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This article cites 55 references, 20 of which can be accessed free at
http://www.jbc.org/content/279/30/30994.full.html#ref-list-1
Vol. 279, No. 30, Issue of July 23, pp. 30994 31001, 2004
Printed in U.S.A.
30994
Epigallocatechin gallate (EGCG) is the major component of green tea extracts and possesses antibacterial,
antiviral, and antitumor activity. Our study focused on
validating the inhibition of the bacterial type II fatty
acid synthesis system as a mechanism for the antibacterial effects of EGCG and related plant polyphenols.
EGCG and the related tea catechins potently inhibited
both the FabG and FabI reductase steps in the fatty acid
elongation cycle with IC50 values between 5 and 15 M.
The presence of the galloyl moiety was essential for
activity, and EGCG was a competitive inhibitor of FabI
and a mixed type inhibitor of FabG demonstrating that
EGCG interfered with cofactor binding in both enzymes.
EGCG inhibited acetate incorporation into fatty acids in
vivo, although it was much less potent than thiolactomycin, a validated fatty acid synthesis inhibitor, and overexpression of FabG, FabI, or both did not confer resistance. A panel of other plant polyphenols was screened
for FabG/FabI inhibition and antibacterial activity.
Most of these inhibited both reductase steps, possessed
antibacterial activity, and inhibited cellular fatty acid
synthesis. The ability of the plant secondary metabolites
to interfere with the activity of multiple NAD(P)dependent cellular processes must be taken into account when assessing the specificity of their effects.
30995
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fatty-acid synthase. Furthermore, the structure-activity relationship shows that the portion of the EGCG molecule required
for inhibition is defined by the boxed area in Scheme 1.
Mechanism for Tea Catechin Inhibition of FabG and FabI
The kinetic mechanism for the inhibition of FabG and FabI was
determined using EGCG as the model compound (Fig. 2). Both
FabG (38) and FabI (39) have compulsory ordered mechanisms
with the nucleotide cofactors as the leading substrates. This
knowledge allowed us to design a kinetic analysis that would
distinguish between the three possible outcomes; EGCG could
bind to the free enzyme, the enzyme-substrate complex, or both
to prevent catalysis. In the first case, the inhibition pattern
with respect to the cofactor would be competitive; in the second,
the inhibition pattern would be non-competitive; and in the
third case, mixed-type inhibition would be observed (40). The
inhibition of FabG by EGCG was mixed with respect to NADPH
(Fig. 2A). Thus, EGCG binds to both the free enzyme to prevent
the binding of the nucleotide cofactor and also to the FabG-
30997
TABLE I
Inhibitory effects of green extract compounds on the activity of fatty acid synthetic enzymes (FabG and FabI) and bacterial growth
IC50 and MIC values greater than 100 and 200 M, respectively, were not determined. The IC50 values were rounded to the nearest 5 M.
30998
DISCUSSION
The emergence of multidrug resistance in pathogenic bacteria is a global problem that calls for the development of new
antibiotics with unique cellular targets (44). Consequently, the
differences between bacterial and mammalian fatty acid biosynthesis are being exploited to develop the type II fatty-acid
synthase as a target for novel drug discovery (21, 45, 46). FabG
is ubiquitously expressed in all bacteria, is highly conserved
across species, and is the only known isozyme that catalyzes
the essential keto reduction step in the elongation cycle (47).
Although there are no known FabG inhibitors (21, 45, 46),
FabG represents an ideal focus for the development of new
antibiotics based on the hypothesis that FabG would be vulnerable to inhibitors that interact with its cofactor binding site
(48). Our study identifies a small molecule scaffold with potent
inhibitory effects against FabG as well as FabI, an established
target in bacterial fatty acid synthesis. Green tea catechins
(EGCG) and related plant polyphenolic compounds are selec-
30999
TABLE II
Inhibitory effects of polyphenol compounds on the activity of fatty acid synthetic enzymes (FabG and FabI) and bacterial growth
b
c
IC50 and MIC values greater than 100 and 200 M, respectively, were not determined. The IC50 values were rounded to the nearest 5 M.
The amount of [14C] acetate incorporated into the cells treated with Me2SO was 100%. The compounds were tested at 4 their MICs.
NT, not tested.
tive inhibitors of the FabG and FabI reductase steps of bacterial type II fatty acid synthesis. The galloyl moiety of the
catechins is absolutely essential for inhibition. Two hydroxyphenyl rings connected by a 23-carbon linker is the basic
requirement for inhibition as illustrated in Scheme 1. Aspects
of this core structure are also found in the hydroxydiphenyl
ether class of potent FabI inhibitors (27), except in this case the
two hydroxyphenyl rings are connected by a single bridging
oxygen. The kinetic analysis indicates that EGCG binds to the
ligand-free form of both FabG and FabI and blocks NAD(P)H
binding. This result does not necessarily mean that EGCG
occupies the same pocket as NAD(P)H, so to advance the design
of inhibitors based on this scaffold it is important to determine
the structure of the EGCG-enzyme binary complex. This structure is particularly significant in light of the high degree of
conformational flexibility in FabG (38). The tight binding of
triclosan to FabI is caused by its ability to lock the reductase in
a closed confirmation (31); identifying a slow binding polyphenol derivative with a high affinity interaction and a specific
FabG conformation has the most promise as an antibacterial
lead.
The green tea catechins also inhibit the reductase and condensation reactions of the polyfunctional mammalian type I
fatty-acid synthase (1315). Thus, the same enzymatic steps
31000
FIG. 4. Biochemical and biological effects of 2,2,4-trihydroxychalcone. A, 2,2,4-trihydroxychalcone inhibited E. coli FabB
(E), FabG (), and FabI () activity with apparent IC50 values of 100,
25, and 40 M, respectively. FabH (f) was refractory to this compound.
B, 2,2,4-trihydroxychalcone inhibited E. coli growth with an MIC of
6.25 M. Overexpression of fabG () or fabI () did not make the cells
more resistant to the drug compared with control strain ANS1 (E). C,
[14C]-acetate incorporation was inhibited when cells were treated with
increasing concentrations of 2,2,4-trihydroxychalcone. TLM (a known
fatty acid biosynthesis inhibitor) and chloramphenicol (a known protein
synthesis inhibitor) were used as positive and negative controls. The
assays were performed using the conditions described under Experimental Procedures. Error bars show standard deviations.
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