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INTRODUCTION |
The fatty acid synthase system of Escherichia coli is
the paradigm for the type II or dissociated fatty acid synthase systems (for reviews, see Refs. 1-3). Distinct genes encode each of the individual enzymes, and the same basic chemical reaction is often catalyzed by multiple isozymes. There are four basic reactions that
constitute a single round of elongation. The first step is the
condensation of malonyl-ACP1
with either acetyl-CoA to initiate fatty acid synthesis (FabH) or with
the growing acyl chain to continue cycles of elongation (FabB or FabF).
The
-ketoacyl-ACP is reduced by an NADPH-dependent
-ketoacyl-ACP reductase (FabG). Only a single enzyme is responsible for this step (4). There are two
-hydroxyacyl-ACP dehydrases (FabA
and FabZ) capable of forming trans-2-enoyl-ACP. The product of the fabA gene is specifically involved in the
introduction of a cis double bond into the growing acyl
chain at the
-hydroxydecanoyl-ACP step and most efficiently
catalyzes dehydration of short-chain
-hydroxyacyl-ACPs, whereas the
FabZ dehydratase has a broader substrate specificity (5). The last
reaction in each elongation cycle is catalyzed by enoyl-ACP reductase
(FabI). Contrary to the initial conclusion that there were two
enoyl-ACP reductases, based on assays in crude extracts (6), E. coli cells possess only a single NADH-dependent
enoyl-ACP reductases encoded by the fabI gene that utilizes
all chain lengths (7).
The importance of fatty acid biosynthesis to cell growth and function
makes this pathway an attractive target for the development of
antibacterial agents. Two important control points in the cycle are the
condensing enzymes and the enoyl-ACP reductase (8, 9), and both
reactions are targeted by compounds that effectively inhibit fatty acid
synthesis. Two natural products, cerulenin and thiolactomycin, are
potent antibiotics that function by specifically inhibiting the
condensing enzyme reactions (for reviews, see Refs. 3, 10). The
diazaborines, a class of heterocyclic antibacterials, inhibit fatty
acid biosynthesis by blocking the FabI step (11). Resistance to the
diazaborines arises from a missense mutation in the fabI
gene that leads to the expression of a FabI[G93S] mutant protein (12,
13). Similarly, the fabI analog in Mycobacterium tuberculosis, the inhA gene, encodes a cellular target
for isoniazid and ethionamide. A point mutation in the inhA
gene confers resistance to the drugs (14). Both isoniazid and
diazaborine bind at the substrate site of the respective enoyl-ACP
reductases and covalently react with NAD+ to form tight
binding bi-substrate complexes (15, 16). Triclosan is a broad-spectrum
antibacterial agent that enjoys widespread applications in a multitude
of contemporary consumer products including, soaps, detergents,
toothpastes, skin care products, cutting boards, and mattress pads
(17). Triclosan is widely thought to be a nonspecific biocide that
attacks bacterial membranes (18-20), and if triclosan does not have a
discrete mechanism of action, the acquisition of cellular resistance is
unlikely. However, recent work reveals that resistant E. coli strains arise from missense mutations in the fabI
gene (21, 22) and that triclosan and other 2-hydroxydiphenyl ethers
directly inhibit fatty acid biosynthesis in vivo and FabI
catalysis in vitro (22). The goal of this study was to
investigate the mechanism by which triclosan inhibits FabI.
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EXPERIMENTAL PROCEDURES |
Materials--
Sources of supplies were: American
Radiochemicals, Inc., [adenine-2,8-3H]NAD (specific
activity 25 Ci/mmol); Sigma, NADH and NAD+; and Millipore
Corp., Ultrafree Probind centrifugal filtration units. The substrate
analogs, 4:1-NAC and 8:1-NAC, were the generous gift of Rocco Gogliotti
and John Domagala (Parke-Davis). Triclosan was the gift of KIC
Chemicals, Inc. (Armonk, NY). All other chemicals were of the best
grade available.
Purification of FabI and FabI[G93V]--
The expression and
purification of His-tag FabI was described previously (7). The
fabIG93V allele was amplified from the
chromosome of the triclosan-resistant strain RJH108 (22) and ligated
into the pET15b expression vector. DNA sequencing of the expression
construct confirmed the presence of the single point mutation. The
His-tagged FabI[G93V] was then expressed and purified to homogeneity
by Ni2+ affinity chromatography using the same methods as
described for the wild-type FabI (7). Protein was determined by the
method of Bradford (23).
Spectrophotometric Assay of FabI--
FabI activity was assayed
spectrophotometrically by monitoring the decrease in absorption at 340 nm using an adaptation of the spectrophotometric assay used by Bergler
et al. (24). Standard reactions contained 100 µM 8:1-NAC, 12 µg of homogeneous FabI (7, 8), 200 µM NADH, 0.1 M sodium phosphate, pH 7.5, in a
final volume of 300 µl. The reactions were performed at 24 °C in
semimicro quartz cuvettes. The change in optical density was continuously monitored for 1 min, and the reaction rate was calculated from the slope of the trace. Triclosan was added to the final concentrations indicated in the figure legend from serially diluted stock solutions in Me2SO. The Me2SO
concentration in all assays was maintained at 1.66%, which did not
significantly affect FabI activity. The FabI specific activity in the
absence of drugs under these experimental conditions was 0.36 µmol/min/mg. Data points were the mean of duplicate assays, and the
individual values were within ± 5% of the average.
Analysis of [3H]NAD+ Binding to
FabI--
Two methods were used to measure the binding of
NAD+ to FabI in the presence and absence of triclosan. The
first method employed gel filtration chromatography to separate
[3H]NAD+ bound to FabI from free
[3H]NAD+. A 0.7 × 8-cm column (3 ml) of
Sephadex G-25 was equilibrated in 0.1 M sodium phosphate,
pH 7.5, at 24 °C. Samples (50 µl) were applied, the column eluted
with the same buffer at a flow rate of approximately 0.25 ml/min, and
5-drop (110 µl) fractions were collected. The second method used
binding of FabI to a polyvinylidene difluoride membrane to separate
free from FabI-bound [3H]NAD+. Assays were
placed into a Ultrafree-Probind centrifugal filtration unit containing
a 0.2-cm (2), 0.45-µm polyvinylidene difluoride membrane (Millipore,
Corp.). The filters were washed once with 100 µl of 0.1 M
sodium phosphate and counted in 5 ml of ScintSafe scintillation
solution. Both assays contained 0.1 M sodium phosphate, 1 µM [3H]NAD+ (specific activity
4 Ci/mmol), 2% Me2SO, the indicated concentrations of
triclosan, and 7.6 µg of FabI or FabI[G93V] in a final volume of 50 µl. Samples were incubated at 24 °C for 15 min and then separated
using one of the two methods outlined above.
Structural Biology--
Crystals of FabI were grown from hanging
drops (6 µl) containing 5 mg/ml FabI, 25 mM Tris, 150 mM NaCl, 0.5 mM dithiothreitol, 5 mg/ml NADH,
50 mM sodium acetate, 4% polyethylene glycol 4000 (w/v),
pH 4.6. The drops were equilibrated against a reservoir (600 µl)
containing 100 mM sodium acetate, pH 4.6, and 8%
polyethylene glycol 4000 (w/v). Large hexagonal bipyrimidal crystals
appeared after equilibration for 1 day at 26 °C and reached
dimensions of 1.0 × 0.5 × 0.5 mm.
Crystals of the FabI-NAD+-triclosan ternary complex were
prepared by soaking pregrown crystals of FabI in solutions containing 10 mg/ml NAD+, 10 mg/ml triclosan, 100 mM
sodium acetate, pH 4.6, and 10% polyethylene glycol 4000 (w/v) for 7 days. X-ray diffraction data were collected on the IMCA beamline
(17-ID) at the Advanced Photon Source at the Argonne National
Laboratory. The crystals used for data collection were first
transferred to a cryoprotectant buffer containing 100 mM
sodium acetate, pH 4.6, 10% polyethylene glycol 4000 (w/v), 25%
glycerol (v/v) for 1 h and then flash-frozen in a stream of liquid
nitrogen. X-ray data to 1.8-Å resolution were collected at 100 K using
synchrotron X-radiation at a 1.0-Å wavelength and a Bruker 2 × 2 mosaic CCD x-ray detector. The x-ray data were then processed using the
SAINT (Bruker x-ray, Madison, WI) program package.
Crystals of the FabI-NAD+-triclosan complex were hexagonal,
space group P6122 (number 178) with unit cell dimensions
a = b = 79.70 and c = 327.7 Å and two molecules of the ternary complex in the asymmetric
unit. These crystals were isomorphous to those of the
FabI-NAD+-diazaborine complex reported by Baldock et
al. (15). The FabI structure was initially refined as a rigid body
using XPLOR, and the positions and conformation of the bound
NAD+ and triclosan molecules were determined from Fo-Fc
difference electron density maps. In addition, the positions of the 185 tightly bound water molecules were determined from subsequent residual electron density maps. The entire structure containing 2 molecules of
the FabI-NAD+-triclosan complex and 185 water molecules was
refined to a crystallographic R-factor of 0.232 using data from 8.0 to
1.8 Å resolution. Residues 194-210 were disordered in the structure.
Solvent-accessible surface areas were calculated using a 1.4-Å probe
with the SOLVATION module of the InsightII software (version 97.1, Molecular Simulations, Inc.).
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RESULTS |
Biochemical Characterization of the Triclosan-resistant Mutant,
FabI[G93V]--
Previous work showed that FabI was inhibited by
triclosan (22) and that a mutation in the fabI gene that
results in the expression of a FabI[G93V] protein exhibited high
level triclosan resistance (21, 22). FabI[G93V] was cloned into the
pET15b expression vector and expressed and purified as described under "Experimental Procedures." The apparent Km
values for the two substrates were determined for both the wild-type
and mutant protein. The wild-type protein had an apparent
Km for NADH of 15 ± 1 µM,
whereas the FabI[G93V] exhibited an apparent Km
for NADH of 8 ± 2 µM. Thus, there were not major
differences between the kinetic constants for NADH in the wild-type and
mutant protein. We also measured the apparent Km for
the enoyl substrate using 4:1-NAC. This substrate analog was selected
for these experiments because it had the higher solubility in the assay
buffer than 8:1-NAC. The apparent Km for 4:1-NAC was
25 ± 5 mM for FabI compared with 15 ± 2.2 mM for FabI[G93V]. The Vmax for
FabI was 4.4 µmol/min/mg, and for FabI[G93V], the Vmax was 3.6 µmol/min/mg. These data
illustrated that FabI and FabI[G93V] had similar specific activities
and substrate and cofactor binding characteristics.
Triclosan Inhibition of FabI and FabI[G93V]--
The observation
that cells expressing the FabI[G93V] protein have a minimum
inhibitory concentration for triclosan that was 64-fold higher than the
wild-type strain (0.5 compared with 32 µg/ml) (22) suggested that the
FabI[G93V] protein would be refractory to triclosan inhibition
in vitro. We directly tested this assumption using the
spectrophotometric FabI assay and found that both FabI and FabI[G93V]
were inhibited by triclosan (Fig. 1).
The IC50 for FabI (2 µM) was lower than
the IC50 for FabI[G93V] (10 µM). This
5-fold increase in the IC50 for FabI[G93V] was
consistent with increased resistance of the enzyme to triclosan
inhibition; however, the magnitude of this difference was significantly
less than was predicted from the 64-fold difference in the
effectiveness of triclosan against the mutant strain.

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Fig. 1.
Triclosan inhibition of FabI and
FabI[G93V]. The spectrophotometric assay described under
"Experimental Procedures" was used to determine the inhibition of
the initial rate of FabI ( ) or FabI[G93V] ( ) catalysis by
triclosan using 8:1-NAC. The points are the average of
duplicate determinations from one experiment, and the experiment was
replicated with the same results.
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This unexpected discrepancy between the minimum inhibitory
concentration and IC50 data led us to examine the mechanism
of FabI inhibition by triclosan in more detail. The data in Fig. 1 was
obtained by starting the assay with the addition of FabI and measuring
the initial rate of NADH oxidation from the linear portion of the trace
within the first 1 min of the reaction. Examination of the time course
of the reaction over a longer period of time (15 min) revealed a clear
difference between the inhibition of FabI and FabI[G93V] by triclosan
(Fig. 2). Although there was an initial
burst of activity in the presence of triclosan, within 3 min of the
start of the experiment, FabI catalysis essentially ceased (Fig.
2A). Thus, the inhibition of FabI by triclosan became irreversible as a function of time. In contrast, triclosan inhibition of FabI[G93V] was consistent throughout the 15-min time course, yielding a slower rate and no indication of increasing inhibition as a
function of time (Fig. 2B). These data suggested that the difference between the effectiveness of triclosan against wild-type strains compared with strain RJH108 (fabIG93V)
was the ability of triclosan to irreversibly inhibit FabI, but not
FabI[G93V], as a function of time.

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Fig. 2.
Time-dependent inhibition of FabI
and FabI[G93V] by triclosan. The spectrophotometric assay
described under "Experimental Procedures" was used to determine the
effect of triclosan on the extended time course of the reaction FabI
(panel A) or FabI[G93V] (panel B). The
lines in the figures represent the continuous monitoring of
the absorbance at 340 nm for 15 min. A triclosan concentration of 2 µM was used in panel A in the experiment with
FabI, and a concentration of 10 µM was used in
panel B for the experiment with FabI[G93V]. These
traces are representative of three independent
measurements.
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Triclosan and NAD+ Binding to FabI--
The data in
Figs. 1 and 2 suggested that the irreversible inhibition of FabI by
triclosan arose from the tight binding of the drug to the enzyme in
conjunction with another component in the assay. This prediction was
experimentally tested by measuring the association of
[3H]NAD+ with either FabI or FabI[G93V] in
the presence of triclosan by gel filtration chromatography (Fig.
3). FabI bound approximately 80% of the
[3H]NAD+ (0.2 mol/mol of FabI subunit) when
triclosan was present, as evidenced from the shift in radioactivity
from the included volume (fractions 18-40) to the excluded volume
(fractions 11-17) of the column. In contrast, FabI[G93V] did not
displace [3H]NAD+ from the included to the
void volume of the column. The column was not equilibrated with
triclosan; thus the appearance of label associated with FabI in the
excluded volume indicated a stable association with the enzyme during
the course of the separation (about 30 min). The binding of
[3H]NAD+ to the two enzymes in the absence of
triclosan was barely detectable and equivalent to the binding of
[3H]NAD+ to FabI[G93V] in the presence of
triclosan shown in Fig. 3 (not shown, see below). These data
demonstrated that triclosan induced the high affinity binding of
NAD+ to FabI but not to FabI[G93V].

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Fig. 3.
Triclosan-induced binding of
[3H]NAD+;1 to FabI and
FabI[G93V]. The ability of FabI ( ) or FabI[G93V] ( ) to
bind [3H]NAD+ in the presence of 8 µM triclosan was determined by gel filtration
chromatography as described under "Experimental Procedures." The
FabI proteins were located in the excluded volume of the Sephadex G-25
column (fractions 12-17), and the free
[3H]NAD+ eluted in the included volume
(fractions 20-35).
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This concept was experimentally verified using a filter binding assay
developed to quantitate the binding of
[3H]NAD+ to FabI in the presence and absence
of triclosan. FabI exhibited saturable NAD+ binding in the
presence of 4 µM triclosan (Fig.
4A). An apparent association
constant (K) of 2.5 µM-1 for
NAD+ in the presence of triclosan was calculated by
analysis of the data using a double-reciprocal plot (Fig.
4B). There was no indication of cooperative binding or the
existence of more than a single class of sites by analysis of the data
using a Scatchard plot (not shown). In contrast, we detected virtually
no binding of [3H]NAD+ to FabI in the absence
of triclosan (Fig. 4A). The affinity of FabI for
NAD+ was so low that we measured only trace levels of
[3H]NAD+ binding to the free enzyme by this
technique. Therefore, we examined the effectiveness of NAD+
as a product inhibitor of FabI using the spectrophototmetric assay.
NAD+ was a poor inhibitor of the FabI reaction, with 50%
inhibition occurring between 10 and 20 mM NAD+.
These data indicated a very low apparent affinity of FabI for NAD+ in the 10-50 mM range (not shown). The
filter binding assay was next used to examine the effect of triclosan
concentration on NAD+ binding to FabI (Fig.
5). The association of
[3H]NAD+ with FabI was dependent on the
concentration of triclosan and saturable (Fig. 5A). The
apparent K for triclosan binding was calculated as 4.2 µM-1 (Fig. 5B). Thus, in the
presence of triclosan, the affinity of FabI for NAD+
increased by approximately 4 orders of magnitude (>10 mM
to 2.5 µM-1).

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Fig. 4.
Triclosan increases the affinity of FabI for
NAD+;1. Panel A, binding of
[3H]NAD+ to FabI in the presence ( ) and
absence ( ) of 4 µM triclosan. Panel B,
double-reciprocal plot of the data in panel A. The apparent
NAD+ association constant (K) was 2.5 µM. The filter binding assay was performed as described
under "Experimental Procedures."
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Fig. 5.
Triclosan-dependent
[3H]NAD+;1 binding to FabI.
Panel A, dependence of [3H]NAD+
binding to FabI on the concentration of triclosan. Panel B,
double-reciprocal plot of the data in panel A. The apparent
triclosan association constant (K) was 4.2 µM.
The filter binding assay was performed as described under
"Experimental Procedures."
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Structure of the FabI-NAD+-triclosan Ternary
Complex--
The binding studies suggested that the basis for the
potency of triclosan as a FabI inhibitor was because of the formation of a high affinity FabI-NAD+-triclosan ternary complex.
Determining the structure of the FabI-NAD+-triclosan
ternary complex by x-ray crystallography directly tested this idea (see
"Experimental Procedures"). Triclosan bound noncovalently in a
location adjacent to the nicotinamide portion of NAD+ (Fig.
6A). There was 494.7 Å2 of surface area buried between triclosan and the
protein and 238.5 Å2 of surface area buried at the
interface with NAD+. The diphenyl ether of triclosan
adopted a conformation with a dihedral angle of about 90° between the
two phenyl rings of the inhibitor. The 2-hydoxy-3-chlorophenyl ring
formed a parallel stack with the nicotinamide ring of the co-factor
with an interplanar stacking distance of 3.4 Å. In addition to the
nicotinamide ring, the hydroxychlorophenyl ring was surrounded by
hydrophobic side chains on the protein including Tyr-146 and Tyr-156.
The 2'-hydroxyl group of triclosan was involved in two strong hydrogen
bonds. One bond was with the 2'-hydroxyl group of the nicotinamide
ribose, and the second was to the phenolic hydroxyl group of Tyr-156. The 2,4-dichlorophenyl ring of triclosan was rotated 90° of the plane
of the hydroxychlorophenyl group. The chlorine substituent in the
4-position of the phenyl ring accepted a hydrogen bond from the
backbone amide of Ala-95 and formed hydrophobic contacts with the side
chain of Met-159. These data supported the concept that the formation
of a stable, ternary FabI-NAD+-triclosan complex accounted
for the effective inhibition of the FabI reaction by this drug.

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Fig. 6.
Structure of
FabI-NAD+;1-inhibitor ternary complexes.
Panel A, the active site region of the
FabI-NAD+-triclosan ternary complex. The
hydroxychlorophenyl ring stacks with the nicotinamide ring of the
NAD+ with an interplanar distance of 3.4 Å and contacts
Tyr-146 and Tyr-156 on the protein. The hydroxyl group of the ligand
forms hydrogen bonds with phenol of Tyr-156 and with the 2'-hydroxyl of
the NAD+ ribose. The 2,4-dichlorophenyl ring of triclosan
sits in a hydrophobic pocket in contact with Met-159. The 4-chloro
substituent accepts a hydrogen bond from the amide backbone amide
nitrogen of Ala-95. Panel B, binding of the
diazaborine-NAD+ complex to FabI as described by Baldock
et al. (15). The bicyclic ring of the diazaborine compound
stacks with the NAD+ nicotinamide ring in the same manner
as the hydroxychlorophenyl ring of triclosan. The diazaborine compounds
form a covalent complex with NAD+, with the boron atom
covalently attached to the 2'-hydroxyl of the NAD+
ribose.
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DISCUSSION |
The formation of a ternary FabI-NAD+-triclosan complex
accounts for the effectiveness of triclosan as an antibacterial agent. Triclosan binds to the enoyl substrate site on FabI, and tight binding
of the drug requires interactions between both the protein and the
NAD+ cofactor (Fig. 6A). There is a strong
similarity between the mode of triclosan NAD+ binding to
FabI (Fig. 6A) and the structure of the
FabI-NAD+-diazaborine complex described by Baldock et
al. (15) (Fig. 6B). The diazaborine compounds form
covalent adducts with NAD+ at the FabI active site. The
boron atom of the diazaborine is attached to the 2'-hydroxyl of the
NAD+ ribose, and this is the same 2'-hydroxyl group that
forms a hydrogen bond with the hydroxychlorophenyl hydroxyl of
triclosan. The FabI-NAD+-triclosan ternary complexes can be
superimposed with the diazaborine complex with a root mean square
deviation of 0.3 Å for the observed
-carbons. Residues 194-210
were not well defined in the electron density map of the
FabI-NAD+-triclosan complex. These residues are also not
observed in the native structure of FabI, but the positions of these
residues are evident in the FabI-NAD+-diazaborine structure
(15). If the loop seen in the FabI-NAD+-diazaborine complex
is modeled onto the triclosan complex structure, there are no
conflicting contacts between triclosan and the protein. In this model
Ile-200 is in van der Waals contact with the 2,4-dichlorophenyl ring of
triclosan, and Phe-203 is in contact with the hydroxychlorophenyl group. These data support the conclusion that triclosan forms a ternary
complex that acts as a dead-end inhibitor that effectively removes
protein from the catalytic cycle. These results lead to the general
conclusion that all 2-hydroxydiphenyl ether FabI inhibitors (22) block
enzyme activity through the formation of noncovalent bisubstrate
complexes with NAD+.
Missense mutations, like FabI[G93V], confer resistance by preventing
the formation of the FabI-NAD+-triclosan ternary complex.
Strains expressing either FabI[G93S] and FabI[G93V] have
significantly elevated minimum inhibitory concentrations for triclosan
(21, 22). Although FabI[G93V] activity is still inhibited at high
triclosan concentrations (Fig. 2B), the drug is not capable
of forming a stable complex with the reductase. Triclosan is neither a
dead-end inhibitor of FabI[G93V] (Fig. 2) nor does it form a
FabI[G93V]-NAD+-triclosan ternary complex based on the
inability of FabI[G93V] to bind [3H]NAD+
(Fig. 3). These biochemical results are interpreted by modeling the
Val-93 into the Gly-93 position in the structure of the triclosan complex. Gly-93 lies on one side of the substrate binding pocket, and
the branched hydrophobic side chain of Val protrudes into the pocket
and occupies the same space as the dichlorophenyl ring of triclosan
(see Fig. 6). Therefore, we conclude that the substitution of Gly-93
with bulkier amino acid residues imparts resistance to
2-hydroxydiphenyl ethers because of steric interference.
Our identification of a ternary FabI-NAD+-triclosan complex
reinforces the conclusion that FabI is a specific intracellular target
for triclosan. Triclosan permeabilizes the bacterial envelope (18-20),
and these findings have justified the widespread and increasing use of
triclosan in consumer personal care products based on the reasoning
that bacteria would not acquire resistance to a nonspecific membrane
disrupter. However, this type of evidence is not sufficient to conclude
that a compound has nonspecific membrane effects, because agents that
block fatty acid biosynthesis perturb membrane assembly by stopping
phospholipid production. For example, the temperature-sensitive
fabI mutant was initially designated envM because
it was isolated through a genetic selection for strains with
envelope-permeability defects (25). Also, the diazaborine class of FabI
inhibitors are known to perturb membrane function as well (12). The
first evidence that triclosan has a specific cellular target was
provided by McMurray et al. (21) who found that
triclosan-resistant strains have missense mutations in the fabI gene. The biochemical analysis of FabI inhibition by
triclosan (22) and the delineation of its mechanism of binding (this
study) provide definitive evidence that enoyl-ACP reductase is the
primary site for triclosan action. Thus, the reported effects of
triclosan on membrane structure and function are a consequence of its
specific inhibition of fatty acid biosynthesis at the FabI step.
The ability of E. coli to acquire genetic resistance to
triclosan and related compounds through missense mutations in the fabI gene suggests that the widespread use of this drug will
lead to the appearance of resistant organisms that will compromise the
usefulness of triclosan. The ubiquitous occurrence of type II fatty
acid synthase systems in bacteria and the essential nature of the FabI
reaction make this enzyme an attractive target for antibacterial drugs.
Accordingly, triclosan is effective against a broad spectrum of
bacteria (17), including multi-drug-resistant Staphylococcus
aureus (26, 27). The design and development of second generation
FabI inhibitors based on their ability to form ternary
FabI-NAD+-drug complexes will supplement the arsenal
against a broad spectrum of bacteria.