From the
The mechanism of inhibition of DNA gyrase by cyclothialidine, a
novel gyrase inhibitor isolated from Streptomyces filipinensis NR0484, has been studied further by using
[
DNA gyrase is a type II DNA topoisomerase that catalyzes the
negative supercoiling of DNA in prokaryotes, and its function is
essential for cell growth. The enzyme is implicated in the process of
DNA replication, transcription, and recombination and in a number of
other cellular processes(1, 2, 3) . DNA gyrase
from Escherichia coli consists of two subunits, A and B, with
molecular masses of 97,000 and 90,000 Da, respectively. The active
enzyme is an A
From our
screening of natural products for DNA gyrase inhibitors, we isolated a
novel gyrase inhibitor, cyclothialidine, from Streptomyces
filipinensis NR0484(20) . Cyclothialidine contains a unique
12-membered lactone ring that is partly integrated into a pentapeptide
chain(21) . Cyclothialidine exhibited the highest inhibitory
activity against DNA gyrases from several bacterial species, including Escherichia coli and Staphylococcus aureus, with high
selectivity in its biological activity(22) .
Our previous
studies (23) indicated that cyclothialidine inhibits, under
steady-state conditions, the ATPase activity of E. coli DNA
gyrase competitively with a K
[
The characterization of the mode of inhibition of DNA gyrase
by cyclothialidine promises to yield important information on the
mechanism of DNA supercoiling and especially on the role of the ATPase
activity of DNA gyrase during this process, since we have shown that
cyclothialidine inhibits the ATPase activity of the B subunit. One
possible means of characterizing the mode of inhibition would be to
study the binding of [
Our data presented here (Fig. 2) show that
[
Furthermore, our observations agreed
well with the ``model of ATP hydrolysis by DNA gyrase'' as
described by Maxell and Gellert (29) in which they say that ATP
hydrolysis requires the binding of DNA to two sites on the enzyme and
that when both DNA binding sites on the enzyme are occupied by DNA the
enzyme is proposed to undergo a conformational change whereby it
becomes an active ATPase. It is possible that the conformation of the
ATP binding site is changed more dramatically than those for
cyclothialidine and for the coumarin antibiotics (Fig. 4).
The
mechanism of inhibition of DNA gyrase by cyclothialidine may be almost
the same as those by coumarin antibiotics(23) . However, two
important differences were observed between cyclothialidine and
coumarin antibiotics. 1) Cyclothialidine is much more selective toward
DNA gyrase than are coumarin antibiotics(22) . 2)
Cyclothialidine is active against a DNA gyrase isolated from a
novobiocin-resistant E. coli gyrB mutant strain(23) .
Furthermore, our data presented in Fig. 4and showed
that the profile of displacement curves of
[
Wigley et al. reported on the crystallization of the 43-kDa
N-terminal fragment of the E. coli DNA gyrase B subunit, which
comprises positions 2-393 of the intact protein in the presence
of ADPNP(10) . They have described the 43-kDa N-terminal
fragment containing two distinct subdomains: an N-terminal subdomain
(residues 2-220) containing the bound ADPNP and a C-terminal
subdomain (residues 221-393), forming the sides of a proposed
DNA-binding site. Ali et al.(8) have reported on
steady-state ATPase experiments by using the 43-kDa N-terminal fragment
of the B subunit; their results are consistent with the hypothesis of a
noncompetitive mechanism for the inhibition of the ATPase activity of
the B subunit by the coumarin antibiotics novobiocin and coumermycin
A1. This indicates that coumarin antibiotics bind close to the ATP
binding site of the protein with low affinity for ATP. We also suggest
that, similar to coumarin antibiotics, cyclothialidine binds close to
the ATP-binding site of the gyrase B subunit and stabilizes a
conformation of the protein that is unable to bind ATP. However, our
results further suggest that cyclothialidine recognizes a site that is
also different from that recognized by the coumarin antibiotics.
To
sum up, two models with the currently available data are proposed as
follows: 1) ATP, cyclothialidine, and coumarins bind to the same site,
but the precise interactions that are important for ATP binding are
different from those that are involved in cyclothialidine or coumarin
binding. 2) The binding sites for ATP, cyclothialidine, and coumarins
are completely separate but interactive and, to some extent, exclusive.
Thus occupancy of the cyclothialidine site would prevent binding of ATP
(or coumarins) and vice versa. The connectivity between the
sites is dependent on the tertiary structure of the protein, and one
could easily imagine that it would be influenced by the oligomeric
state of the protein. The three-dimensional structure of the 43-kDa
N-terminal fragment of the B subunit together with cyclothialidine
should provide precise clues about the binding site of cyclothialidine
and should determine whether cyclothialidine recognizes the same amino
acids as do ATP or the coumarin antibiotics.
Gilbert et al. have reported that the production and properties of a 24-kDa
N-terminal fragment of the B subunit (residues 2-220) is shown to
contain the coumarin antibiotics-binding site(30) . Lewis et
al.(31) have recently reported on their succeeding in the
co-crystallization of the 24-kDa N-terminal fragment and novobiocin and
of the 24-kDa fragment and GR122222X, an inhibitor that is structurally
related to cyclothialidine. Our results are consistent with those
findings, meaning that the binding sites of cyclothialidine and
novobiocin probably lie within this 24-kDa N-terminal fragment of the B
subunit.
However, our results did not allow us to determine whether
conformational changes of the binding site of cyclothialidine on the B
subunit or in its vicinity occur before cyclothialidine binding or
after. By using the electric dichroism method, as previously reported
by Rau et al.(32) , we may obtain the necessary
information for determining the time point of the structural changes.
We thank Dr. Malcolm Page for critical reading of the
manuscript, Dr. Martin Gellert for providing the E. coli strains N4186 and MK47, and Dr. F. Hermann for carrying out the
purification of the 43-kDa N-terminal fragment.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
C]benzoylcyclothialidine and a reconstituted Escherichia coli gyrase system consisting of the A subunit,
the B subunit and relaxed ColE1 DNA. The mechanism of inhibition was
also studied with the 43-kDa N-terminal fragment of the B subunit. The
[
C]benzoylcyclothialidine could bind to the B
subunit alone but not to the A subunit nor to the plasmid DNA alone.
Furthermore, the compound also bound to the 43-kDa N-terminal fragment
of the B subunit. Scatchard analysis of
[
C]benzoylcyclothialidine binding to DNA gyrase
showed that the binding affinity of the compound increased, depending
on the assembly of the gyrase (A
B
)
DNA
complex. This suggests that the binding site of cyclothialidine on the
B subunit or its vicinity causes a conformational change during the
assembly of the gyrase
DNA complex (increase in affinity: B
A
B
A
B
DNA).
Furthermore, displacement curves of
[
C]benzoylcyclothialidine binding by nonlabeled
cyclothialidine, ATP analogues, and coumarin antibiotics indicated that
cyclothialidine, coumarins, and ATP share a common (or overlapping)
site of action on the B subunit of DNA gyrase; however, the
microenvironment of the binding sites may differ.
B
tetramer complex. The mechanism
of DNA supercoiling by gyrase involves the wrapping of a segment of DNA
around a protein core, cleavage of this DNA, and passage of another
piece of DNA through the double-stranded break, which is then
rejoined(4) . The reaction cycle normally requires the
hydrolysis of ATP. However, replacement of ATP by a nonhydrolyzable ATP
analogue, ADPNP,
(
)results in limited
supercoiling by gyrase, suggesting that ATP binding can promote a
single round of supercoiling, but that the hydrolysis step is required
to regenerate the enzyme in its active form(5) . The A subunit
contains the site of the DNA breakage and rejoining of the DNA
supercoiling, while the B subunit contains the site of ATP hydrolysis.
Both subunits are thought to consist of two distinct functional
domains. A subunit contains an N-terminal domain (58-64 kDa),
thought to be involved in the DNA breakage-rejoining reactions, and a
C-terminal domain involved in the DNA-subunit
interactions(6, 7) . The B subunit contains a 43-kDa
N-terminal domain, which involves the site of ATP
hydrolysis(8) , and a 47-kDa C-terminal domain, which interacts
with the A subunit and DNA(9) . The crystal structure of this
43-kDa N-terminal fragment protein complexed with ADPNP has been
reported(10) . The genes for the gyrase subunits, gyrA and gyrB, have been sequenced and cloned such that the A
and B subunits can be overproduced, up to 40% of soluble cell
proteins(11) . DNA gyrase is known to be the target of two
classes of antibiotics: the synthetic quinolones, typified by nalidixic
acid and the new fluoro-quinolones, and the natural coumarins such as
novobiocin and coumermycin A1. The quinolones are thought to act at the
A subunit probably by interfering with the DNA-rejoining step of the
gyrase-mediated DNA strand-passing
reaction(12, 13, 14, 15, 16) .
The coumarins are thought to act at the B subunit probably by competing
with ATP for binding to the B subunit of the
enzyme(5, 17, 18, 19) .
of 6
nM. Therefore, cyclothialidine, being a B subunit inhibitor of
DNA gyrase, shows the same mode of action as the coumarin antibiotics
novobiocin and coumermycin A1. However, cyclothialidine was also active
against a DNA gyrase resistant to novobiocin, suggesting that the
residues required for novobiocin binding are not involved in
cyclothialidine binding. In addition, there is no obvious structural
resemblance among cyclothialidine, novobiocin, and ATP (Fig. 1).
Here, we report on a further characterization of the DNA
gyrase-mediated ATPase activity by cyclothialidine. We performed
[
C]benzoylcyclothialidine binding experiments to
correlate the inhibition of the ATPase activity of DNA gyrase by
cyclothialidine with binding of the compound to the B subunit. We also
compared the binding of cyclothialidine with that of coumarin
antibiotics and ATP analogues. For these studies we used a
reconstituted E. coli DNA gyrase system that contained the A
subunit, the B subunit, and relaxed ColE1 plasmid DNA. Some studies
were also performed with the 43-kDa N-terminal fragment of the B
subunit(8) , which contains the ATP- and the coumarin-binding
site(s).
Figure 1:
Structures of
cyclothialidine, [C]benzoylcyclothialidine,
novobiocin, and ATP.
Materials
Cyclothialidine was purified from a
culture broth of S. filipinensis NR0484 having a >98%
purity detected by HPLC analysis(21) . Novobiocin, coumermycin
A1, distamycin A, ATP, ADPNP, and ATPS were purchased from Sigma.
Phosphoenolpyruvate, NADH, and pyruvate kinase/lactate dehydrogenase
mix were purchased from Boehringer Mannheim.
DNA Gyrase
Gyrase subunits A and B were purified
separately from the E. coli overproducing strains N4186 and
MK47 by the method of Mizuuchi et al.(24) . The
fraction containing the gyrase A subunit, after it had been subjected
to valine-Sepharose chromatography, still contained minor amounts of
the gyrase B subunit. Therefore, the fraction was applied to a
novobiocin-Sepharose column, as described by Staudenbauer and
Orr(25) , and the flow-through fraction was collected. The
gyrase B subunit was eluted from a hydroxylapatite column. Each sample
was stored in 25 mM Hepes-KOH (pH 8.0) containing 1 mM dithiothreitol, 0.2 mM EDTA, and 50% (w/v) ethylene
glycol at -70 °C. Purified A subunit was judged to be >90%
pure by SDS-polyacrylamide gel electrophoresis; similarly, the purified
B subunit was >95% pure. Gyrase activity was measured using a
standard supercoiling assay(26) . One unit was defined as the
minimum amount of reconstituted gyrase that will maximally supercoil
0.5 µg of relaxed ColE1 DNA at 30 °C in 30 min. The specific
activities of the A subunit and B subunit were 6.4 10
units/mg and 1.2
10
units/mg, respectively.
Protein concentrations were determined by the Bio-Rad protein assay
using bovine serum albumin as the standard.
Purification of the 43-kDa N-terminal Fragment of the DNA
Gyrase B Subunit
The 43-kDa protein was purified from E.
coli cells containing plasmid pAJ1 essentially as described by
Jackson et al.(27) . Briefly, cells were grown at 37
°C in LB broth containing 50 µg/ml ampicillin to an OD of 0.5 and induced for 4 h by the addition of
isopropyl-
-D-thiogalactopyranoside to 50 µM.
Cells were harvested and resuspended at a concentration of 1 g/ml in 50
mM Tris-HCl (pH 7.6), 10% sucrose. The cell suspension was
adjusted to 1 µg/ml RNase, 1 µg/ml DNase, and 20 µg/ml
lysozyme and incubated for 30 min at room temperature. The cells were
disrupted at 60 megapascals using a French press and centrifuged at
100,000
g
for 60 min at 4 °C. Cell
extracts were dialyzed against 50 mM Tris-HCl (pH 8.0) and
applied to a DEAE-Sepharose CL 6B column (Pharmacia Biotech Inc.).
After washing with 50 mM Tris-HCl (pH 8.0), the column was
eluted with a 100-ml linear gradient of 0.0-0.7 M NaCl
in 50 mM Tris-HCl (pH 8.0). Fractions containing the 43-kDa
protein were identified by SDS-polyacrylamide gel electrophoresis,
pooled, dialyzed against 50 mM Tris-HCl (pH 8.0), and loaded
onto an FPLC Mono Q HR 10/10 column (Pharmacia). After washing the
column with 50 mM Tris-HCl (pH 8.0), we eluted the 43-kDa
protein with a 160-ml linear gradient of 0.0-0.4 M NaCl
in 50 mM Tris-HCl (pH 8.0). Fractions containing the protein
fragment were identified by SDS-polyacrylamide gel electrophoresis,
pooled, and dialyzed against 50 mM Tris-HCl (pH 8.0), 100
mM KCl, 5 mM dithiothreitol, 1 mM EDTA, 10%
(w/v) glycerol, frozen in liquid nitrogen, and stored at -80
°C(28) . [
C]Benzoylcyclothialidine-The
[
C]benzoylcyclothialidine was prepared by the
reaction of cyclothialidine with [
C]benzoic acid
(21.8 mCi/mmol) in the presence of N,N`-disuccinimidyl carbonate in
acetonitrile/pyridine (1:1) having >98% purity. The labeled compound
has a specific activity of 21.8 mCi/mmol. Its mobilities on TLC and
HPLC are identical to those of authentic benzoyl-cyclothialidine.
Benzoyl-cyclothialidine has almost the same inhibitory activity against E. coli DNA gyrase as cyclothialidine in the DNA supercoiling
assay (results not shown). [
C]Benzoylcyclothialidine Binding
Experiment-The binding of
[
C]benzoylcyclothialidine was determined by a
centrifugal filtration method(13) . Centrifree micropartition
devices (Amicon number 4103) were used to separate
[
C]benzoylcyclothialidine bound to DNA gyrase
from the free ligand. Reactions (400 µl) were carried out similar
to that of the supercoiling assay but without the addition of ATP; the
reaction mixture contained an appropriate amount of DNA gyrase and
radioactive ligand in standard buffer (50 mM Tris-HCl (pH
8.0), 20 mM KCl, 10 mM MgCl
, 1 mM EDTA, and 1 mM dithiothreitol). After incubation for 30
min at 30 °C, the mixtures were transferred to the centrifree
devices and centrifuged at 1,600
g in a Kubota KR-180B
(swinging bucket rotor) for 30 min at 4 °C. The membrane disks and
o-rings of the centrifree devices were placed in vials with 1 ml of
standard buffer. The vials were shaken on a rotary shaker for 2 h for
solubilization of the [
C]benzoylcyclothialidine.
Then 15 ml of liquid scintillation fluid (toluene-ethanol (1:1), 0.7%
[2-(4-tert-butylphenyl)5-(4`-biphenylyl)-1,3,4-oxadiazole])
was added per vial for the measurement of radioactivity. For the
determination of nonspecific ligand binding, the assay was run with
excess cyclothialidine. The amount of bound ligand was calculated after
subtracting the nonspecific bound radioactivity.
ATPase Assay
ATPase assays were carried out at 25
°C in the following: 300 µl in 40 mM Tris-HCl (pH
8.0), 25 mM KCl, 2.5 mM spermidine, 4 mM MgCl with phosphoenolpyruvate and NADH at 400 and 250
µM, respectively, and 3 µl of pyruvate kinase/lactate
dehydrogenase mix (in ammonium sulfate solution, 3.2 M)(8) . The ATP was added at concentrations from 0.5 to
3.5 mM; the 43-kDa N-terminal fragment of the DNA gyrase B
subunit at a concentration of 5 µM; and cyclothialidine at
concentrations from 0.025 to 10 µM. Reactions were
initiated by the addition of the protein, and the decrease in A
was continuously measured as a function of
time (up to 10 min). The change in absorbance was related to ADP
production using
= 5100, with the
production of NADH stoichiometrically related to the amount of ADP
released.
C]Benzoylcyclothialidine Binds to
the 43-kDa N-terminal Fragment of the B Subunit-Our previous
studies have shown (23) that, under steady-state conditions,
cyclothialidine competitively inhibits the ATPase activity of the E. coli DNA gyrase B subunit with a K
value of 6 nM and that
[
C]benzoyl-cyclothialidine binds to the DNA
gyrase holoenzyme (A
B
tetramer) in the absence
of DNA. This binding is inhibited by cyclothialidine, novobiocin, and
the ATP analogue ATP
S, but it is not inhibited by ofloxacin,
strongly suggesting that cyclothialidine inhibits the binding of ATP to
the gyrase by acting on the B subunit. However, it is not yet known
whether the binding of [
C]benzoylcyclothialidine
requires both subunits of the DNA gyrase or only the B subunit and what
effect DNA has on the binding. To investigate these aspects more
precisely, we studied [
C]benzoylcyclothialidine
binding by using a system of reconstituted E. coli gyrase
subunits plus relaxed ColE1 DNA and also the 43-kDa N-terminal fragment
of the B subunit. The binding experiments were carried out by a
centrifugal filtration technique. The results of the binding studies of
[
C]benzoylcyclothialidine to the gyrase subunits
and to the plasmid DNA are shown in Fig. 2. There was significant
binding of the compound to the B subunit but not to the A subunit nor
to the relaxed ColE1 plasmid DNA. Furthermore, the compound bound to
the 43-kDa N-terminal fragment of the B subunit, showing that the
binding site of cyclothialidine as well as that for the coumarin
antibiotics is located within the 43-kDa N-terminal fragment.
Figure 2:
The binding of
[C]benzoylcyclothialidine to A subunit, B
subunit of DNA gyrase, or relaxed ColE1 DNA. Reactions were as
described under ``Experimental Procedures'' except for the
addition of the indicated amounts of either a 43-kDa N-terminal
fragment of the B subunit (
), B subunit (
), A subunit
(
), or relaxed ColE1 DNA (
). Reaction mixtures contained
1.3
10
M [
C]benzoyl-cyclothialidine. After 30 min at
30 °C, the levels of bound
[
C]benzoyl-cyclothialidine were determined by
the centrifugal filtration method.
Next,
to study whether the binding of
[C]benzoylcyclothialidine depends only on the
presence of the B subunit alone, we performed our binding experiments
in the presence of 1) the B subunit, 2) the B subunit plus A subunit,
and 3) the B subunit plus the A subunit plus plasmid DNA. The amount of
[
C]benzoylcyclothialidine bound to the B subunit
was somewhat higher in the presence of the A subunit and even higher in
the presence of the A subunit plus plasmid DNA (data not shown). When a
250-fold excess of unlabeled cyclothialidine was added, the amount of
[
C]benzoylcyclothialidine bound to the B subunit
decreased to the level where it could hardly be detected under all
conditions (data not shown). We confirmed that
[
C]benzoylcyclothialidine reversibly binds to
the B subunit even in the presence of the A subunit and plasmid DNA.
Scatchard Analysis
To study the binding of
[C]benzoyl-cyclothialidine to the B subunit
further, including the effect of the A subunit and plasmid DNA on the
binding, we performed Scatchard analysis for the four conditions. The
results in Fig. 3show that the
[
C]benzoylcyclothialidine bound to the B subunit
in the presence of the 43-kDa fragment with a K
value of 2.5 ± 0.40
10
M; in the presence of the B subunit with a value of 2.4
± 0.10
10
M; in the presence
of the B subunit plus the A subunit with a value of 1.5 ± 0.31
10
M; and in the presence of the B
subunit plus the A subunit plus plasmid DNA with a value of 8.3
± 0.12
10
M. This result
suggests that the binding affinity of the compound to the B subunit is
dependent on the stage of the assembly of the gyrase
DNA complex.
The lowest binding affinity to the B subunit could be observed in the
presence of the B subunit alone, and the highest binding affinity could
be observed in the presence of the A
B
DNA
complex (increase in affinity: B
A
B
A
B
DNA), although the binding
capacity (B
) remained invariant, with
approximately 1 pmol of
[
C]benzoylcyclothialidine bound per pmol of the
B subunit.
Figure 3:
Scatchard analysis of
[C]benzoylcyclothialidine binding to the
reconstituted E. coli DNA gyrase subunits plus DNA. Reactions
were as described in the legend to Fig. 2 and under ``Experimental
Procedures'' except that the incubation was done for 30 min.
Reaction mixtures contained 0.11-1.6
10
M [
C]benzoylcyclothialidine. When
added, 100 pmol of A subunit, 50 pmol of B subunit, 50 pmol of the
43-kDa fragment, and 0.5 µg of relaxed ColE1 DNA were present.
Scatchard plots in the presence of the 43-kDa fragment (
), B
subunit (
), B subunit + A subunit (
), and B subunit
+ A subunit + plasmid DNA (
) were
performed.
Inhibition of Binding by Cyclothialidine, ATP Analogues,
and Coumarin Antibiotics
Although the structures are different (Fig. 1), cyclothialidine proved to be similar to novobiocin and
coumermycin A1 in their inhibition of the DNA-dependent ATPase activity
of DNA gyrase. Furthermore, there are no structural similarities
between ATP and cyclothialidine. Therefore, to investigate whether the
binding site for cyclothialidine on the B subunit is different from
that for ATP and for the coumarin antibiotics, we examined the effects
of nonlabeled cyclothialidine, ADPNP, ATPS, noboviocin, and
coumermycin A1 on the binding of
[
C]benzoylcyclothialidine to the 43-kDa
N-terminal fragment of the B subunit, to the B subunit alone, and to
the B subunit in the presence of the A subunit or in the presence of
the A subunit and DNA. The results are shown in Fig. 4and . The displacement curves of
[
C]benzoyl-cyclothialidine binding obtained
after the addition of cyclothialidine had almost the same profiles,
with IC
values of 8.4
10
M for the 43-kDa protein, 9.3
10
M for the B subunit, 1.0
10
M for the B subunit plus A subunit, and 8.1
10
M for the B subunit plus the A subunit plus plasmid DNA,
respectively. However, the displacement curves induced by ADPNP were
surprisingly quite different from those induced by cyclothialidine and
critically depended on the form of the B subunit present. The relative
inhibitory activity of ADPNP is as follows: B subunit plus A subunit
plus DNA > B subunit plus A subunit > B subunit > 43-kDa
N-terminal fragment, with IC
values of 8.0
10
M, 4.5
10
M, 1.5
10
M, and
>1.5
10
M, respectively. The
IC
value of ADPNP for the
A
B
DNA complex is 19-fold lower than that
for the B subunit alone, and similar results were obtained with
ATP
S and coumarin antibiotics (). The coumarin
antibiotic coumermycin A1, for example, inhibited
[
C]benzoyl-cyclothialidine binding in the order
B subunit plus A subunit plus DNA > B subunit plus A subunit > B
subunit > 43-kDa N-terminal fragment, with IC
values of
9.5
10
M, 1.8
10
2.4
10
, and 6.3
10
M, respectively. The IC
value for the A
B
DNA complex is
6.6-fold lower than that for only the 43-kDa N-terminal protein.
Figure 4:
Effect of cyclothialidine, ATP analogues,
and coumarin antibiotics on
[C]benzoylcyclothialidine binding to the each B
subunit form of DNA gyrase. Reactions were as described in the legend
to Fig. 3 and under ``Experimental Procedures.'' Reaction
mixtures contained 1.3
10
M [
C]benzoylcyclothialidine and the indicated
amounts of cyclothialidine (A), ADPNP (B), ATP
S (C), novobiocin (D), and coumermycin A1 (E).
The effect of the compounds on
[
C]benzoylcyclothialidine binding to the 43-kDa
fragment (
), B subunit (
), B subunit + A subunit
(
), and B subunit + A subunit + plasmid DNA (
)
was measured. After 30 min at 30 °C, the levels of bound
[
C]benzoylcyclothialidine were determined by the
centrifugal filtration method.
Furthermore, Scatchard analysis of the competitive binding of ADPNP
and novobiocin to the B subunit plus the A subunit were also carried
out (Fig. 5). Although the slope of the Scatchard plot decreased
in the presence of ADPNP and was dependent on its concentration, the
binding capacity (B) remained invariant. The
same result was obtained with novobiocin. These observations suggest
that ATP and coumarin antibiotics competitively inhibit the binding of
[
C]benzoyl-cyclothialidine to the B subunit.
Figure 5:
Scatchard analysis of
[C]benzoylcyclothialidine bind-ing for the
competition studies of ADPNP and novobiocin. Reactions were as
described in the legend to Fig. 3 (B subunit + A subunit). A, reaction mixtures contained no ADPNP (
), 3 mM ADPNP (
), or 10 mM ADPNP (
). B,
reaction mixtures contained no novobiocin (
), 0.3 µM novobiocin (
), or 1.5 µM novobiocin
(
).
Cyclothialidine Is Not a Simple Competitive Inhibitor of
the ATPase Activity of the 43-kDa N-terminal Fragment
The 43-kDa
N-terminal fragment of the DNA gyrase B subunit hydrolyzes ATP and
binds novobiocin and coumermycin A1(8) . We showed that
cyclothialidine binds to the B subunit and inhibits the ATPase activity
of DNA gyrase(23) . To test whether cyclothialidine also
inhibits the ATPase activity of the 43-kDa N-terminal fragment of the
DNA gyrase B subunit, we determined its inhibitory activity in
steady-state kinetic experiments. Cyclothialidine was included in the
ATPase assay, and the results indicate that cyclothialidine is indeed
an inhibitor of the ATPase activity of this N-terminal fragment of the
B subunit (Fig. 6). The V values
decrease with increasing drug concentrations, indicating that
cyclothialidine is not a simple competitive inhibitor. The calculated
K
(mM ATP) and V
(nM ADP/µM 43 kDa
s) from these data are as follows: 2.1 mM and 30.0
nM/µM
s for no cyclothialidine, 2.1
mM and 26.7 nM/µM
s for 1
µM cyclothialidine, and 2.4 mM and 17.4
nM/µM
s for 5 µM cyclothialidine. The values determined for the ATPase activity of
the 43-kDa protein in the presence of novobiocin are
K
= 0.68 mM and V
18.5 nM/µM
s for no novobiocin and K
=
0.51 mM and V
= 10.5
nM/µM
s for 6 µM novobiocin(8) . These results are consistent with the
results of the displacement of the
[
C]benzoyl-cyclothialidine binding by the
compounds, shown in Fig. 4, B and C, in that
the binding of the labeled compound to the 43-kDa N-terminal fragment
of the B subunit is hardly inhibited by the ATP analogues ADPNP and
ATP
S.
Figure 6:
ATPase activity of the 43-kDa N-terminal
fragment of DNA gyrase B subunit (5 µM) in the presence of
cyclothialidine. Reactions were as described under ``Experimental
Procedures.'' The K (mM ATP) and V
(nM ADP/µM 43 kDa
s) are as follows: 2.1 mM and 30.0
nM/µM
s for no cyclothialidine
(
), 2.1 mM and 26.7 nM/µM
s for 1 µM cyclothialidine (
), and 2.4
mM and 17.4 nM/µM
s for 5
µM cyclothialidine (
).
C]benzoylcyclothialidine
to the reconstituted E. coli DNA gyrase with the centrifugal
filtration method.
C]benzoyl-cyclothialidine binds to the B
subunit alone and also to the 43-kDa N-terminal fragment of this
subunit. Scatchard analysis, carried out with the reconstituted gyrase
subunits plus DNA ( Fig. 3and ) showed that the
affinity (K
value) for the binding of
[
C]benzoylcyclothialidine to the B subunit
depended on the association state of the gyrase
DNA complex
(increasing affinity: B
A
B
A
B
DNA). This suggests that the B
subunit undergoes conformational changes as it becomes part of the
complete gyrase
DNA complex. The affinity of cyclothialidine (K
= 8.3 ± 0.12
10
M) with the binding site on the
gyrase
DNA complex (A
B
DNA) was also
in good agreement with the inhibition of the DNA-dependent ATPase
activity of gyrase (K
= 6
10
M). Therefore, there is a close
correlation of the inhibition of the ATPase activity of gyrase by
cyclothialidine with the amount of
[
C]benzoylcyclothialidine bound to the B subunit
in the gyrase
DNA complex.
C]benzoylcyclothialidine binding induced by
ADPNP or coumermycin A1 were significantly different from that induced
by nonlabeled cyclothialidine. However, from the Scatchard analysis for
the competition studies shown in Fig. 5, ADPNP and novobiocin
competitively inhibited the binding of
[
C]benzoylcyclothialidine to the B subunit in
the A
B
form. These results suggest that
cyclothialidine, coumarin antibiotics, and ATP share a common (or
overlapping) site of action on the B subunit of DNA gyrase; however,
the microenvironment of the binding sites may differ. Maxwell et
al. described (8) that the ADPNP inhibition against the
ATPase activity of the 43-kDa fragment is different from those of ADP
and novobiocin because in the presence of ADPNP the protein behaves as
a dimer whereas in the presence of ADP or novobiocin the enzyme is a
monomer. The result shown in Fig. 6also might reflect the
artifactual nature of the protein fragment, because in the subunit
structure of A
B
DNA cyclothialidine
competitively inhibits the ATPase activity of DNA gyrase. It is
possible that the differential competition seen with the 43-kDa
fragment of the B subunit is due to the effects of cyclothialidine and
coumarin antibiotics on the dimerization of the protein fragments. As
shown in Fig. 4, the inhibition by nonlabeled cyclothialidine of
[
C]benzoylcyclothialidine binding to each B
subunit form (B, A
B
, and
A
B
DNA) is essentially the same in each
case. These findings are reasonable because the affinities of
[
C]benzoylcyclothialidine (ligand) and
nonlabeled cyclothialidine (inhibitor) change in parallel.
Table: Competition of substrate analogues and
gyrase inhibitors for [C]benzoylcyclothialidine
binding
-
-imidodiphosphate; ATP
S,
adenosine-5`-O-(thiotriphosphate); HPLC, high performance
liquid chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.