order to calculate
the requisite kinetic constants we have assumed a Ping Pong Bi Bi
mechanism for the enzyme catalyzed conversion of substrates to
products, after the work of Heydanek and et al.(24) .
We have also assumed, based on the above evidence, that mureidomycin A
behaves as a classical dead-end inhibitor (see Fig. 6).
Figure 6: A kinetic model for the inhibition of translocase I by mureidomycin A. A, dansyl-UDP-MurNAc-pentapeptide; B, dodecaprenyl phosphate, P, UMP; and Q, dodecaprenyl-pyrophosphoryl-MurNAc-pentapeptide; E and F are free enzyme and a putative covalent intermediate, respectively(24) . EI* is the tightly associated enzyme-inhibitor complex.
K and K
*, equilibrium
constants for simple competitive and slow-binding inhibition, were
calculated using , where v is the initial velocity
or steady state final velocity for K
or K
* respectively. A, B, and I are the concentrations of dansyl-UDP-MurNAc-pentapeptide,
dodecaprenyl-P, and mureidomycin A, respectively.
Plots of 1/v versus I yielded values of 36 ± 6
nM and 2.0 ± 0.6 nM for K and K
*, respectively (see Fig. 7). In
order to measure K
* and subsequently k
values over a wide range of inhibitor
concentrations, it was necessary to record time courses of inhibition
at three different enzyme concentrations, since the inherent
nonlinearity of the assay time course imposes a time limit for the
observation of the onset of inhibition at any given enzyme
concentration. Diluted enzyme remains linear over an increase of
2.5 fluorescence units. More experimental error is observed in the K
* data compared to the K
data due to the fact that K
* is calculated
from the much lower steady state final rates, rather than initial rates
which can be measured with a high degree of confidence. However, the
observed data is consistent with the slow binding inhibition model, and
a value of 2.0 ± 0.6 nM can be deduced for K
*.
Figure 7:
Determination of slow-binding inhibition
kinetic constants. Panel A, determination of K from initial rates in the presence of
varying [I]. K
was determined
from the slope as described in . Panel B,
determination of K
* from final steady
state rates in the presence of varying [I]. K
* was determined from the slope as
described in . Results comprise three sets of data, each at
different enzyme concentrations. Consequently, rates are expressed as a
percentage of that measured in the absence of inhibitor. Assay mixtures
contained 105 µM dansyl-UDPMurNAc-pentapeptide, 40
µM dodecaprenyl phosphate, and 100 µg/ml
phosphatidylglycerol.
Values of k, the
observed rate constant for the approach to the steady-state final rate,
were determined at each inhibitor concentration using (29) , where v is the velocity at time t, and v
and v
are
the intial and steady-state
velocities.
The rate constant for the isomerization of EI to EI*, k was determined by plotting k
against a function of inhibitor concentration, I, as in (Fig. 8) (29) .
Figure 8:
Determination of the rate constant, k, for the isomerization of E
I to E
I*. Values of k
were measured
over a range of inhibitor concentrations, and calculated using . k
was determined from the slope of
the plot of k
versus f(I)
= (I/K
)/(1 + (A/K
(A)) + (I/K
)) as described in .
From the gradient of this plot, the value obtained for k was 0.92 ± 0.2 min
,
whose magnitude is consistent with the time course of the original
progress curves.
Attempts were made to measure the rate constant for
the dissociation of the tightly bound EI* complex, k
, experimentally by rapid separation of enzyme
from inhibitor by the method of Penefsky(30) . An aliquot of
enzyme was inactivated with 0.4 µM mureidomycin A under
the assay conditions described under ``Experimental
Procedures.'' The desalted enzyme was completely inactive, but no
time-dependent regain of activity was observed. It is likely that the
mureidomycin associates with the detergent micelles and so is not
effectively separated from the enzyme.
In order to investigate in detail the molecular basis for inhibition of translocase I by mureidomycin A we have overexpressed and solubilized the E. coli enzyme activity, and have developed a reproducible continuous assay for this enzyme. It is not known why the level of overexpression (28-fold) is so modest given the well documented strength of the trc promoter.
Mureidomycin A was
found to inhibit the solubilized activity with IC <100
nM using a stopped radiochemical assay. Using the continuous
fluorescence enhancement assay we have identified mureidomycin A as a
slow binding enzyme inhibitor. This provides the first detailed insight
into the molecular mechanism of action of this antibiotic. It joins a
select group of slow binding enzyme inhibitors which include other
examples in peptidoglycan biosynthesis such as the inhibition of D-alanine:D-alanine ligase by aminoalkylphosphinate
transition state analogues(31) . This mode of enzyme inhibition
results from the reversible conversion of the initial E
I complex into a more tightly binding E
I* complex. In
some cases formation of the E
I* complex is due to a
conformational change of the enzyme or inhibitor; in other cases it is
due to a reversible chemical reaction taking place at the enzyme active
site(29) . The observation that mureidomycin A is competitive
with respect to both dansyl-UDP-MurNAc-pentapeptide and dodecaprenyl-P
implies that it acts as a bifunctional inhibitor in the formation of
the E
I complex. However, the nature of the transition to
the E
I* complex remains to be determined. Studies are in
progress to determine the catalytic mechanism of translocase I and the
role of catalytic active site residues which may also participate in
the mechanism of slow binding inhibition by mureidomycin A.
The K value of 36 nM determined for
mureidomycin A is a remarkable 500-fold lower than the K
of the substrate analogue dansyl-UDP-MurNAc-pentapeptide, and the K
* value a further 20-fold lower. This binding
affinity offers an explanation for its antibacterial potency in
vivo, and coupled with an apparent lack of toxicity in mammalian
systems means that the elucidation of the mechanism of slow binding
inhibition by mureidomycin A could be a basis for the rational design
of novel antibacterial agents.