From the Departments of Structural Biology and
§ Biochemistry, St. Jude Children's Research Hospital and
the ¶ Department of Biochemistry, University of Tennessee,
Memphis, Tennessee 38105
Received for publication, August 6, 2000, and in revised form, October 9, 2000
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ABSTRACT |
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The Drug resistance in infectious organisms has become a serious
medical problem, and fatty acid synthesis has emerged as a promising target for the development of novel therapeutic agents. Lipid synthesis
is not only essential to cell viability but specificity for bacteria
and other infectious organisms can be achieved by taking advantage of
the organizational and structural differences that exist in the fatty
acid synthetic systems of different organisms. There are two major
types. The associated, or type I, systems exist in higher organisms
such as mammals and compose a single, multifunctional polypeptide (1).
The dissociated, or type II, fatty-acid synthases exist in bacteria and
plants and are composed of a collection of discrete enzymes that each
carry out an individual step in the cycles of chain elongation (2, 3).
Triclosan (4, 5) and isoniazid (6) are two commonly used antibacterial agents that target fatty acid synthesis.
The type II system has been most extensively studied in
Escherichia coli where the three
The crystal structures of all three condensing enzymes from E. coli (FabB, FabF, and FabH) have now been determined (19-22). Their primary structures are clearly related, and these translate into
similar dimeric structures and active site architectures. The
structures of the monomers compose an internally duplicated helix-sheet-helix motif, and the active site is located at the convergence of the pseudo dyad-related Two natural products inhibit type II fatty acid synthesis by blocking
the activity of one or more of the We report the structures of the FabB-TLM and FabB-cerulenin complexes,
and we identify structural features that define the differences in the
biochemical mode of action and target selectivity of the two
antibiotics. We have further validated our understanding of the
mechanisms of antibiotic binding through the mutagenesis of a key
residue involved in the protein-drug interaction, and the subsequent
assay of the mutant. This work contributes not only to the development
of new antibacterials that target the condensation step in type II
fatty acid synthesis but also to the understanding of the condensation
reaction mechanism.
Materials--
Sources of supplies are as follows:
[14C]malonyl-CoA (specific activity, 55.0 Ci/mol) and
[14C]acetyl-CoA (specific activity, 52.0 Ci/mol) from
Amersham Pharmacia Biotech; microbiological media from Difco; molecular
reagents from Promega; cerulenin and ACP from Sigma;
Ni2+-agarose resin from Qiagen; pET vector and expression
strains from Novagen; and pCR2.1 vector from Invitrogen. Proteins were quantitated by the Bradford method (38) unless otherwise indicated. Acyl-ACP was prepared using an established acyl-ACP synthetase method
(14, 39, 40). All other supplies were reagent grade or better.
Purification and Assay of Condensing Enzymes--
The three
condensing enzymes of E. coli and malonyl-CoA:ACP
transacylase (FadD) were expressed and purified to homogeneity as
described previously (13, 14, 41, 42). Purified enzymes were then
dialyzed against 20 mM Tris-HCl, pH 7.6, 1 mM
dithiothreitol, concentrated with an Amicon stirred cell, and stored in
50% glycerol at
A filter disc assay was used to assay FabH activity with
[1-14C]acetyl-CoA as described previously (15, 18). The
assays contained 50 µM ACP, 1 mM
FabB and FabF radiochemical assay was performed using the scheme
devised by Garwin et al. (11) using myristoyl-ACP as the substrate. The assays contained 100 µM ACP, 0.3 mM dithiothreitol, 1 mM EDTA, 0.1 M
potassium phosphate buffer, pH 6.8, 50 µM
[14C]malonyl-CoA (specific activity 55 Ci/mol), 100 µM myristoyl-ACP, FabD (0.3 µg of protein), in a final
volume of 20 µl. A mixture of ACP, 0.3 mM dithiothreitol,
1 mM EDTA, and the buffer was incubated at 37 °C for 30 min to ensure complete reduction ACP, and then the remaining components
(except the condensing enzyme) were added. The mixture was then
aliquoted into the assay tubes, and the reaction was initiated by the
addition of FabB or FabF. The reaction mixture was incubated at
37 °C for 20 min, and then 400 µl of reducing agent (0.1 M K2HPO4, 0.4 M KCl,
30% tetrahydrofuran, and 5 mg/ml sodium borohydride) was added into
the reaction tubes and incubated for 40 min. Finally, 400 µl of
toluene was added and vigorously mixed, and 300 µl of upper phase
solution was counted in 3 ml of scintillation mixture.
Assay for TLM Binding--
Kd values for TLM
and the condensing enzymes were measured by fluorescence spectroscopy
on a SLM-Amicon 8100 spectrofluorimeter (43). Quenching of the
intrinsic protein fluorescence was measured with excitation at 280 nm
and emission at 337 (FabB), 332 (FabF), or 321 nm (FabH). The
concentration of each enzyme was 1 µM in 0.1 M sodium phosphate buffer, pH 7.5, and TLM was titrated in 2-µl aliquots from stock solutions in water. Each curve was corrected for the nominal absorption by TLM at both excitation and emission wavelengths and normalized. Data was then fitted to Equation 1 (43),
where Fo is the initial fluorescence;
Construction of the FabB[H333N] Mutant--
A portion of the
fabB gene was amplified using the polymerase chain reaction
with two specific primers. The first primer introduced the desired
mutation and extended over a unique AgeI site
(5'-AAAGCCATGACCGGTAACTCTC-3'). The second primer created a
BamHI site downstream of the stop codon
(5'-GCAGGATCCGGCGATTGTCAATGATG-3'). The resulting fragment was
sequenced to confirm that the mutation had been correctly introduced
and then was digested with AgeI and BamHI and
cloned into the pET-15b-FabB expression vector that had been digested with the same enzymes. The FabB[H333N] protein was expressed and purified as described above.
Structure Determination of the FabB-Cerulenin and FabB-TLM
Complexes--
Both structures were solved using electron density
difference maps. Pure FabB protein was dialyzed (10 mM
Tris-HCl, pH 8.0, 1 mM dithiothreitol, 1 mM
EDTA) and concentrated to 15 mg/ml. The inhibitors (TLM and cerulenin)
were added directly to separate aliquots of the protein solution and
were gently agitated for 1 h. The ratio of inhibitor molecules to
FabB monomers was about 10:1. The FabB-antibiotic complexes were
crystallized by the same procedure as followed for the native crystals
(21). Crystals measuring 0.1 × 0.3 × 1.0 mm grew in 1-2
weeks. The crystals were mounted on standard nylon loops, passed
through a cryoprotectant of 50% paratone-N, 50% mineral oil and were
frozen directly in liquid nitrogen. Data were collected at 100 K using
a Nonius FR591 x-ray generator and DIP 2030H detector system. All
diffraction data were integrated using the HKL software package (44).
Integrated data were merged and scaled using SCALEPACK. Crystals of
both complexes have space group
P212121 and cell dimensions similar to the native crystals (Tables I and II).
All refinements of models against our data were carried out using XPLOR
(version 3.851) (45). First, the native FabB structure (21) was refined
against our data, and then 2 mFo Inhibition of Condensing Enzymes by TLM and Cerulenin--
The
relative sensitivities of the condensing enzymes to TLM and cerulenin
are known from the inhibition of the pathway in crude cell extracts and
from analyses of growth inhibition in genetically modified E. coli strains. However, the activities of the purified enzymes have
not been compared using natural substrates. Therefore, we determined
the IC50 values for all three condensing enzymes for both
TLM and cerulenin (Fig. 1). Each of the
three condensing enzymes was inhibited by TLM (Fig. 1A).
Under the in vitro assay conditions employed, FabF was the
most sensitive enzyme (IC50 = 6 µM) followed
by FabB (IC50 = 25 µM) and FabH, which was
considerably less sensitive (IC50 = 110 µM).
These data are consistent with genetic experiments that show that
overexpression of FabB confers TLM resistance, whereas FabH
overexpression does not (49). Increased expression of FabF blocks
growth (50) precluding a similar experiment with this condensing
enzyme. However, since FabF is not essential for the growth of E. coli (11), FabB is the physiologically relevant TLM target in this
bacterium.
As expected, both FabB and FabF were inhibited by cerulenin, with FabB
being the most sensitive enzyme under our assay conditions (Fig.
1B). Since cerulenin forms an irreversible covalent complex with the FabB and FabF, the differences noted in Fig. 1B
reflect the rate of complex formation under our assay conditions, and eventually both FabB and FabF will be completely inactivated by cerulenin. However, our experiments are consistent with previous work
that examined the effect of cerulenin on fatty acid production in
either normal E. coli strains, strains lacking FabF
activity, or strains that overexpressed FabB, which all indicated that
FabB was the more sensitive of the two enzymes in vivo (51,
52). FabH was not effectively inhibited by cerulenin, as reported
previously for cell extracts (17).
TLM Is a Reversible Inhibitor--
The thiolactone structure in
TLM suggested that it may form a covalent adduct with the condensing
enzyme via a thioester exchange reaction. We examined this hypothesis
by comparing the kinetics of TLM inhibition to that of cerulenin (Fig.
2), which is known to form a covalent
complex with the active site cysteine of the condensing enzymes. The
kinetics of cerulenin inhibition exhibited the hallmarks of a slow
binding, irreversible inhibitor (53). When the reaction was initiated
with enzyme, there was an initial burst of product formation, but the
FabB reaction rate rapidly decreased and ceased by 20 min (Fig. 2).
When cerulenin was preincubated with FabB and the reaction was
initiated by the addition of the substrates, there was no discernible
product formation. This indicates the formation of the irreversible
FabB-cerulenin binary complex. The same two types of time course
experiments were performed with TLM (Fig. 2), and there was no evidence
for the formation of an irreversible or slow-binding FabB-TLM complex.
To verify that TLM was a reversible inhibitor, we performed a dilution
experiment in which FabB was first exposed to 100 µM TLM,
which inhibits the enzyme about 95%, and then the TLM concentration
was diluted to 1 µM, and the enzyme activity was
recovered to the same value as when FabB was continually exposed to 1 µM TLM. These data verify the reversibility of TLM
inhibition that was shown in previous experiments with cell extracts
(32).
Direct Measurement of TLM Affinities--
The kinetic experiments
reported in Fig. 1 illustrate the relative effectiveness of TLM as an
inhibitor under standard assay conditions for the three condensing
enzymes. However, the in vitro assays are different as are
the substrate specificities of the enzymes, which precludes using the
IC50 values as a direct comparison of the affinities of the
enzymes for TLM. Therefore, we used fluorescence spectroscopy to
measure directly the binding of TLM to the enzymes (Fig.
3). FabB exhibited the highest affinity
(26 µM), FabF intermediate affinity (60 µM), and FabH (158 µM) the lowest affinity.
Whereas the actual affinities of TLM for FabB and FabH were consistent with the kinetic experiments, the TLM affinity for FabF was lower than
would be extrapolated from the IC50 determination.
The Structure of the FabB-TLM Binary Complex--
The final
refined electron density of the complex clearly shows the conformation
of the entire TLM molecule and all of its important interactions. The
statistics of the final model are good (Table
I). TLM makes a number of specific but
noncovalent interactions within the FabB active site (Fig.
4). The C-9 and C-10 methyl groups are
nestled within two hydrophobic pockets comprising phenylalanines 229 and 392 and Pro-272 and Phe-390, respectively. The isoprenoid moiety is
wedged between two peptide bonds, 391-392 "below" and 271-272
"above," and this intercalated stacking interaction between three
delocalized systems is clearly an important element of specificity. The
ring of Pro-272 participates in this molecular sandwich by van der
Waals interactions. The O-1 and O-2 exocyclic oxygens are both involved
in hydrogen bonding interactions. O-1 interacts with the N
The exquisite fit of TLM into the FabB active site is reflected in the
minimal distortion it causes to the native FabB structure. The
movements that do occur involve local changes in the FabB main chain
positions, but these do not extend beyond the immediate vicinity of the
active site (Fig. 4). The side chain of Cys-163 shifts by 2.1 Å to
avoid a clash with the TLM sulfur, and His-298 moves by 2 Å to improve
the hydrogen bonding geometry to the O-1 of TLM. Finally,
phenylalanines 390 and 392, and the associated loop comprising residues
388-394, all move by about 1.0 Å, and the side chain of Phe-392 also
rotates ~40°. These latter movements correlate with the new
location of His-298. A hydrogen bonding interaction between the N The Structure of the FabB-Cerulenin Complex--
In the final,
refined electron density map, the conformation of the attached end of
the cerulenin molecule is unambiguous (Fig.
5B). The electron density
corresponding to the acyl chain of the inhibitor is weaker. This
may be due to the conformation flexibility of the chain. The final
model has good statistics (Table II). In
contrast to TLM, cerulenin forms a covalent complex with FabB (Fig. 5).
Also, with one major exception, it primarily occupies a different
region of the active site than TLM. The covalent bond is formed between
the central C-2 of cerulenin and the active site Cys-163. The O-2 and
O-3s are crucial specificity determinants that form important hydrogen
bonds. O-2 interacts with the N
Like TLM, cerulenin occupies the active site with minimal distortion of
the surrounding FabB structure, and the slight movements that do occur
are similar to those observed in the TLM complex. Thus, Cys-163 moves
to form the covalent bond; histidines 298 and 333 move to optimize the
hydrogen bond interactions with the O-2, and phenylalanines 390 and 392 and their associated loop shift by ~1.5 Å in concert with the
movement of His-298. Notably, residues in the deep hydrophobic pocket
of FabB do not move upon cerulenin binding, whereas Ile-108 must rotate
to accommodate the cerulenin chain in the FabF-cerulenin structure
(54).
Importance of His-His Active Site Configuration in TLM
Inhibition--
The structural information indicates that the strong
hydrogen bond interactions between the two active site histidines and the O-1 of TLM are important determinants of high affinity TLM binding.
The FabH condensing enzyme has a His-Asn configuration and was much
less sensitive to TLM (Figs. 1 and 3). The importance of the two
histidines was directly addressed by creating the FabB[H333N] mutant
that converts the FabB active site into a FabH configuration. The
specific activity of FabB[H333N] was reduced compared with FabB, but
it still retained significant condensation activity (Fig.
6A). However, FabB[H333N]
was significantly less sensitive to inhibition by both TLM (Fig.
6B) and cerulenin (Fig. 6C) in the standard
biochemical assay. Accordingly, the affinity of FabB[H333N] for TLM
was an order of magnitude lower than FabB (Fig. 3) supporting the
importance of the two-histidine motif in TLM binding. These data
indicate that the two-histidine active site architecture is an
important determinant of the reactivity of condensing enzymes toward
both TLM and cerulenin and that it contributes to the observed resistance of the FabH class of enzymes to these two antibiotics (Fig.
1).
The structural and biochemical analyses of the binding of TLM and
cerulenin to FabB provide the framework for understanding the
specificity of these antibiotics and provide clues for the development
of more potent compounds that target type II fatty acid synthesis.
Although the condensation enzymes have similar active sites, subtle
structural variations define their differential response to these
molecules. The sensitivity of the condensing enzymes to TLM inhibition
is FabF > FabB The isoprenoid moiety in TLM takes advantage of a specific hydrophobic
crevice that is present in the active sites of both FabB and FabF.
However, these hydrophobic pockets extend further back into the
interior of the proteins and are not optimally filled by the TLM side
chain. This would explain the results of inhibition studies against
plant (55) and mycobacterial (56) fatty-acid synthase systems that used
TLM analogs in which the isoprenoid was replaced by various acyl
chains. Analogs with longer, more flexible chains showed increased
activity against FAS II in these organisms, and these longer chains may
more completely fill the available space. Also, TLM analogs with
shorter chains lacking the double bond are less active. It is worth
noting that the analog studies assumed that the isoprenoid chain binds
in the natural substrate pocket, and our structural studies clearly
show that this is not the case. This structural insight has important
implications for the design of more potent inhibitors against these enzymes.
In the case of cerulenin, the order of inhibition is FabB > FabF
It is clear that FabB is the physiologically important TLM target in
E. coli since FabF is not an essential enzyme (11), and
elevated expression of the fabB gene confers TLM resistance whereas increased levels of FabH do not (49). In contrast, FabF-like proteins are the only elongation condensing enzymes expressed in many
pathogens, such as Streptococcus pneumoniae and
Staphylococcus aureus, and it is likely that FabF is the
relevant TLM target in these organisms. Bacillus subtilis
stands out as an organism that is uniquely resistant to TLM (58, 59).
Accordingly, TLM is a very weak inhibitor of B. subtilis
FabF in vitro.2
The nature of the subtle differences among these enzymes that determine
TLM sensitivity are under investigation.
The structures of the FabB-TLM and FabB-cerulenin complexes support the
division of the common active site of the enzymes into a transacylation
half and a decarboxylation half based on our site-directed mutagenesis
work (20). The location of the two antibiotics with respect to the two
sides of the FabB active site is clearly seen in an overlay of the
FabB-TLM and FabB-cerulenin structures (Fig.
7). The tail of cerulenin occupies the
hydrophobic cavity that accommodates the fatty acid chain of the
acyl-enzyme intermediate. On the other hand, the ring of TLM takes the
place of the incoming malonyl group that participates in the
decarboxylation reaction. The O-1 of TLM and O-2 of cerulenin are the
only two atoms that occupy the same space in the two structures. In
both cases, these carbonyl oxygens form strong hydrogen bonds with the
active site histidine dyad, an important component of antibiotic interactions with the enzyme (Fig. 7). It is interesting to note that
the movements in the FabB active site caused by TLM and cerulenin are
very similar. The fact that these movements directly relate to the
hydrogen bonding interactions of the inhibitors with the histidine dyad
suggests that they also occur during substrate binding and are part of
the active site mechanism. One possibility is that they represent
communication between the two half-sites that promotes the ping-pong
mechanism.
-ketoacyl-acyl carrier protein (ACP)
synthases are key regulators of type II fatty acid synthesis and are
the targets for two natural products, thiolactomycin (TLM) and
cerulenin. The high resolution structures of the FabB-TLM and
FabB-cerulenin binary complexes were determined. TLM mimics malonyl-ACP
in the FabB active site. It forms strong hydrogen bond interactions
with the two catalytic histidines, and the unsaturated alkyl side chain interaction with a small hydrophobic pocket is stabilized by
stacking interactions. Cerulenin binding mimics the condensation transition state. The subtle differences between the FabB-cerulenin and
FabF-cerulenin (Moche, M., Schneider, G., Edwards, P., Dehesh, K., and
Lindqvist, Y. (1999) J. Biol. Chem. 244, 6031-6034)
structures explain the differences in the sensitivity of the two
enzymes to the antibiotic and may reflect the distinct substrate
specificities that differentiate the two enzymes. The FabB[H333N]
protein was prepared to convert the FabB His-His-Cys active site triad
into the FabH His-Asn-Cys configuration to test the importance of the two His residues in TLM and cerulenin binding. FabB[H333N] was significantly more resistant to both antibiotics than FabB and had an
affinity for TLM an order of magnitude less than the wild-type enzyme,
illustrating that the two-histidine active site architecture is
critical to protein-antibiotic interaction. These data provide a
structural framework for understanding antibiotic sensitivity within
this group of enzymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ketoacyl-ACP1 synthases
have emerged as important regulators of the initiation and elongation
steps in the pathway. These enzymes catalyze the Claisen condensation
reaction, transferring an acyl primer to malonyl-ACP and thereby
creating a
-ketoacyl-ACP that has been lengthened by two carbon
units. Two of these synthases are elongation condensing enzymes.
Synthase I (FabB) is required for a critical step in the elongation of
unsaturated fatty acids. Mutants (fabB) lacking synthase I
activity require supplementation with exogenous unsaturated fatty acids
to support growth (7, 8). Synthase II (FabF) controls the
temperature-dependent regulation of fatty acid composition
(9, 10). Mutants lacking synthase II (fabF) are deficient in
the elongation of palmitoleate to cis-vaccenate but grow
normally under standard culture conditions (9, 11, 12). The third
synthase functions as the initiation condensing enzyme. Synthase III
(FabH) catalyzes the first condensation step in the pathway and is thus
ideally situated to govern the rate of fatty acid synthesis (13-16).
Unlike FabB and FabF, FabH enzymes use an acyl-CoA rather than an
acyl-ACP as the primer (15-18). FabH is further distinguished by a
His-Asn-Cys (19, 20) catalytic triad in contrast to the His-His-Cys
triad in the FabB (21) and FabF (22) enzymes.
-helices at the center of the
molecule. The buried active site is accessed by a tunnel that
accommodates the 4'-phosphopantetheine prosthetic group of ACP (and
also CoA in the case of FabH). The active site is functionally and
architecturally divided into halves, and each half is associated with
one of the duplicated motifs (20). The initial transacylation half-reaction, which attaches the acyl primer to the active site cysteine, is facilitated by an
-helix dipole and an oxyanion hole.
The decarboxylation half-reaction, which transfers the acyl primer to
malonyl-ACP, is accelerated by the formation of two adjacent hydrogen
bonds to the thioester carbonyl of the incoming malonyl-ACP. The
hydrogen bond donors are two histidines in the FabB/FabF class and a
histidine and an asparagine in the FabH class. Also, the side chain of
a conserved phenylalanine promotes the decarboxylation step in both
types of enzymes. This scheme is supported by mutagenesis studies of
FabH (20) and differs somewhat from the mechanisms proposed by others
(19, 22).
-ketoacyl-ACP synthases. Cerulenin is an irreversible inhibitor of
-ketoacyl-ACP synthases I
and II (23-25) and forms a covalent adduct with the active site cysteine (26). Cerulenin is not a selective antibacterial because it is
also a potent inhibitor of the condensation reaction catalyzed by the
mammalian multifunctional (type I) fatty-acid synthase (27, 28).
However, cerulenin and related compounds have antineoplastic activity
(29) and reduce food intake and body weight in mice (30). TLM is a
unique thiolactone molecule that reversibly inhibits type II, but not
type I, fatty-acid synthases (31, 32) and is effective against many
pathogens. The antibiotic is not toxic to mice and affords significant
protection against urinary tract and intraperitoneal bacterial
infections (33). TLM is active against Gram-negative anaerobes
associated with periodontal disease (34) and exhibits antimycobacterial
action by virtue of its inhibition of mycolic acid synthesis (35). TLM
also has activity against malaria (36) and trypanosomes (37), extending
the potential for using this template as a platform to develop more antimicrobials.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C.
mercaptoethanol, 45 µM [14C]acetyl-CoA
(specific activity, 52.0 Ci/mol), and 50 µM malonyl-CoA, E. coli FabD (0.3 µg) and 0.1 M sodium
phosphate buffer, pH 7.0, in a final volume of 40 µl. The reaction
was initiated by the addition of FabH, and the mixture was incubated at
37 °C for 12 min. A 35-µl aliquot was removed and deposited on a
Whatman 3MM filter disc. The discs were washed with three changes (20 ml/disc for 20 min) of ice-cold trichloroacetic acid. The concentration of the trichloroacetic acid was reduced from 10 to 5 to 1% in each
successive wash. The filters were dried and counted in 3 ml of
scintillation mixture.
(Eq. 1)
F is the change in fluorescence; [E] is the
enzyme concentration; and [TLM] is the drug concentration. Each
experiment was repeated several times and with the same results.
DFc maps were calculated using CCP4 programs (46). To optimize the maps, the program DM (47) was used to perform histogram
matching, solvent flattening, and 4-fold NCS averaging (there are 2 dimers in the asymmetric unit). Maps were examined using the program O
(48) and were determined to be of good quality. Both maps clearly
showed the presence of an antibiotic in the active site. The
three-dimensional structures of the inhibitors were fit by hand into
the electron density of one monomer and then extended by NCS operators
into the other sites. For both antibiotics, all four sites showed a
good fit between the map and the hand-fit molecule. Waters were picked
using XPLOR and were visually inspected for good electron density and
for sensible H-bonding geometry. Incorrectly assigned waters were
rejected, and some additional waters were added by hand. Full scale
refinements using NCS restraints were then performed. The residues to
include in NCS restraints were chosen to be those residues not involved in crystal contacts, as determined by the XPLOR script "geomanal" and by a visual inspection of crystal packing. Two to three cycles of
refinement followed by manual rebuilding of each model completed the
structure determinations. The statistics of the final models are shown
in Tables I and II.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Inhibition of E. coli
condensing enzymes by TLM and cerulenin. A, the
inhibition of FabF ( ), FabB (
), and FabH (
) by TLM. The
IC50 values are as follows: FabF, 6 µM; FabB,
25 µM; and FabH, 110 µM. B, the
inhibition of FabF (
), FabB (
), and FabH (
) by cerulenin. The
IC50 values are as follows: FabF, 20 µM;
FabB, 3 µM; and FabH, >700 µM. The
activities of the three condensing enzymes were compared using the
radiochemical assays described under "Experimental
Procedures."
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[in a new window]
Fig. 2.
FabB is reversibly inhibited by TLM and
irreversibly inhibited by cerulenin. The time course for the
inhibition of FabB activity under five experimental conditions was
determined to address the reversibility of the antibiotics. Time course
for the FabB reaction in the absence of inhibitors ( ) and in the
presence of 20 µM TLM (
or 3 µM
cerulenin (
) is shown. FabB was preincubated with either 20 µM TLM (
) or 3 µM cerulenin (
) for 30 min prior to initiation of the reaction by the addition of the other
substrates. FabB activity was measured using the radiochemical assay
with myristoyl-ACP as the substrate as described under "Experimental
Procedures."
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Fig. 3.
Direct measurement of TLM binding to the
condensing enzymes. Binding of TLM to the condensing enzymes was
measured by quenching of the intrinsic tryptophan fluorescence of the
proteins as described under "Experimental Procedures." Data are
presented as the observed minus initial fluorescence, normalized to the
total change. The calculated Kd values are as
follows: FabB ( ), 26 µM; FabB[H333N] (
), 256 µM; FabF (
), 60 µM; and FabH (
), 158 µM.
-2
nitrogens of the two active site histidines 298 and 333, and O-2 bonds
to the carbonyl oxygen of residue 270 and the amide nitrogen of residue
305 through a lattice of water molecules. These water molecules are at
the base of the active site tunnel, and the O-2 is appropriately
oriented toward the tunnel. Finally, the thiolactone sulfur does not
make any obvious specific interactions, but it is adjacent to the
active site cysteine 163.
Statistics of data collection and refinement for the FabB-TLM binary
complex
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Fig. 4.
The structure of the FabB-TLM binary
complex. A, stereo diagram of the complex. TLM is shown
with magenta bonds. Secondary structural elements are shown
in orange, as are all carbons. The atoms in TLM, the
atoms in the active site residues (His-298, His-333, Cys-163, Phe-392),
and important protein backbone atoms are color-coded as follows:
carbon, black; nitrogen, blue; oxygen,
red; and sulfur, yellow. Hydrophobic residues are
shown green. Water molecules are shown as cyan.
Hydrogen bonds are shown as dotted red lines. The native,
unbound conformations of the active site residues are shown in
purple. TLM forms hydrogen bonds with the two active site
histidines, His-298 and His-333, and to a network of waters which is
held in place by the carbonyl oxygen of Val-270 and by the amine group
of Gly-305. TLM is further stabilized by the intercalation of its
isoprenoid tail into the space between Pro-272 and its associated
peptide bond and the peptide bond between Gly-391 and Phe-392.
B, electron density of bound TLM. The electron density is
contoured at the one
level.
-2
nitrogen and the carbonyl oxygen of residue 390 fixes the orientation
of the histidine. When the histidine moves, the loop also moves to
maintain this important interaction.
-2s of the active site histidines 298 and 333 and is in the same position as the O-1 of TLM. O-3 is
hydrogen-bonded to the amide nitrogens of residues 163 and 392. The O-1
and N-1 do not form hydrogen bonds but likely interact with the
electrons of the adjacent Phe-292 side chain. Finally, the extended
acyl chain of cerulenin is located within a deep hydrophobic pocket at
the dimer interface comprising Gly-107, Pro-110, Val-134' (prime refers
to the other monomer), alanines 137' and 162, methionines 138' and 197, Phe-201, and Leu-335. No electron density was observed for C-12,
indicating that the end of the cerulenin chain may be flexible.
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Fig. 5.
The structure of the FabB-cerulenin
covalent complex. A, stereo diagram of the complex.
Cerulenin is shown with yellow bonds. The rest of the
coloring scheme in this figure is the same as in Fig. 3. Cerulenin
forms a covalent bond with the active site cysteine (Cys-163). Its O-2
forms hydrogen bonds with the two active site histidines, His-298 and
His-333. The O-3 sits in the oxyanion hole, forming hydrogen bonds to
the amide of Phe-392 and the amide of Cys-163. The tail of cerulenin
occupies a long hydrophobic cavity, which normally contains the growing
acyl chain of the natural substrate. B, electron density of
bound cerulenin. The electron density is contoured at the one level.
Statistics for data collection and refinement of the FabB-cerulenin
binary complex
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[in a new window]
Fig. 6.
FabB[H333N] is resistant to TLM.
A, the specific activities of FabB and FabB[H333N]. The
specific activity of FabB ( ) was 425 ± 6 pmol/min/µg,
whereas FabB[H333N] (
) had 1.6% of the condensation activity of
the wild-type protein (6.6 ± 0.4 pmol/min/µg). B, a
comparison of the inhibition of FabB[H333N] (
) and FabB (
) by
TLM. The IC50 for FabB was 25 µM compared
with the higher IC50 of FabB[H333N] (350 µM). C, a comparison of the inhibition of
FabB[H333N] (
) and FabB (
) by cerulenin. The IC50
for FabB was 3 µM compared with the higher
IC50 of FabB[H333N] (30 µM). The assays
were performed using 14:0-ACP as the primer for the radiochemical assay
described under "Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FabH. The interactions that account for the
slight difference in TLM binding to FabF and FabB is not clear. In
contrast, there are two reasons why TLM should be a poor inhibitor of
FabH. First, histidines 298 and 333 in FabB are replaced by His-244 and
Asn-274 in FabH, and our FabB-TLM structure reveals that the two
histidines form strong hydrogen bonds with the antibiotic (Fig. 4). To
test the importance of this interaction, we converted the FabB active
site into a FabH active site by constructing the FabB[H333N] mutant,
and we showed that the absence of the histidine dyad decreases the
sensitivity to TLM (Figs. 3 and 6). However, the exact mechanism for
the lower TLM affinity is not entirely clear since the asparagine is
capable of promoting the condensation reaction and could presumably
donate a hydrogen bond to O-1 of TLM. Second, the peptide bond
"sandwich" that binds the isoprenoid group may not be able to form
in FabH. Specifically, there is no equivalent in FabH to the loop
containing Pro-272 in FabB, and the sandwich cannot form.
FabH. One reason cerulenin is a poor inhibitor of FabH is the
fact that FabH lacks the substrate hydrophobic pocket to accommodate
the acyl chain of the drug. This pocket is not required in E. coli FabH where the substrate acyl group is simply the initiating acetyl moiety. However, long chain acyl groups are accommodated by
Mycobacterium tuberculosis FabH, and this enzyme is still
resistant to cerulenin (16). This supports our finding that the His-His active site, as opposed to the FabH His-Asn active site configuration, is crucial for optimal cerulenin inhibition (Fig. 6). The reason why
FabB is more susceptible than FabF is understood by comparing the
FabB-cerulenin complex (Fig. 5) with the FabF-cerulenin complex (54).
In the FabB-cerulenin complex, Gly-107 and Met-197 face each other in
the substrate hydrophobic pocket and direct the cerulenin acyl chain
toward strand
4 (to the back in Fig. 5). However, in the
FabF-cerulenin complex, the steric configuration is reversed, and
Ile-108 and Gly-198 direct the cerulenin tail away from strand
4 (to
the front in Fig. 5). Furthermore, Ile-108 must swing around to
accommodate the acyl chain in FabF, but Met-197 does not move in FabB.
This required movement in FabF probably explains why cerulenin is a
better inhibitor of FabB than of FabF. It is also possible that this
structural difference between FabF and FabB relates to the differences
in their substrate specificities and physiological functions. When the
cerulenin structures of FabB and FabF are superimposed, they are
identical except for the acyl chain that adopts these different
positions. FabB catalyzes reactions in the elongation of short chain
unsaturated fatty acid intermediates, and its active site must
accommodate acyl chains with the characteristic kink imposed by the
cis double bond. In contrast, FabF does not accept these
intermediates. Thus, these cerulenin-bound structures provide a
framework for modifying the substrate binding pocket by site-directed
mutagenesis to define the important differences in the elongation
condensing enzymes that define their physiological functions (57).
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Fig. 7.
Overlay of TLM and cerulenin in the
FabB active site. The FabB-TLM and FabB-cerulenin structures were
superimposed to illustrate the differences in the binding of the
antibiotics in the active site. The coloring scheme in this figure is
the same as in Figs. 3 and 4. TLM binds on the malonyl-ACP side and
cerulenin occupies the acyl-enzyme intermediate half. The O-1 of TLM
and O-2 of cerulenin are the only portions of the antibiotics that
overlap in the structure, and they form hydrogen bonds with the His-His
dyad in the active site. Note that the protein structure shown is that
of the FabB-TLM complex. Binding of the two antibiotics results in
essentially identical changes in the conformations of the active site
residues.
Cerulenin mimics the condensation transition state (Fig.
8, lower panel). The C-2 forms
a covalent bond with the active site cysteine; the O-3 mimics the
substrate oxygen in the oxyanion hole formed by the amide nitrogens of
residues 163 and 392, and the O-2 represents the carbonyl oxygen of the
incoming malonyl group. The acyl chain mimics the location of the
acyl-enzyme intermediate, and its location identifies the hydrophobic
substrate-binding pocket.
|
In contrast, TLM mimics the noncovalently bound thiomalonate that
enters the active site after the formation of the acyl-enzyme intermediate to participate in the decarboxylation/condensation half-step of the reaction (Fig. 8, upper panel). During the
decarboxylation reaction, we proposed (20) that the thiomalonate is
oriented such that the thioester carbonyl oxygen is hydrogen-bonded to the two acceptors (histidines 298 and 333 in the case of FabB), and the
terminal carboxyl group is adjacent to a conserved phenylalanine (Phe-229 in the case of FabB). This promotes the movement of electrons away from the carboxyl group during decarboxylation and the formation of a carbanion at C-2 of malonate. This scheme requires that the thiomalonate be bent within the active site, and the TLM thiolactone ring mimics this bent structure. Thus, we propose that the ring sulfur,
the O-1, and the C-9 of TLM corresponds to the positions of the thiol,
the carbonyl oxygen, and the carboxylate carbon of the malonyl-ACP
substrate, respectively. The isoprenoid TLM side chain nestles into a
hydrophobic side pocket in the active site tunnel and does not
represent a structural feature of the malonyl-ACP substrate. The
function, if any, of the hydrophobic pocket and stacking
interaction exploited by TLM for high affinity binding in the
condensing enzyme reaction cycle remains enigmatic.
![]() |
ACKNOWLEDGEMENT |
---|
We thank C. Hornsby for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM34496 (to C. O. R.) and GM44973 (to S. W. W.), Cancer Center (CORE) Support Grant CA 21765, and the American Lebanese Syrian Associated Charities.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1FJ4 and 1FJ8) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed: Dept. of
Biochemistry, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. Tel.: 901-495-3491; Fax:
901-525-8025; E-mail: charles.rock@stjude.org.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M007101200
2 K.-C. Choi and C. O. Rock, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ACP, acyl carrier
protein;
FabB, -ketoacyl-ACP synthase I;
FabF,
-ketoacyl-ACP
synthase II;
FabH,
-ketoacyl-ACP synthase III;
TLM, thiolactomycin,
[(4S)(2E,5E)]-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene-4-thiolide;
cerulenin, (2R,3S)-2,3-epoxy-4-oxo-7,10-dodecandienolyamide.
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