THE JOURNAL OF BIOLOGICAL CHEMISTRY
11110 –11114, 1999
© 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanism of Triclosan Inhibition of Bacterial Fatty Acid
Synthesis*
(Received for publication, December 9, 1998, and in revised form, January 13, 1999)
Richard J. Heath‡, J. Ronald Rubin§, Debra R. Holland§, Erli Zhang§, Mark. E. Snow§, and
Charles O. Rock‡¶i
From the ‡Department of Biochemistry, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105,
the §Department of Biomolecular Structure and Drug Design, Parke-Davis Pharmaceutical Research,
Ann Arbor, Michigan 48105, and the ¶Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163
Triclosan is a broad-spectrum antibacterial agent that
inhibits bacterial fatty acid synthesis at the enoyl-acyl
carrier protein reductase (FabI) step. Resistance to tri-
closan in Escherichia coli is acquired through a mis-
sense mutation in the fabI gene that leads to the expres-
sion of FabI[G93V]. The specific activity and substrate
affinities of FabI[G93V] are similar to FabI. Two differ-
ent binding assays establish that triclosan dramatically
increases the affinity of FabI for NAD1. In contrast,
triclosan does not increase the binding of NAD1 to
FabI[G93V]. The x-ray crystal structure of the FabI-
NAD1-triclosan complex confirms that hydrogen bonds
and hydrophobic interactions between triclosan and
both the protein and the NAD1 cofactor contribute to
the formation of a stable ternary complex, with the drug
binding at the enoyl substrate site. These data show that
the formation of a noncovalent “bi-substrate” complex
accounts for the effectiveness of triclosan as a FabI in-
hibitor and illustrates that mutations in the FabI active
site that interfere with the formation of a stable FabI-
NAD1-triclosan ternary complex acquire resistance to
the drug.
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 reac-
tion 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 b-ketoacyl-ACP is reduced by an NADPH-depend-
ent b-ketoacyl-ACP reductase (FabG). Only a single enzyme is
responsible for this step (4). There are two b-hydroxyacyl-ACP
dehydrases (FabA and FabZ) capable of forming trans-2-enoyl-
ACP. The product of the fabA gene is specifically involved in
* This work was supported by National Institutes of Health Grant
GM 34496, Cancer Center (CORE) Support Grant CA 21765, and the
American Lebanese Syrian Associated Charities. The costs of publica-
tion of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
i To whom correspondence should be addressed: Dept. of Biochemis-
try, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Mem-
phis, TN 38105-2794. Tel.: 901-495-3491; Fax: 901-525-8025; E-mail:
charles.rock@stjude.org.
1
The abbreviations used are: ACP, acyl carrier protein; FabI, enoyl-
acyl carrier protein reductase; triclosan, 2,4,49-trichloro-29-hydroxydi-
phenyl ether; IC50, concentration giving 50% inhibition of activity;
4:1-NAC, crotonyl-N-acetylcysteamine; 8:1-NAC, trans-2-octadecenoyl-
N-acetylcysteamine.
11110
This is an Open Access article under the CC BY license.
Vol. 274, No. 16, Issue of April 16, pp.
Printed in U.S.A.
the introduction of a cis double bond into the growing acyl
chain at the b-hydroxydecanoyl-ACP step and most efficiently
catalyzes dehydration of short-chain b-hydroxyacyl-ACPs,
whereas the FabZ dehydratase has a broader substrate speci-
ficity (5). The last reaction in each elongation cycle is catalyzed
by enoyl-ACP reductase (FabI). Contrary to the initial conclu-
sion that there were two enoyl-ACP reductases, based on as-
says 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 de-
velopment 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 com-
pounds that effectively inhibit fatty acid synthesis. Two natu-
ral products, cerulenin and thiolactomycin, are potent
antibiotics that function by specifically inhibiting the condens-
ing enzyme reactions (for reviews, see Refs. 3, 10). The diaza-
borines, 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 tubercu-
losis, the inhA gene, encodes a cellular target for isoniazid and
ethionamide. A point mutation in the inhA gene confers resist-
ance to the drugs (14). Both isoniazid and diazaborine bind at
the substrate site of the respective enoyl-ACP reductases and
covalently react with NAD1 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 bio-
cide 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-hydroxy-
diphenyl 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.
EXPERIMENTAL PROCEDURES
Materials—Sources of supplies were: American Radiochemicals, Inc.,
[adenine-2,8-3H]NAD (specific activity 25 Ci/mmol); Sigma, NADH and
NAD1; 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 purifica-
This paper is available on line at http://www.jbc.org
 FabI-NAD1 Triclosan Ternary Complex 11111
G93V
tion of His-tag FabI was described previously (7). The fabI 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 Ni21 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 spec-
trophotometrically 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 mM 8:1-NAC, 12 mg of homo-
geneous FabI (7, 8), 200 mM NADH, 0.1 M sodium phosphate, pH 7.5, in
a final volume of 300 ml. The reactions were performed at 24 °C in
semimicro quartz cuvettes. The change in optical density was continu-
ously 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 mmol/min/mg. Data points were the mean of duplicate assays,
and the individual values were within 6 5% of the average. FIG. 1. Triclosan inhibition of FabI and FabI[G93V]. The spec-
Analysis of [3H]NAD1 Binding to FabI—Two methods were used to trophotometric assay described under “Experimental Procedures” was
measure the binding of NAD1 to FabI in the presence and absence of used to determine the inhibition of the initial rate of FabI (E) or
triclosan. The first method employed gel filtration chromatography to FabI[G93V] (●) catalysis by triclosan using 8:1-NAC. The points are the
separate [3H]NAD1 bound to FabI from free [3H]NAD1. A 0.7 3 8-cm average of duplicate determinations from one experiment, and the
column (3 ml) of Sephadex G-25 was equilibrated in 0.1 M sodium experiment was replicated with the same results.
phosphate, pH 7.5, at 24 °C. Samples (50 ml) were applied, the column
eluted with the same buffer at a flow rate of approximately 0.25 ml/min, RESULTS
and 5-drop (110 ml) fractions were collected. The second method used Biochemical Characterization of the Triclosan-resistant Mu-
binding of FabI to a polyvinylidene difluoride membrane to separate
tant, FabI[G93V]—Previous work showed that FabI was inhib-
free from FabI-bound [3H]NAD1. Assays were placed into a Ultrafree-
Probind centrifugal filtration unit containing a 0.2-cm (2), 0.45-mm ited by triclosan (22) and that a mutation in the fabI gene that
polyvinylidene difluoride membrane (Millipore, Corp.). The filters were results in the expression of a FabI[G93V] protein exhibited
washed once with 100 ml of 0.1 M sodium phosphate and counted in 5 ml high level triclosan resistance (21, 22). FabI[G93V] was cloned
of ScintSafe scintillation solution. Both assays contained 0.1 M sodium into the pET15b expression vector and expressed and purified
phosphate, 1 mM [3H]NAD1 (specific activity 4 Ci/mmol), 2% Me2SO, the as described under “Experimental Procedures.” The apparent
indicated concentrations of triclosan, and 7.6 mg of FabI or FabI[G93V]
Km values for the two substrates were determined for both the
in a final volume of 50 ml. Samples were incubated at 24 °C for 15 min
and then separated using one of the two methods outlined above. wild-type and mutant protein. The wild-type protein had an
Structural Biology—Crystals of FabI were grown from hanging drops apparent Km for NADH of 15 6 1 mM, whereas the FabI[G93V]
(6 ml) containing 5 mg/ml FabI, 25 mM Tris, 150 mM NaCl, 0.5 mM exhibited an apparent Km for NADH of 8 6 2 mM. Thus, there
dithiothreitol, 5 mg/ml NADH, 50 mM sodium acetate, 4% polyethylene were not major differences between the kinetic constants for
glycol 4000 (w/v), pH 4.6. The drops were equilibrated against a reser- NADH in the wild-type and mutant protein. We also measured
voir (600 ml) containing 100 mM sodium acetate, pH 4.6, and 8% poly-
the apparent Km for the enoyl substrate using 4:1-NAC. This
ethylene glycol 4000 (w/v). Large hexagonal bipyrimidal crystals ap-
peared after equilibration for 1 day at 26 °C and reached dimensions of substrate analog was selected for these experiments because it
1.0 3 0.5 3 0.5 mm. had the higher solubility in the assay buffer than 8:1-NAC. The
Crystals of the FabI-NAD1-triclosan ternary complex were prepared apparent Km for 4:1-NAC was 25 6 5 mM for FabI compared
by soaking pregrown crystals of FabI in solutions containing 10 mg/ml with 15 6 2.2 mM for FabI[G93V]. The Vmax for FabI was 4.4
NAD1, 10 mg/ml triclosan, 100 mM sodium acetate, pH 4.6, and 10% mmol/min/mg, and for FabI[G93V], the Vmax was 3.6 mmol/min/
polyethylene glycol 4000 (w/v) for 7 days. X-ray diffraction data were
mg. These data illustrated that FabI and FabI[G93V] had
collected on the IMCA beamline (17-ID) at the Advanced Photon Source
at the Argonne National Laboratory. The crystals used for data collec- similar specific activities and substrate and cofactor binding
tion were first transferred to a cryoprotectant buffer containing 100 mM characteristics.
sodium acetate, pH 4.6, 10% polyethylene glycol 4000 (w/v), 25% glyc- Triclosan Inhibition of FabI and FabI[G93V]—The observa-
erol (v/v) for 1 h and then flash-frozen in a stream of liquid nitrogen. tion that cells expressing the FabI[G93V] protein have a min-
X-ray data to 1.8-Å resolution were collected at 100 K using synchrotron imum inhibitory concentration for triclosan that was 64-fold
X-radiation at a 1.0-Å wavelength and a Bruker 2 3 2 mosaic CCD x-ray
higher than the wild-type strain (0.5 compared with 32 mg/ml)
detector. The x-ray data were then processed using the SAINT (Bruker
x-ray, Madison, WI) program package. (22) suggested that the FabI[G93V] protein would be refractory
Crystals of the FabI-NAD1-triclosan complex were hexagonal, space to triclosan inhibition in vitro. We directly tested this assump-
group P6122 (number 178) with unit cell dimensions a 5 b 5 79.70 and tion using the spectrophotometric FabI assay and found that
c 5 327.7 Å and two molecules of the ternary complex in the asymmetric both FabI and FabI[G93V] were inhibited by triclosan (Fig. 1).
unit. These crystals were isomorphous to those of the FabI-NAD1- The IC50 for FabI (2 mM) was lower than the IC50 for FabI[G93V]
diazaborine complex reported by Baldock et al. (15). The FabI structure
(10 mM). This '5-fold increase in the IC50 for FabI[G93V] was
was initially refined as a rigid body using XPLOR, and the positions and
conformation of the bound NAD1 and triclosan molecules were deter- consistent with increased resistance of the enzyme to triclosan
mined from Fo-Fc difference electron density maps. In addition, the inhibition; however, the magnitude of this difference was sig-
positions of the 185 tightly bound water molecules were determined nificantly less than was predicted from the 64-fold difference in
from subsequent residual electron density maps. The entire structure the effectiveness of triclosan against the mutant strain.
containing 2 molecules of the FabI-NAD1-triclosan complex and 185 This unexpected discrepancy between the minimum inhibi-
water molecules was refined to a crystallographic R-factor of 0.232
tory concentration and IC50 data led us to examine the mech-
using data from 8.0 to 1.8 Å resolution. Residues 194 –210 were disor-
dered in the structure. Solvent-accessible surface areas were calculated anism of FabI inhibition by triclosan in more detail. The data in
using a 1.4-Å probe with the SOLVATION module of the InsightII Fig. 1 was obtained by starting the assay with the addition of
software (version 97.1, Molecular Simulations, Inc.). FabI and measuring the initial rate of NADH oxidation from
11112 FabI-NAD1 Triclosan Ternary Complex
FIG. 2. Time-dependent inhibition of FabI and FabI[G93V] by
triclosan. The spectrophotometric assay described under “Experimen-
tal 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
mM was used in panel A in the experiment with FabI, and a concentra-
tion of 10 mM was used in panel B for the experiment with FabI[G93V].
These traces are representative of three independent measurements.
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 be-
tween the inhibition of FabI and FabI[G93V] by triclosan (Fig.
2). Although there was an initial burst of activity in the pres-
ence 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.
Triclosan and NAD1 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 pre-
diction was experimentally tested by measuring the association
of [3H]NAD1 with either FabI or FabI[G93V] in the presence of
triclosan by gel filtration chromatography (Fig. 3). FabI bound
approximately 80% of the [3H]NAD1 (0.2 mol/mol of FabI sub-
unit) 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]NAD1 from the included to
the void volume of the column. The column was not equili-
brated 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]NAD1 to the two enzymes in the
absence of triclosan was barely detectable and equivalent to the
FIG. 3. Triclosan-induced binding of [3H]NAD1 to FabI and
FabI[G93V]. The ability of FabI (E) or FabI[G93V] (●) to bind
[3H]NAD1 in the presence of 8 mM triclosan was determined by gel
filtration chromatography as described under “Experimental Proce-
dures.” The FabI proteins were located in the excluded volume of the
Sephadex G-25 column (fractions 12–17), and the free [3H]NAD1 eluted
in the included volume (fractions 20 –35).
binding of [3H]NAD1 to FabI[G93V] in the presence of triclosan
shown in Fig. 3 (not shown, see below). These data demon-
strated that triclosan induced the high affinity binding of
NAD1 to FabI but not to FabI[G93V].
This concept was experimentally verified using a filter bind-
ing assay developed to quantitate the binding of [3H]NAD1 to
FabI in the presence and absence of triclosan. FabI exhibited
saturable NAD1 binding in the presence of 4 mM triclosan (Fig.
4A). An apparent association constant (K) of 2.5 mM–1 for NAD1
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]NAD1 to FabI in the absence of triclosan (Fig. 4A). The
affinity of FabI for NAD1 was so low that we measured only
trace levels of [3H]NAD1 binding to the free enzyme by this
technique. Therefore, we examined the effectiveness of NAD1
as a product inhibitor of FabI using the spectrophototmetric
assay. NAD1 was a poor inhibitor of the FabI reaction, with
50% inhibition occurring between 10 and 20 mM NAD1. These
data indicated a very low apparent affinity of FabI for NAD1 in
the 10 –50 mM range (not shown). The filter binding assay was
next used to examine the effect of triclosan concentration on
NAD1 binding to FabI (Fig. 5). The association of [3H]NAD1
with FabI was dependent on the concentration of triclosan and
saturable (Fig. 5A). The apparent K for triclosan binding was
calculated as 4.2 mM–1 (Fig. 5B). Thus, in the presence of tri-
closan, the affinity of FabI for NAD1 increased by approxi-
mately 4 orders of magnitude (.10 mM to 2.5 mM–1).
Structure of the FabI-NAD1-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-NAD1-triclosan ternary complex. Determin-
ing the structure of the FabI-NAD1-triclosan ternary complex
by x-ray crystallography directly tested this idea (see “Experi-
mental Procedures”). Triclosan bound noncovalently in a loca-
tion adjacent to the nicotinamide portion of NAD1 (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 NAD1. 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
 FabI-NAD1 Triclosan Ternary Complex
FIG. 4. Triclosan increases the affinity of FabI for NAD1. Panel
A, binding of [3H]NAD1 to FabI in the presence (●) and absence (E) of
4 mM triclosan. Panel B, double-reciprocal plot of the data in panel A.
The apparent NAD1 association constant (K) was 2.5 mM. The filter
binding assay was performed as described under “Experimental
Procedures.”
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 29-hydroxyl group of tri-
closan was involved in two strong hydrogen bonds. One bond
was with the 29-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-NAD1-
triclosan complex accounted for the effective inhibition of the
FabI reaction by this drug.
DISCUSSION
The formation of a ternary FabI-NAD1-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 NAD1 cofactor (Fig. 6A). There is a strong
similarity between the mode of triclosan NAD1 binding to FabI
(Fig. 6A) and the structure of the FabI-NAD1-diazaborine com-
plex described by Baldock et al. (15) (Fig. 6B). The diazaborine
compounds form covalent adducts with NAD1 at the FabI
active site. The boron atom of the diazaborine is attached to the
29-hydroxyl of the NAD1 ribose, and this is the same 29-hy-
droxyl group that forms a hydrogen bond with the hydroxychlo-
rophenyl hydroxyl of triclosan. The FabI-NAD1-triclosan ter-
nary complexes can be superimposed with the diazaborine
complex with a root mean square deviation of 0.3 Å for the
observed a-carbons. Residues 194 –210 were not well defined in
the electron density map of the FabI-NAD1-triclosan complex.
11113
FIG. 5. Triclosan-dependent [3H]NAD1 binding to FabI. Panel
A, dependence of [3H]NAD1 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 mM. The filter bind-
ing assay was performed as described under “Experimental Procedures.”
These residues are also not observed in the native structure of
FabI, but the positions of these residues are evident in the
FabI-NAD1-diazaborine structure (15). If the loop seen in the
FabI-NAD1-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 ter-
nary 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 NAD1.
Missense mutations, like FabI[G93V], confer resistance by
preventing the formation of the FabI-NAD1-triclosan ternary
complex. Strains expressing either FabI[G93S] and
FabI[G93V] have significantly elevated minimum inhibitory
concentrations for triclosan (21, 22). Although FabI[G93V] ac-
tivity 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]-NAD1-
triclosan ternary complex based on the inability of FabI[G93V]
to bind [3H]NAD1 (Fig. 3). These biochemical results are inter-
preted 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). There-
fore, 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-NAD1-triclosan complex
11114 FabI-NAD1 Triclosan Ternary Complex
FIG. 6. Structure of FabI-NAD1-inhibitor ternary complexes.
Panel A, the active site region of the FabI-NAD1-triclosan ternary
complex. The hydroxychlorophenyl ring stacks with the nicotinamide
ring of the NAD1 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 29-hydroxyl
of the NAD1 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-NAD1 complex to FabI as
described by Baldock et al. (15). The bicyclic ring of the diazaborine
compound stacks with the NAD1 nicotinamide ring in the same manner
as the hydroxychlorophenyl ring of triclosan. The diazaborine com-
pounds form a covalent complex with NAD1, with the boron atom
covalently attached to the 29-hydroxyl of the NAD1 ribose.
reinforces the conclusion that FabI is a specific intracellular
target for triclosan. Triclosan permeabilizes the bacterial en-
velope (18 –20), and these findings have justified the wide-
spread 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. How-
ever, 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 temper-
ature-sensitive fabI mutant was initially designated envM be-
cause 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 func-
tion 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 tri-
closan (22) and the delineation of its mechanism of binding
(this study) provide definitive evidence that enoyl-ACP reduc-
tase 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 na-
ture 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-NAD1-drug complexes will
supplement the arsenal against a broad spectrum of bacteria.
Acknowledgments—We thank Amy Sullivan and Magdalena
Kaminska for technical assistance, Rocco Gogliotti and John Domagala
for the trans-2-acyl-NAC substrate analogs, Tom Mueller and Craig
Banotai for the protein used in the crystal structure studies, Steve
VanderRoestand for the fabI clone, and our colleagues for their help in
editing the manuscript.
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