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J. Biol. Chem., Vol. 275, Issue 51, 40128-40133, December 22, 2000
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,¶
From the Department of Biochemistry, St. Jude
Children's Research Hospital, and the ¶ Department of
Biochemistry, University of Tennessee, Memphis, Tennessee 38105-2794 and § Millennium Pharmaceuticals, Incorporated,
Cambridge, Massachusetts 02139
Received for publication, June 27, 2000, and in revised form, September 22, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Enoyl-[acyl-carrier-protein] (ACP)
reductase is a key enzyme in type II fatty-acid synthases that
catalyzes the last step in each elongation cycle. The FabI component of
Bacillus subtilis (bsFabI) was identified in the genomic
data base by homology to the Escherichia coli protein.
bsFabI was cloned and purified and exhibited properties similar to
those of E. coli FabI, including a marked preference for
NADH over NADPH as a cofactor. Overexpression of the B. subtilis
fabI gene complemented the temperature-sensitive growth phenotype
of an E. coli fabI mutant. Triclosan was a slow-binding inhibitor of bsFabI and formed a stable
bsFabI·NAD+·triclosan ternary complex. Analysis of
the B. subtilis genomic data base revealed a second open
reading frame (ygaA) that was predicted to encode a protein
with a relatively low overall similarity to FabI, but contained the
Tyr-Xaa6-Lys enoyl-ACP reductase catalytic architecture.
The purified YgaA protein catalyzed the
NADPH-dependent reduction of
trans-2-enoyl thioesters of both
N-acetylcysteamine and ACP. YgaA was reversibly inhibited
by triclosan, but did not form the stable ternary complex
characteristic of the FabI proteins. Expression of YgaA complemented
the fabI(ts) defect in E. coli and
conferred complete triclosan resistance. Single knockouts of the
ygaA or fabI gene in B. subtilis
were viable, but double knockouts were not obtained. The
fabI knockout was as sensitive as the wild-type strain to
triclosan, whereas the ygaA knockout was 250-fold more
sensitive to the drug. YgaA was renamed FabL to denote the discovery of
a new family of proteins that carry out the enoyl-ACP reductase step in
type II fatty-acid synthases.
Fatty acid synthesis in bacteria is carried out by a collection of
individual enzymes that are known as the type II, or dissociated, fatty-acid synthases (for reviews, see Refs. 1 and 2). The trans-2-enoyl-[acyl-carrier-protein]
(ACP)1 reductase component of
the type II system catalyzes the last step in each elongation cycle and
plays a key role in the regulation of the pathway (3, 4). In
Escherichia coli, there is a single, NADH-dependent isoform of this enzyme that catalyzes all of
the steps in the pathway and that is essential to cell survival (3, 5,
6). FabI is a distant member of an extended superfamily of proteins
termed the short-chain alcohol reductases/dehydrogenases (7). Members
of this family are small (~30 kDa) proteins with ~20-30% overall
identity and utilize nicotinamide cofactors (7, 8). Most short-chain
alcohol reductases/dehydrogenases contain a conserved catalytic triad
of Ser-Xaa10-Tyr-Xaa4-Lys (9). The Lys hydrogen
bonds to the 2'-hydroxyl of the cofactor, whereas the Tyr and/or the
Ser acts as a proton donor (8). FabI is an atypical short-chain alcohol
reductase/dehydrogenase in that the key residues are a diad consisting
of the motif Tyr-Xaa6-Lys (7, 10). Genes encoding homologs
of FabI are easily identified in most of the available bacterial
genomic data bases based on a high degree of overall identity and the
conserved spacing in the Tyr-Lys diad.
The FabI protein is the target of the broad-spectrum antibacterial
agent triclosan (11-13). Triclosan acts as a product mimic and
inhibits the enzyme by forming a tight, noncovalent complex with the
oxidized cofactor in the active site (13-16). The hydroxyl of
triclosan forms a strong hydrogen bond with the catalytic
Tyr156 and the 2'-hydroxyl of NAD+; the
hydroxychlorophenyl ring stacks with the nicotinamide ring of the
cofactor; and the 4'-chlorine accepts a hydrogen bond from the backbone
amide of Ala95. A key feature of the interaction is that
there is a conformational change in the protein that brings the
hydrophobic residues Ile200 and Phe203 in the
flexible substrate-binding loop close enough to the drug to form
additional van der Waals interactions, further stabilizing the complex.
Mutations in the active site of FabI that lead to triclosan resistance
sterically interfere with the ability of the drug to optimally interact
with the protein and cofactor (11, 13). The InhA protein of
Mycobacterium tuberculosis, which is 40% identical to FabI
and catalyzes the identical reaction, is the target of the
anti-tubercular drug isoniazid (17-19), which, when activated, forms a
covalent bisubstrate complex that tightly binds to InhA (19).
Recently, we characterized the NADPH-dependent FabI from
Staphylococcus aureus (20). This reductase is 48%
identical to the E. coli protein, functionally replaces FabI
in E. coli, and forms a high-affinity
FabI·NADP+·triclosan complex (20). The genome of
Bacillus subtilis also contains a gene with very high
similarity to the E. coli and S. aureus FabI
proteins. We cloned this open reading frame (yjbW, B. subtilis fabI) to determine if the NADPH cofactor preference was a common feature of Gram-positive enoyl-ACP reductases.
Furthermore, S. aureus and B. subtilis display a
10-fold difference in their susceptibilities to triclosan, and we
wished to establish if this difference in drug sensitivity was an
intrinsic property of the FabI proteins. We also discovered a new gene
that encodes an NADPH-dependent enoyl-ACP reductase. This
unique protein product is termed FabL, and its expression confers
triclosan resistance to type II fatty-acid synthases.
Materials--
trans-2-Enoyl-NAC derivatives were the
generous gift of Rocco Gogliotti and John Domagala (Parke-Davis
Pharmaceutical Research). Sigma supplied ACP, NADH, NAD+,
NADPH, NADP+, and crotonoyl-CoA. Triclosan
(2,4,4'-trichloro-2'-hydroxydiphenyl ether) was the gift of KIC
Chemicals (Aramonk, NJ). [2-14C]Malonyl-CoA (specific
activity of 55 mCi/mmol) was from Amersham Pharmacia Biotech. All other
reagents were of the highest available quality.
Cloning B. subtilis fabI and fabL--
The entire predicted open
reading frame (yjbW, GenBankTM/EBI Data Bank
accession number Z99110) for the B. subtilis fabI gene was amplified from genomic DNA using primers bs-xho
(5'-CTCGAGATGTCTTTATTAAATATAGGAGGC) and bs-bam
(5'-GGATCCTGCTTAGCGGGCAGTGATATGGAA) and cloned into the
XhoI and BamHI sites of the pET-15b vector
(Novagen). The resultant plasmid, pET-YjbW, was then digested with
NdeI and religated to remove the coding region for the first
11 residues of non-homologous sequences and to fuse the His tag to the
proposed initiator methionine (Met12 of accession number
Z99110). This plasmid, pETbsFabI, expresses the predicted bsFabI
protein with an NH2-terminal His tag. The ygaA
open reading frame was amplified from B. subtilis
chromosomal DNA with primers ygaa-nde
(5'-CCATATGGAACAAAATAAATGTGCACTCGTAAC) and ygaa-bam
(5'-GGATCCTTAAACGAGCAGTGAGCGTCCGCCGTC). The gene fragment was
cloned, using NdeI and BamHI, into pET-15b to
form pAN9. Plasmids pET-YjbW, pETbsFabI, and pAN9 were each transformed into E. coli strain BL21(DE3), and the proteins were
expressed and purified by Ni2+ chelation chromatography as
described previously (3, 21). The B. subtilis fabI and
ygaA His-tagged fusion open reading frames were subcloned as
XbaI-BamHI fragments into pBluescript KS II(+) (Stratagene), forming pAN1 and pAN10, respectively, for constitutive expression in E. coli. The genes were sequenced to ensure
that no artifacts had been introduced during the PCR or cloning steps.
Assays for Enoyl Reductase Activity--
Reduction of the
trans-2-enoyl-CoA and -NAC substrate analogs was measured
spectrophotometrically by following the utilization of NADH or NADPH at
340 nm in quartz semi-microcuvettes at 25 °C as described previously
(3, 11). Triclosan was added from stocks in Me2SO to the
concentrations indicated, and the Me2SO concentration was
kept constant at 1.6% throughout these assays. Oxidized cofactor
(NAD+ or NADP+, 100 µM) was added
to the preincubated assays as indicated.
The rate of reduction of the 4:1-ACP2 substrate was
measured in a reconstituted fatty acid synthesis assay (3) in which the
substrate was generated by an excess of purified E. coli
FabD, FabH, FabG, and FabA proteins (~1 µg each/reaction) with 100 µM acetyl-CoA, [2-14C]malonyl-CoA (50 µM; specific activity of 55 mCi/mmol), 100 µM ACP, 100 µM NADH, and 100 µM NADPH in 40 µl of 0.1 M sodium phosphate (pH 7.0). Enoyl reductase protein was added to initiate the reaction. The reaction was incubated at 37 °C for 15 min and then stopped by
placing in an ice slurry. Products were separated by conformationally sensitive gel electrophoresis on 13% polyacrylamide gels containing 0.5 M urea (3) and quantitated by a PhosphorImager
calibrated with a standard curve of
[2-14C]malonyl-CoA.
Bacterial Growth and Inhibition--
E. coli
cells were grown on LB medium, with ampicillin (100 µg/ml) where
appropriate, at 37 °C, except the fabI(ts) mutant strain
RJH13 (22), which was cultivated at 30 °C for normal growth or at
42 °C for the nonpermissive conditions. B. subtilis cells were grown on LB medium at 37 °C. MIC testing in E. coli was performed by spotting multiple individual colonies onto
plates containing triclosan. Experiments were repeated several times with similar results. MIC testing in B. subtilis was
performed by adding 5 µl of a dilution series of triclosan in 10%
Me2SO to the wells of a sterile 96-well microtiter plate.
Next, 95 µl of a cell suspension (5 × 104 cells/ml)
in LB medium was added to the plates, which were incubated for 48 h. The MIC was the most dilute concentration that resulted in no growth
of the strain of interest.
Gene Deletions--
Nonpolar, precise, in-frame deletions of
fabI (yjbW), yrpB (a
fabK-like gene), and ygaA (fabL) were
generated in B. subtilis PY79 (23) in a manner similar to
that described for Saccharomyces cerevisiae (24). Briefly,
two sets of oligonucleotide primers were designed to PCR-amplify ~1
kilobase of sequence immediately upstream and downstream of the target
gene (see Table I). PCR amplification was carried out using the Expand
High Fidelity PCR system (Roche Molecular Biochemicals) according to
the manufacturer's instructions. Two primers, B/C, anneal to just
outside of the 5'- and 3'-ends, respectively, of the target gene's
coding sequence and catalyze DNA polymerization away from the target
gene in PCR. The remaining primers in each set, A/D, anneal to ~1
kilobase upstream and downstream of primers B/C, respectively, and
allow polymerization toward the target gene. In initial PCRs with
strain PY79 genomic DNA as the template, products are formed using the A/B and C/D primer combinations. Because primers B/C also contain "tails" that anneal to a variety of antibiotic resistance cassettes (see below), the A/B and C/D PCR products can be used as primers in a secondary PCR with an antibiotic resistance cassette. The resulting second PCR product thus encodes sequence upstream of the
target gene, an antibiotic resistance gene in place of the target gene,
and sequence downstream of the target gene. These PCR products were
used to transform B. subtilis strain PY79 to antibiotic
resistance by standard techniques (25). Nonessential genes can be
replaced in this way with the antibiotic resistance marker by double
crossover recombination. Typical transformations yielded at least 20 antibiotic-resistant colonies. The genotypes of transformants were
verified by PCR of genomic DNA from candidate clones.
Antibiotic resistance cassettes were synthesized in PCRs using
universally "tailed" primers and the Expand Long Template PCR system (Roche Molecular Biochemicals). These 5'- and 3'-tails are
complementary to the tails of primers B and C, respectively, and allow
the AB and CD PCR products to act as primers for amplification of the
cassette. Two different cassettes were used: spectinomycin resistance
(the spc gene, GenBankTM/EBI Data Bank accession
number U46203) and chloramphenicol resistance (cat,
accession number V01277). Spectinomycin was used at 150 µg/ml and
chloramphenicol at 5 µg/ml (both from Sigma) in LB medium.
Identification of the Reductases--
A BLAST (26) search of the
B. subtilis genome (27) using the ecFabI sequence
(Swiss-Prot accession number P29132) as the bait revealed an open
reading frame (yjbW, GenBankTM/EBI Data Bank
accession number Z99110) that was predicted to encode a FabI homolog
(bsFabI). The predicted protein product was 51% identical to FabI from
E. coli and 48% identical to FabI from the Gram-positive
S. aureus (20). Two potential start codons were
present in yjbW, the second of which (encoding
Met12 of the predicted YjbW protein) aligned with that of
other FabI proteins (Fig. 1). The open
reading frame encoding both the long and shorter (first 11 codons
deleted) versions of the protein were fused to the pET-15b vector for
expression as NH2-terminal 6-His-tagged proteins, which
were purified to homogeneity by metal chelation affinity chromatography
as described under "Experimental Procedures." The two proteins had
identical activities in the spectrophotometric assay with 8:1-NAC as
substrate (specific activities of 0.08 and 0.14 ± 0.004 nmol/min/µg for the long and short versions, respectively), and we
have utilized the short version of the protein for our studies on
bsFabI.
The second highest score to ecFabI in the B. subtilis genome
was an open reading frame termed ygaA
(GenBankTM/EBI Data Bank accession number Z82044). The
predicted YgaA protein displayed 25% identity to ecFabI (Fig. 1),
which is too low to classify it as a FabI homolog. For example,
short-chain alcohol reductase/dehydrogenase family members that carry
out the unrelated reactions such as reduction of a keto group (E. coli 7- Characterization of the Reductases--
Both bsFabI and YgaA
possessed enoyl reductase activity. The bsFabI protein selectively
utilized NADH and was inactive with NADPH (Fig.
2). YgaA, on the other hand, did not
utilize NADH and catalyzed the reduction of the 8:1-NAC substrate only
in the presence of NADPH. The apparent Km value of
bsFabI for NADH was 7 µM, whereas YgaA had a
Km of 16 µM for NADPH. Neither protein
exhibited the cooperative binding characteristic of S. aureus FabI for NADPH (20). The proteins were further characterized by measuring their specific activities with the 8:1-NAC
substrate (Fig. 3A). The
bsFabI and YgaA proteins had activities of 0.14 ± 0.004 and
0.18 ± 0.01 nmol/min/µg, respectively. Under identical assay
conditions, ecFabI was more active (1.02 ± 0.006 nmol/min/µg).
These data establish that both bsFabI and YgaA are enoyl
reductases.
Intermediates in fatty acid biosynthesis are carried in the cell as
thioesters of ACP. The 8:1-NAC is thus an analog of the true
substrate for FabI, and so we also tested the ability of the bsFabI and
YgaA proteins to reduce a trans-2-enoyl-ACP. 4:1-ACP was
generated in situ by incubating purified E. coli
enzymes of fatty acid synthesis with acetyl-CoA, malonyl-CoA, E. coli ACP, and cofactors as described under "Experimental
Procedures." Enoyl reductase protein was added to initiate the
reaction, and the products were separated by conformationally sensitive
gel electrophoresis. Using these assay conditions (Fig. 3B),
bsFabI was as active as ecFabI (2.3 ± 0.4 and 2.9 ± 0.6 nmol/min/µg), whereas the YgaA protein was somewhat less active
(0.3 ± 0.02 nmol/min/µg). These data clearly demonstrate that
both bsFabI and YgaA have enoyl-ACP reductase activity.
Triclosan Inhibition of the Enoyl-ACP Reductases--
We next
determined whether bsFabI and YgaA are inhibited by triclosan, a drug
known to target ecFabI (11-13) and S. aureus FabI (20).
Initial rate measurements showed that bsFabI was ~8-fold more
resistant to triclosan than ecFabI (IC50 = 16 and 2 µM, respectively) (Fig. 4).
bsFabI contains an Ala at the position equivalent to Gly93
in E. coli (Fig. 1), and substitutions at this position are
known to increase resistance to this inhibitor due to steric
interference with drug binding in the substrate pocket (11-13, 28,
29). YgaA also contains an Ala at a position equivalent to
Gly93 in ecFabI (Fig. 1) and exhibited the same
IC50 (16 µM) as bsFabI (Fig. 4).
The key to the effectiveness of triclosan as an antibacterial agent
lies in its ability to form a stable ternary complex with the protein
and the oxidized cofactor (13). We thus tested whether this complex
forms with bsFabI and YgaA proteins. First, we monitored the
consumption of the cofactor over an extended time course using drug
concentrations that caused 50% inhibition of the initial rate and
initiated the reaction with the protein (Fig.
5A). The rate of reaction with
bsFabI slowed after several minutes and eventually ceased. This is the
behavior of a slow, tight-binding inhibitor (30) and is due to the
time-dependent formation of a stable
bsFabI·NAD+·triclosan ternary complex. This kinetic
pattern is the same as observed previously for ecFabI and S. aureus FabI (13, 20). Time-dependent complex formation
was not observed with YgaA, although the reaction proceeded at a
reduced rate (compared with the uninhibited control) until all of the
substrate was exhausted (Fig. 5B). Thus, triclosan behaves
as a simple, reversible inhibitor of YgaA. Next, enzyme, drug, and
oxidized cofactor were preincubated for 15 min prior to initiating the
reaction with substrate. All other factors (enzyme, drug, and substrate
concentrations and temperature) were constant. The stable ternary
complex formed with the bsFabI enzyme during this preincubation,
inactivating the enzyme such that virtually no reaction was observed
following addition of substrate (Fig. 5A). With YgaA, the
preincubated sample catalyzed the reaction at the same rate as the
reaction initiated with enzyme (Fig. 5B), confirming the
absence of ternary complex formation. Thus, triclosan inhibited bsFabI
by the formation of a stable bsFabI·NAD+·drug ternary
complex, but was a reversible inhibitor of the YgaA enoyl-ACP
reductase.
Biological Activities in E. coli--
The ability of bsFabI and
YgaA to function as enoyl-ACP reductases in vivo was
assessed by transforming constitutive expression plasmids into the
temperature-sensitive E. coli strain RJH13
(fabI(ts)) (22). Strain RJH13 grows at 30 °C, but cannot
grow at 42 °C due to a defective FabI protein (5, 31). Plasmids
overexpressing ecFabI, bsFabI, and YgaA all complemented this growth
defect, indicating that these genes all encode a functional enoyl-ACP reductase (Table II). The same plasmids
were transformed into the wild-type E. coli strain W3110,
and the resultant strains were tested for their sensitivity to
triclosan (Table II). As expected, strain W3110/pBluescript (empty
vector) was sensitive to triclosan, and no growth was observed on
plates containing 0.2 µg/ml drug. Expression of ecFabI increased
resistance to triclosan by ~10-fold, as demonstrated previously (11,
20). Expression of bsFabI conferred a 20-fold increase in resistance on
E. coli (Table II), consistent with the in vitro
data that bsFabI is more resistant than ecFabI (Fig. 4). In sharp
contrast, E. coli cells expressing YgaA grew strongly on
plates containing 2000 µg/ml triclosan, a >10,000-fold increase in
resistance over wild-type E. coli cells. These data
demonstrate the ability of the bsFabI and YgaA proteins to function as
enoyl-ACP reductases in vivo, performing all of the
reactions normally catalyzed by ecFabI. However, the YgaA protein was
unable to form a stable ternary complex with triclosan, conferring
complete triclosan resistance to E. coli cells expressing
YgaA.
Function of FabI and FabL (YgaA) in B. subtilis--
The
physiological function of the two enoyl-ACP reductases in B. subtilis was examined by knocking out the genes and determining the effect of the gene disruptions on cell growth and triclosan sensitivity. Neither the fabI nor ygaA gene was
essential (Table III). We were
unable to generate a fabI ygaA double knockout,
suggesting that the two genes serve redundant functions (Table
III). We next examined the sensitivity of the strains to triclosan
(Table III). The fabI knockout strain had the same MIC for
triclosan as the wild-type strain. However, the ygaA
knockout strain exhibited a 250-fold decrease in the triclosan MIC.
These data demonstrate that the expression of FabL confers resistance
to triclosan in B. subtilis and are consistent with
the in vitro and heterologous expression experiments
described above.
We also knocked out yrpB, a gene that is related to the
recently described enoyl-ACP reductase II from Streptococcus
pneumoniae (32). This gene was not essential singly or in
combination with either fabI or ygaA (Table III),
and the purified protein did not exhibit enoyl-ACP reductase
activity.3 Thus, this gene
does not appear to be involved in fatty acid synthesis in B. subtilis.
The characterization of YgaA defines a distinct class of enoyl
reductases that are usually classified within the short-chain alcohol
reductase/dehydrogenase family. Analysis of the public data bases with
the YgaA protein returned the highest scoring match (38% identical) to
a Streptomyces collinus protein termed ChcA (GenBankTM/EBI
Data Bank accession number AAC44655) that has enoyl-CoA
reductase activity (33). Similar matches were uncovered in
Campylobacter jejuni (40% identical; annotated as putative
oxidoreductase; GenBankTM/EBI Data Bank accession number CAB73072) and Helicobacter pylori (39% identical;
annotated as a 7- E. coli has a single enoyl-ACP reductase isoform (FabI) that
reduces all of the intermediates in the pathway and was considered a
ubiquitous component of type II fatty-acid synthases. Earlier work
suggested the presence of both NADH- and NADPH-dependent enoyl-ACP reductases in E. coli (34). However, the cloning
of the fabI gene (6), the fact that it is essential (6, 31), the demonstration that FabI carries out the reduction of all enoyl-ACP intermediates (3), and the identification of homologs in most bacteria
established that a single enzyme is all that is necessary to perform
this step in the pathway. This contrasts with other steps in the
pathway where multiple isoforms are known. For example, there are two
dehydratases (FabA and FabZ) and three condensing enzymes (FabH, FabB,
and FabF) (see Refs. 1 and 2). Our recent identification of a
flavoprotein from S. pneumoniae as trans-2-enoyl-ACP reductase II (FabK) (32) and the discovery of reductase III (FabL) described in this study add two new isoforms of
this enzyme to the list of type II fatty-acid synthase components in bacteria.
Although FabL is inhibited by triclosan, the inability to form a stable
FabL·NADP+·drug complex makes triclosan an ineffective
inhibitor of this enzyme in vivo. Triclosan is a
slow-binding inhibitor of FabI, and the formation of the high-affinity
ternary complex accounts for its effectiveness as an
antibacterial agent (Fig. 6A)
(13). Triclosan is a reversible inhibitor of FabL (Figs. 5 and
6B), and expression of this gene confers complete resistance
of E. coli to triclosan (Table
II). This observation confirms
that FabI is the only triclosan target in E. coli.
However, FabI is not the only triclosan target in B. subtilis. Whereas fabI knockout strains have the
same sensitivity to triclosan as the wild-type parent, fabL
knockout strains are 250-fold more sensitive to triclosan (Table III).
Thus, the expression of fabL in B. subtilis
explains the higher level of triclosan resistance in this organism
compared with S. aureus, which only expresses
fabI (20). S. aureus has a MIC of 0.2 µg/ml,
and expression of a triclosan-resistant S. aureus FabI
mutant or overexpression of wild-type S. aureus FabI increases the triclosan MIC to only 2 µg/ml (20). The triclosan MIC
for B. subtilis of 2 µg/ml, and not the complete
resistance observed in E. coli cells expressing
fabL, leads to the conclusion that, as in S. aureus, there is another triclosan target in this bacterium.
S. pneumoniae, which expresses the triclosan-resistant FabK
flavoprotein instead of FabI, also has a triclosan MIC of 2 µg/ml,
consistent with the existence of a second triclosan target in this
Gram-positive organism (32). These experiments indicate that
bacteria that express FabL would be refractory to inhibitors that are
specifically tailored to FabI, although the similarity between these
proteins (Fig. 1) suggests the potential for the discovery of compounds
that are effective against both enzymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (52K):
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Fig. 1.
Alignment of FabI and FabL (YgaA) protein
sequences. ecFabI and bsFabI were aligned with FabL (YgaA) from
B. subtilis. The key catalytic residues of ecFabI,
Tyr156, Lys163, and two prolines that flank the
substrate-binding loop, are indicated (+). Gly93 of FabI is
indicated by the asterisk. Residues present in at least two
of the three sequences are boxed.
-hydroxysteroid dehydrogenase (GenBankTM/EBI Data Bank accession number AE000257) and 
-ketoacyl-ACP reductase (FabG; GenBankTM/EBI Data Bank accession number AE000210)) were 28 and
24% identical, respectively, to ecFabI. However, YgaA possessed a
Tyr-Xaa6-Lys motif, suggesting that it may be an enoyl
reductase rather than a short-chain alcohol reductase/dehydrogenase.
FabI also contains a flexible substrate-binding loop delimited by two prolines, one of which was present in YgaA (Fig. 1). Based on these
similarities to FabI, we cloned the ygaA gene to determine if it encodes an enoyl-ACP reductase. The protein was expressed and
purified to homogeneity as described under "Experimental Procedures."
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Fig. 2.
Cofactor utilization of the enoyl-ACP
reductases. The bsFabI ( 
) and YgaA (FabL) (
) proteins were
assayed spectrophotometrically with different concentrations of NADH
(A) or NADPH (B) in the presence of 100 µM 8:1-NAC as described under "Experimental
Procedures."
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Fig. 3.
Specific activity of the enoyl-ACP
reductases. The ecFabI ( 
), bsFabI (), and FabL (YgaA) ()
proteins were assayed spectrophotometrically with the substrate analog
8:1-NAC (A) or in a coupled assay system with the
physiological substrate 4:1-ACP (B) as described under
"Experimental Procedures."
View larger version (14K):
[in a new window]
Fig. 4.
Inhibition of the enoyl-ACP reductases by
triclosan. Inhibition of the initial rate of the reduction of
8:1-NAC by the ecFabI ( ), bsFabI (), and FabL (YgaA) ()
enoyl-ACP reductases by triclosan was measured spectrophotometrically.
Rates are plotted as a percentage of the rate obtained with no
drug.
View larger version (16K):
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Fig. 5.
YgaA (FabL) is reversibly inhibited by
triclosan. Spectrophotometric assays of the bsFabI (A)
and FabL (YgaA) (B) enoyl reductases included 100 µM oxidized cofactor (NAD+ for bsFabI and
NADP+ for FabL). Reactions contained no drug ( ); 12 µM triclosan and were initiated by addition of protein
(); or 12 µM triclosan and were initiated with
substrate following a 15-min preincubation of the protein with drug
(
). The oxidation of NAD(P)H was monitored for 15 min.
Sequences of PCR primers used in the construction of the B. subtilis
knockout strains
Properties of the B. subtilis fabI and fabL (ygaA) genes expressed
in E. coli
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxysteroid dehydrogenase; GenBankTM/EBI Data
Bank accession number F64646). Each of these predicted proteins possesses the Tyr-Xaa6-Lys spacing rather than the
Ser-Tyr-Lys catalytic triad of short-chain alcohol
reductases/dehydrogenases. The S. collinus chcA gene is
in an operon encoding enzymes involved in the synthesis of the
polyketide antibiotic ansatrienin and functions as an enoyl-CoA
reductase in this pathway (33). Genes related to the S. collinus ansatrienin biosynthetic genes are not found in B. subtilis, which does not make this antibiotic (33); and the region
on the B. subtilis chromosome where ygaA resides
does not appear to contain genes related to polyketide synthases. Thus,
the YgaA protein appears to be an enoyl reductase different from ChcA.
YgaA was active with both NAC and ACP substrates and can fulfill all of
the functions of FabI in E. coli. Both the fabI
and ygaA knockouts are viable, whereas our inability to
obtain the double knockouts points to the two proteins having redundant
functions. Thus, we propose that YgaA functions in B. subtilis fatty acid biosynthesis and that YgaA be named FabL
(trans-2-enoyl-ACP reductase III). It is possible that FabL
(YgaA) may also have a physiological function auxiliary to the main
fatty acid biosynthetic pathway or plays a specialized role in this pathway.
View larger version (17K):
[in a new window]
Fig. 6.
Model for mechanism of triclosan
inhibition. A, the FabI family of enoyl-ACP reductases
form noncovalent, high-affinity ternary complexes with triclosan and
NAD(P)+, preventing the enzyme from participating in the
biosynthetic pathway. In this scheme, triclosan functions as a
slow-binding inhibitor, and the stable ternary complex takes several
minutes to form. B, the FabL enoyl-ACP reductase does not
form a high-affinity complex with triclosan and cofactor and is
reversibly inhibited by the drug. Thus, the reaction rate is decreased
by the presence of triclosan, but is not stopped. Triclosan is an
effective antibacterial agent against E. coli and other
bacteria that express only FabI, but FabL expression in E. coli is associated with high levels of resistance to the
drug.
Growth phenotypes and triclosan sensitivity of knockout strains of
B. subtilis
| |
ACKNOWLEDGEMENTS |
|---|
We thank Amy Sullivan and Pam Jackson for excellent technical assistance and Dr. Suzanne Jackowski for informative discussions.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grant GM34496, 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.
To whom correspondence should be addressed: Dept. of
Biochemistry, St. Jude Children's Research Hospital, 332 N. Lauderdale St., Memphis, TN 38105-2794. Tel.: 901-495-3491; Fax: 901-525-8025; E-mail: charles.rock@stjude.org.
Published, JBC Papers in Press, September 27, 2000, DOI 10.1074/jbc.M005611200
3 R. J. Heath and C. O. Rock, unpublished observations.
2 n:n, ratio of number of carbon atoms to double bonds.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: ACP, acyl carrier protein; NAC, N-acetylcysteamine; bsFabI, Bacillus subtilis FabI; ecFabI, Escherichia coli FabI; PCR, polymerase chain reaction; MIC, minimal inhibitory concentration.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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