From the Departments of Biochemistry and
§ Medicinal Chemistry, Institute for Structural Biology and
Drug Discovery, Virginia Commonwealth University,
Richmond, Virginia 23219
Received for publication, November 29, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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Mycolic acids ( An estimated annual incidence rate of 8 million people and an
annual mortality rate of 3 million (1992) continue to make infection by
Mycobacterium tuberculosis a serious worldwide health
problem (1). The appearance of drug-resistant strains of M. tuberculosis and the human immunodeficiency virus pandemic have
exacerbated this situation (2, 3). Effective treatment of tuberculosis infections requires the identification of both new drugs and drug targets. Fatty acid biosynthesis in pathogenic microorganisms is
essential for cell viability and has recently attracted considerable interest as a target for development of new therapeutic agents (4-6).
In these organisms, de novo fatty acid biosynthesis from an
acetyl-CoA or related starter unit is typically catalyzed by a type II
or dissociated fatty-acid synthase, composed of discrete enzymes (7).
In contrast, de novo fatty acid biosynthesis in mammals and
other higher organisms is catalyzed by a type I or associated
fatty-acid synthase, composed of one or more multifunctional polypeptides (8).
Mycobacteria are unusual in that they possess both a type I and a type
II fatty-acid synthase (Fig. 1) (9, 10).
The type I fatty-acid synthase is responsible for formation of
16-24-carbon length fatty acids, which are then elongated to form long
chain high molecular mass mycolates (11). These acids are high
molecular mass -alkyl-
-hydroxy long chain
fatty acids) cover the surface of mycobacteria, and inhibition of their
biosynthesis is an established mechanism of action for several key
front-line anti-tuberculosis drugs. In mycobacteria, long chain
acyl-CoA products (C14-C26) generated by
a type I fatty-acid synthase can be used directly for the
-branch of
mycolic acid or can be extended by a type II fatty-acid synthase to
make the meromycolic acid (C50-C56))-derived
component. An unusual Mycobacterium tuberculosis
-ketoacyl-acyl carrier protein (ACP) synthase III (mtFabH)
has been identified, purified, and shown to catalyze a Claisen-type condensation between long chain acyl-CoA substrates such as
myristoyl-CoA (C14) and malonyl-ACP. This enzyme, presumed
to play a key role in initiating meromycolic acid biosynthesis, was
crystallized, and its structure was determined at 2.1-Å resolution.
The mtFabH homodimer is closely similar in topology and active-site
structure to Escherichia coli FabH (ecFabH), with a
CoA/malonyl-ACP-binding channel leading from the enzyme surface to the
buried active-site cysteine residue. Unlike ecFabH, mtFabH contains a
second hydrophobic channel leading from the active site. In the ecFabH
structure, this channel is blocked by a phenylalanine residue, which
constrains specificity to acetyl-CoA, whereas in mtFabH, this residue
is a threonine, which permits binding of longer acyl chains. This same
channel in mtFabH is capped by an
-helix formed adjacent to a
4-amino acid sequence insertion, which limits bound acyl chain length
to 16 carbons. These observations offer a molecular basis for
understanding the unusual substrate specificity of mtFabH and its
probable role in regulating the biosynthesis of the two different
length acyl chains required for generation of mycolic acids. This
mtFabH presents a new target for structure-based design of novel
antimycobacterial agents.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-alkyl-
-hydroxy fatty acids with the general
structure R-CH(OH)-CH(R')-COOH (where R is a meromycolate chain (50-56
carbons) and R' is a significantly shorter chain (22-26 carbons)),
which are key components of the mycobacterium cell wall. Triclosan and isoniazid are commonly used antibacterial agents that target mycolate biosynthesis (12). In the case of isoniazid, prevention of mycolate biosynthesis results from inhibition of the enoyl-acyl carrier protein
(ACP)1 reductase (InhA) and
possibly the ketoacyl-ACP synthase (KasA) (13-15). This latter enzyme
is apparently responsible for catalyzing the decarboxylative
condensation between an acyl-ACP and a malonyl-ACP in the carbon chain
extension steps in mycolate biosynthesis and has also been
shown to be inhibited by thiolactomycin (4, 15). A crystal structure
has not been reported for KasA, but a hypothetical structure has been
presented as an aid in drug design (4).
View larger version (12K):
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Fig. 1.
Proposed role of mtFabH in initiation of
mycolate biosynthesis in M. tuberculosis. Carbon
chain lengths are as follows: R1 = 13-15
(saturated), R2 = 48-54 (containing double bonds
and cyclopropyl groups), and R3 = 22-24
(saturated). The acyl-CoA substrate for the initiation of
-ketoacyl-ACP synthase III (mtFabH) is released from the type
I fatty-acid synthase (FAS) and is presumed to be used for
both mycolate and phospholipid biosynthesis. Subsequent elongation
steps are catalyzed by different ketoacyl-ACP synthase (KAS)
enzymes (KasA and KasB), which have been shown to be targets for
both thiolactomycin and isoniazid.
A ketoacyl-ACP synthase activity is also presumably needed to initiate
the first decarboxylative condensation in mycolate biosynthesis. In
other type II systems, this activity is provided by a -ketoacyl
synthase III (FabH, ketoacyl-ACP synthase III), which catalyzes a
decarboxylative condensation between an acetyl-CoA or similar substrate
and malonyl-ACP (16, 17). By analogy, a ketoacyl-ACP synthase III
activity that utilizes longer chain acyl-CoA substrates would be a link
between the type I and type II fatty-acid synthases of M. tuberculosis.
In this study, we report that we have identified an M. tuberculosis FabH (mtFabH) that is able to preferentially use long chain acyl-CoA substrates such as myristoyl-CoA over acetyl-CoA. We
have crystallized and determined the structure of this mtFabH and shown
that it has several unique structural features that allow it to utilize
longer chain acyl-CoA substrates and that may help in rational design
of new drugs. Such drugs may be valuable in the treatment of
drug-resistant M. tuberculosis since none of the current
therapies target this enzyme, which appears to occupy a significant
regulatory role in initiating mycolate biosynthesis.
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EXPERIMENTAL PROCEDURES |
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Materials-- The following reagents were used: N-hydroxysuccinimidobiotin (Pierce); Escherichia coli acyl carrier protein, imidazole, dithiothreitol, and malonyl-CoA (Sigma); [3H]acetyl-CoA (specific activity, of 60 Ci/mmol; Moravek Biochemicals, Inc.); [9,10-3H]myristoyl-CoA (specific activity, 60 Ci/mol; American Radiochemical Chemicals); streptavidin-coated yttrium silicate scintillation proximity fluorospheres (SPA beads; Amersham Pharmacia Biotech); microbiological media (Difco); restriction enzymes and T4 DNA ligase (New England Biolabs Inc.); pET vector and expression strains (Novagen); Ni2+-agarose resin (QIAGEN Inc.); and crystal screen kits 1 and 2 and polyethylene glycol (PEG) 4000 (Hampton Research).
Expression Plasmid of the M. tuberculosis fabH Gene in E. coli-- The putative fabH gene (Rv0533c) was amplified from M. tuberculosis (H37Rv) chromosomal DNA. The forward primer 5'-CAGATAGGACGCATATGACGGAGATCG-3' was designed to introduce an NdeI restriction site (underlined) at the start of the 5'-end of fabH. A BamHI site was created (underlined) downstream of the fabH stop codon in the reverse primer 5'-ATCCCTGGCTGGATCCGATCTTCGC-3'. Polymerase chain reaction was performed using the GeneAmp® XL polymerase chain reaction kit (PerkinElmer Life Sciences). The resulting polymerase chain reaction product was eluted from agarose gel using Qiax (QIAGEN Inc.), digested with NdeI and BamHI, and ligated into NdeI/BamHI-digested pET15b to create pXH8. The insert coding sequence of FabH was verified by DNA sequence analysis.
Purification of His-tagged mtFabH--
The pXH8 plasmid was used
to transform E. coli BL21(DE3) pLysS cells (Novagen), and
transformants were grown in LB medium to an absorbance at 600 nm of
0.35-0.4, induced with 0.5 mM
isopropyl--D-thiogalactopyranoside, and incubated for an
additional 3 h at 37 °C. Cells were harvested by centrifugation
at 10,000 × g for 10 min at 4 °C and stored at
20 °C overnight. Lysis was performed with lysozyme according to
the QIAexpressionist protocol, and lysate was then frozen at
70 °C until used. Thawed lysate suspension was centrifuged at 12,000 × g for 30 min at 4 °C, and the supernatant
was loaded onto a Ni2+-nitrilotriacetic acid-agarose
column, which was then washed with 50 mM imidazole in 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10%
glycerol, and 3 mM
-mercaptoethanol. mtFabH was eluted
with 200 mM imidazole in the same buffer and dialyzed
overnight against 50 mM Tris-HCl (pH 8.0), 10% glycerol,
and 3 mM
-mercaptoethanol with either 300 or 50 mM NaCl at 4 °C. Purified protein was concentrated in a
Centricon tube (Amicon, Inc.) with a molecular mass cutoff of 30,000 Da
to 4-8 mg/ml as determined by the Bradford assay.
Dithiothreitol was added to protein concentrates to 2 mM,
and these concentrates were stored at 4 °C for 3-5 days. For longer
storage of the enzyme, glycerol was added to 40-50%, and the
aliquoted protein was stored at
20 °C.
Molecular Mass Determination-- The purity and molecular mass of His-tagged mtFabH were estimated by SDS-polyacrylamide gel electrophoresis and gel exclusion chromatography under native conditions using a Sephacryl S-200-HR column.
mtFabH Assays-- Radioactive FabH assays using an SPA format and biotinylated malonyl-ACP were conducted essentially as described previously (18) using radioactive myristoyl-CoA. The standard reaction mixture contained the following components in a final volume of 20 µl: 0.15 µg of mtFabH or sgFabH (from Streptomyces glaucescens), 100 mM sodium phosphate buffer, 1% Triton X-100 (pH 7.0), 2.2 µM biotinylated malonyl-ACP, and 0.17 µM [9,10-3H]myristoyl-CoA (0.20 µCi; specific activity, 60 Ci/mmol). The reaction was initiated by the addition of [9,10-3H]myristoyl-CoA and incubated at 37 °C for various time periods. For each assay, 50 µl of the SPA bead solution (10 mg/ml) was added once the reaction was terminated.
Nonradioactive assays were carried out by monitoring a loss of malonyl-ACP. Malonyl-ACP (20 µM) was combined individually with different putative substrates (250 µM acetyl-CoA, propionyl-CoA, butyryl-CoA, octanoyl-CoA, lauroyl-CoA, myristoyl-CoA, or palmitoyl-CoA) in 0.1 M sodium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol, 0.1% Triton X-100 in a final volume of 20 µl. The reaction was initiated by the addition of 0.8 µg of mtFabH, incubated at 37 °C for 60 min, and terminated on ice. The reaction mixture was then analyzed on a conformationally sensitive 13-15% polyacrylamide gel containing 2.5 M urea, on which malonyl-ACP and the corresponding 3-ketoacyl-ACP products of a FabH-catalyzed reaction are readily resolved (19).
Enzyme Crystallization and Characterization-- Crystallization trials were performed by the vapor diffusion method, initially using Hampton crystal screen kits (20). Quasi-crystalline aggregates were obtained with PEGs as precipitant, and further refinement of conditions yielded two crystal forms suitable for x-ray analysis from PEG 4000, Tris-HCl, pH 8.0, and 300 mM NaCl. Form 1 crystals grew after mixing equal volumes of enzyme solution (4 mg/ml in 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, and 2 mM dithiothreitol) with 30% PEG 4000, 100 mM Tris-HCl (pH 8.0), and 300 mM NaCl and equilibrating against a reservoir containing 17-26% PEG 4000 in the same buffer. Conglomerates, multiples, and some single crystals (0.15 × 0.15 × 0.05 mm) with a rhomboid shape grew in 2-3 days. Form 2 crystals were obtained using the same precipitant, but with the enzyme solution containing up to 40% glycerol and 10 mM CaCl2. These crystals grew as very thin rhomboid plates (0.1 × 0.1 × 0.01 mm) in 3-5 days.
X-ray Intensity Data Collection--
Form 1 crystals were
flash-cooled in a cryoprotectant solution containing 80% reservoir
solution and 20% PEG 400. Diffraction intensity data were
collected at 170 °C on a Raxis II image plate detector with osmic
confocal optics and a rotating anode source at 50 kV and 100 mA at a
detector-to-crystal distance of 80 mm. Oscillation data frames were
reduced, integrated, scaled, and merged with the HKL package (21).
Merged intensity data were converted to structure factor amplitudes
using the program Truncate (22). Form 1 crystals diffracted to 2.2-Å
resolution, but were inferred to be twinned upon failure of convergence
of refinement of the model obtained by molecular replacement using the
E. coli FabH (ecFabH) structure as a search model (see below).
Form 2 crystals were cryoprotected in 15% glycerol and 20% PEG 400 in
crystallization reservoir solution and flash-frozen, and a half-sphere
of data was collected and reduced as described above. These data
extended to 2.1-Å resolution; showed no evidence of twinning; and were
indexed in space group P21 with unit cell dimensions
a = 64.1, b = 54.8, and
c = 89.2 Å and = 90.3o. The
Matthews coefficient was consistent with a dimer of FabH in the
asymmetric unit.
Structure Determination-- A polyalanine chain based on residues 1-317 2 of a monomer of the refined structure of ecFabH (6, 23) (Protein Data Bank code 1EBL) was used as a search model for molecular replacement. In constructing the search model, the 4-residue insertion following residue 202 and the 1-residue insertion at position 263 of the E. coli sequence were excluded. A cross-rotation search was carried out using data between 15 and 4 Å with the fast direct protocol (24) as implemented in CNS Version 1.0 (25). The solutions corresponding to the 15 highest peaks from the cross-rotation search were used as input in a translation search (26) as implemented in CNS Version 1.0. The presence of a dimer in the asymmetric unit was confirmed by cross-rotation and translation searches using a polyalanine chain based on the ecFabH dimer structure. In these searches, we noted that the orientations of the best monomer and dimer solutions were nearly identical and that the values of the monitor function in the translation search for the dimer were significantly higher for the best dimer solutions than for the best monomer solutions.
Model Building and Refinement--
All model building was done
using O Version 7.0 (27). An initial 2.8-Å SIGMAA weighted
2mFo dFc (28) electron density
map calculated using phases based on the best molecular replacement
dimer solution was not readily interpretable. Two cycles of real space
2-fold averaging of this map using a mask based on the E. coli monomer with the Ave program (29) resulted in significant
improvements in the quality of the map. The resulting map was readily
interpretable and permitted placement of many side chains that were not
present in the initial search model.
The model was iteratively refined via simulated annealing based on
torsion-angle dynamics and a maximum likelihood target function (30)
using CNS Version 1.0. Each refinement cycle was followed by manual
rebuilding into mFo dFc SIGMAA
weighted cross-validated maps and mFo
dFc SIGMAA weighted cross-validated composite omit
maps (31). Non-crystallographic symmetry was enforced via positional
restraints between symmetry-related molecules. These restraints were
initially assigned a weight of 300 kcal/mol/Å, which was reduced to
37.5 kcal/mol/Å in the final stages of the refinement. In the final
stages of the refinement, non-crystallographic symmetry
restraints on temperature factors were omitted from the restrained
atomic B-factor refinement. Model phases were iteratively
extended in steps to 2.1 Å over several cycles of refinement. During
iterative rebuilding, residue geometries were monitored with the
programs OOPs (32) and WHATCHECK (33). In the final stages of the
refinement, 278 solvent molecules, 7 glycerol molecules, and a ligand
modeled as lauric acid (in monomer A) were added based on the
presence of peaks with intensity
3
in a SIGMAA weighted difference
Fourier map. In the final stages of iterative rebuilding, SIGMAA
weighted cross-validated difference Fourier maps calculated in the
absence of solvent molecules contoured at 3
were used to assist in
locating model errors. Coordinate errors in the refined structure were
estimated using Cruickshank's diffraction data precision indicator as
implemented in the SFCHECK program (34). Refinement statistics are
summarized in Table I.
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RESULTS |
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Catalytic Activity of mtFabH with Long Chain Acyl-CoA
Substrates--
The translated open reading frame (Rv05366) of the
M. tuberculosis genome (35) was identified as having high
sequence similarity to the identified FabH proteins of E. coli (ecFabH) and S. glaucescens FabH (sgFabH) (Fig.
2) (16, 17). The sequence of
putative mtFabH contained all of the signature sequences for FabH
enzymes, leading to the prediction that the protein would catalyze the condensation of an acyl-CoA substrate with a malonyl-ACP substrate. The
phenylalanine residue (Phe87) proposed from the crystal
structure of the E. coli ketoacyl-ACP synthase III to be
important in restricting the acyl-CoA substrate specificity to carbon
chain lengths of 2 or 3 (6) is a threonine in both mtFabH and sgFabH.
sgFabH has been shown to accept a much greater range of acyl-CoA
substrates than ecFabH (17), indicating that mtFabH might similarly be
able to utilize longer acyl-CoA substrates.
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To test this hypothesis, mtFabH was expressed, purified, and
characterized as a recombinant protein with an N-terminal His tag.
SDS-polyacrylamide gel electrophoresis analysis of the recombinant protein showed a molecular mass of 37,000 Da, and gel exclusion chromatography showed a native molecular mass of 76,600 ± 2500 Da, indicating that, like ecFabH and sgFabH, mtFabH is a homodimer. The
recombinant protein was shown in an SPA to be active with the long
chain myristoyl-CoA (Fig. 3). This
activity appears to be specific for mtFabH since no activity could be
detected carrying out the same assay using sgFabH. The ability of the
mtFabH enzyme to process a range of acyl-CoA substrates was examined by
monitoring the loss of malonyl-ACP substrate over time (Fig.
4). No significant loss of malonyl-ACP
substrate was observed either with the enzyme alone or in the presence
of short chain acyl-CoA substrates (C2-C4). In
a control experiment using sgFabH, a loss of malonyl-ACP could be
observed using these shorter chain acyl-CoA substrates (data not
shown). However, a loss of malonyl-ACP substrate was observed when
longer chain substrates (C6-C16) were provided
in the mtFabH enzyme assay. An independent analysis recently carried
out using a coupled assay methodology has reported a similar substrate
range for mtFabH, with the apparent preference for the substrate
lauroyl-CoA (36). These observations are consistent with the proposed
role of this enzyme in initiating mycolate fatty acid biosynthesis and
prompted attempts to crystallize and solve the structure of this unique
FabH.
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Structure of mtFabH-- The refined electron density map for mtFabH was well resolved and continuous in all regions except for fragmented density in molecule B from residues 24 to 50. The amino-terminal extensions carrying the histidine tag, five side chains in monomer A, and 32 side chains (14 between residues 24-50) in monomer B have weak or missing electron density.
As implied by the successful use of the ecFabH structure as a search
model in the molecular replacement solution of the mtFabH structure,
the backbone folds of these two molecules are closely similar (Fig.
5). Excluding the two interior sequence
insertions at residues 202 and 263 and the amino- and carboxyl-terminal
extensions of mtFabH (Fig. 2), the root mean square deviation in main
chain coordinates for the monomer structures of ecFabH and mtFabH is 1.37 Å. The active-site residues Cys112,
His244, and Asn274 implicated in catalysis are
similarly disposed in mtFabH and ecFabH, as is the oxyanion hole. The
CoA/malonyl-ACP-binding channel and the interactions that stabilize its
structure are also almost identical in the two enzymes. Differences
occur at residues 144 and 210, which are both prolines in mtFabH and
arginine and asparagine, respectively, in ecFabH. The side chain of
Arg144 is spatially replaced by the side chain of
Lys141 in mtFabH, thereby maintaining stabilization of the
loops defining the channel.
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The amino-terminal 10-residue extension of mtFabH, which is absent in
ecFabH, extends to make stabilizing contacts both with the other
monomer of each dimer as well as with its own monomer (Fig. 5). The
amino-terminal segments of each monomer lie in a surface depression of
the opposite monomer and make several hydrogen bonds and a hydrophobic
contact. Duplicate hydrogen bonds of each subunit with the other are as
follows: Thr9-Gln237,
Arg316-Asn
5, and
Arg316-Ile174 mediated by a water molecule.
Ile
7 of each monomer amino terminus lies in a
hydrophobic pocket of the juxtaposed monomer created by the side chains
of Leu298, Val314, Val233, and
Ala231.
The 4-residue insertion at position 202 in mtFabH relative to ecFabH
distorts the local conformation of this loop in a surprising way and
creates a number of stabilizing intermonomer contacts. The alteration
of this loop L9 (see nomenclature in Ref. 23), consisting of residues
191-204, by the 4-residue insertion at position 202 has the effect of
inducing a new -helix at positions 194-202 in mtFabH (Fig. 5). This
new
-helix lies at the distal end of the putative acyl-binding
channel inferred from the position of the acetyl group in the ecFabH
crystal structure, and its possible functional significance is
discussed below (Fig. 6). Differences in
sequence between ecFabH and mtFabH upstream of the insertion site are
consistent with the existence of an
-helix at this position in the
latter, but not in the former. In ecFabH, Asn193,
Asn198, and Pro199 would inhibit helix
formation, whereas in mtFabH, these residues are Ile, Phe, and Ala,
respectively.
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The extended loops created as a result of the 4-residue sequence
insertion converge at the non-crystallographic 2-fold symmetry axis
relating the two monomers to make a number of interactions that would
stabilize the mtFabH dimer (Fig. 6). At their nexus, they create a
small hydrophobic core about the 2-fold symmetry axis consisting of
Phe198, Ile196, and Trp195 from
each monomer, with the two Trp195 indole rings stacking on
each other. The sequence differences relative to ecFabH at the
positions creating this intermonomer hydrophobic locus in mtFabH are as
follows: Asn198 Phe, Arg196
Ile, and
Asp195
Trp. These changes, plus the extra interactions
at the amino termini of the mtFabH dimer, result in an additional 1384 Å2 of contact area between the two monomers relative to
ecFabH.
The single alanine insertion in mtFabH at residue 263 causes a local
difference in conformation at a -turn, which results in two less
hydrogen bonds relative to ecFabH:
Asn264(N
2) to the peptide oxygen
of Asp239 and Asp239 to the peptide nitrogen of
Leu298. These differences are compensated by formation of
an ion pair between Arg261 and the carboxylate of
Asp239. These changes are far from both the active site and
the binding site of the enzyme and from the dimer interface, and their
functional and biological significance, if any, is not obvious.
The electron density map of mtFabH shows significant continuous density
in the site of monomer A corresponding to the location of the
pantothenic acid moiety of CoA observed in the ecFabH structure (denoted binding channel 1) (Fig. 7). We
have modeled this density as a lauric acid group extending from the
active-site Cys112 to the open mouth of this binding
channel. If this density represents an acyl group, it could possibly
form a linkage with the active-site Cys112 sulfur, but both
tenuous electron density and suboptimal stereochemistry argue against
this. We believe that this group is a hydrophobic molecule taken up by
mtFabH during purification, and its identity is currently under
investigation.
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Electron density in the other binding channel (binding channel 2) for
long chain fatty acid, inferred by analogy to the
Cys112-acetylated ecFabH complex, contains five discrete
peaks assigned as solvent molecules. Two features of this site can
explain the distinct substrate specificities of mtFabH and ecFabH. The
presence of Phe87B (where B is monomer B) in this fatty
acyl-binding site of ecFabH obstructs binding of straight fatty acid
chains longer than ~4 carbons, thereby accounting for the selectivity
of the E. coli enzyme for acetyl over longer chain
substrates (6). In mtFabH, residue 87B is a threonine, whose smaller
size permits binding of longer chain fatty acids (Fig.
8). The -OH of the Thr87B
side chain is hydrogen-bonded to a bound solvent, thereby orienting the
side chain methyl group toward the position of the acyl substrate and
contributing to the hydrophobicity of its environment. This channel is
also blocked in ecFabH by Arg196B and Leu191A
(where A is monomer A), which in mtFabH are isoleucine and glutamine, respectively, and by Ile203A and Leu205A, which
are displaced in mtFabH by the changes around the insertion at position
202.
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The end of the putative acyl-binding channel (channel 2) distal to the
active-site Cys112 in mtFabH is capped by the -helix at
positions 194-202 induced just before the 4-residue insertion (Fig.
9). The Arg2024A side chain,
which is hydrogen-bonded to the peptide carbonyl oxygen of
Pro144A, blocks the end of this substrate channel, as do,
to a lesser extent, the side chains of Gln191A,
Ile196B, Phe198A, Ala199B, and
Gln200B. In the ecFabH structure, this area is open to
solvent, but the inner part of the channel proximal to the active site
is blocked by other residues as described above, preventing binding of
longer chains.
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DISCUSSION |
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Initiation of fatty acid biosynthesis in all type II systems studied to date requires the action of a specialized condensing enzyme, FabH (7, 17, 38). This enzyme catalyzes the condensation of an acyl-CoA substrate with malonyl-ACP to generate a 3-ketoacyl-ACP product. This product is reduced to an acyl-ACP and is extended by condensation catalyzed by one or more different ketoacyl-ACP synthase isozymes. To date, there have been no successful reports of the generation of any FabH knockout mutants despite extensive effort in both our laboratory and others. FabH has recently attracted significant interest as a target for new antibacterials, as all evidence to date indicates that FabH is essential for cell viability (6, 36).
mtFabH is unusual in that the substrate specificity of this enzyme indicates that it does not play a role in de novo fatty acid biosynthesis, which is carried out by a type I fatty-acid synthase, but rather in initiating biosynthesis of very long chain fatty acids used in mycolate biosynthesis (Fig. 1). Inhibition of mycolate biosynthesis is known to be effective in the treatment of mycobacterial infections, although none of the existing therapies, or even compounds known to inhibit mycobacterial growth, appears to target FabH specifically (4, 14, 15). mtFabH therefore offers a unique opportunity to develop new therapeutic agents that could be effective against drug-resistant M. tuberculosis strains. A key step in designing inhibitors specific to this unusual FabH is the determination of the structure of the protein and an understanding of the key features that differentiate it from similar enzymes involved in de novo fatty acid biosynthesis.
The crystal structure of mtFabH reported here confirms its close similarity to the structure of ecFabH, which has recently been determined (6, 23). The active-site regions of both proteins are very similar, and both have a CoA/malonyl-ACP-binding site (binding channel 1). These observations are consistent with the fact that, in both enzymes, the latter half of the catalytic process involves release of CoA and a decarboxylative condensation between an acylated enzyme and malonyl-ACP. There are, however, several notable differences between the ecFabH and mtFabH dimer structures. The latter has a larger number of stabilizing intermonomer interactions than ecFabH as a result of sequence extensions at the amino terminus and of a 4-residue internal insertion. The amino terminus creates an arm that extends from each monomer and makes contacts with the opposite monomer of the dimer, whereas the internal insertion creates an added contact area in the dimer interface with stabilizing hydrogen bond and hydrophobic contributions. Although the significance of these observations is unclear, it is worth noting that a similar amino acid extension and internal insertion are observed in sgFabH (Fig. 2).
Finally, there are several unique features of mtFabH that have bearing
on its distinct specificity and its potential role in regulating
mycolate biosynthesis. In particular, sequence differences in the
inferred binding channel 2 for long chain fatty acids between ecFabH
and mtFabH explain the inability of ecFabH to utilize acyl-CoAs with
chains longer than ~4 carbons in the acyl group. The mtFabH crystal
structure now offers one plausible explanation why this enzyme can
accept longer acyl-CoA substrates and also a structural basis for its
apparent upper limit of 16 carbons in acyl-CoA chain length (36). As
described above, the type I fatty-acid synthase in mycobacteria
produces a bimodal (C14:0-C16:0 and
C24:0-C26:0) distribution of acyl-CoA fatty
acids. It has been unclear whether one or both of these fatty acid
products act as substrates for the synthesis of the long chain
meromycolic acid (C50-C56)-derived component
of mycolates by the type II fatty-acid synthase (36). The specificity
of mtFabH and the crystal structure now indicate that only the shorter
acyl-CoA products (C14:0-C16:0) obtained from
the type I fatty-acid synthase are elongated (Fig. 1). The longer chain
acyl-CoA products (C24:0-C26:0) would thus be
excluded from chain elongation and would remain available to be
utilized, presumably in the coenzyme A form, as substrates for
formation of the -alkyl chain of the mycolates (Fig. 1). The
availability of these substrates might be markedly reduced if mtFabH
used them to initiate meromycolic acid biosynthesis. Thus, we speculate that a combination of the bimodal distribution of fatty acids made by
the type I fatty-acid synthase and the substrate specificity of mtFabH
ensure the appropriate equimolar distribution of dramatically different
chain length acyl thioester substrates required for mycolate biosynthesis.
It is widely accepted that mycolate biosynthesis is the main target of
several front-line therapies for treating mycobacterial infections and
that all of the enzymes in the unusual type II fatty-acid synthase
involved in this process represent attractive targets for drug
development (14). An important component of this system yet to be
specifically targeted is mtFabH, which appears likely to play a key
role in initiating and regulating mycolate biosynthesis. The
availability of the structure of this enzyme and an SPA suitable for
high throughput screening should facilitate the design, discovery, and
development of much needed novel antimycobacterial agents.
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ACKNOWLEDGEMENT |
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We thank Clifton Barry III (National Institutes Health) for providing the M. tuberculosis (H37Rv) chromosomal DNA and for helpful discussions.
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FOOTNOTES |
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* This work was supported by NIAID Grant AI44772 from the National Institutes of Health (to K. A. R. and H. T. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1HZP) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ To whom correspondence should be addressed: ISBDD, Suite 212, Virginia Biotechnology Research Park, 800 East Leigh St., Richmond, VA 23219. Tel.: 804-828-6139; Fax: 804-827-3664; E-mail: xrdproc@ hsc.vcu.edu.
Published, JBC Papers in Press, March 8, 2001, DOI 10.1074/jbc.M010762200
2
Sequence numbering of mtFabH is that of
ecFabH, with amino-terminal extension residues numbered negatively in
reverse beginning with position 1 and carboxyl-terminal extensions
added to the terminal sequence number of ecFabH. Internal insertions
are denoted by integer suffixes to the preceding residue in sequence.
Suffixes A and B to sequence numbers denote the two monomers of the
crystallographic and presumed dimer.
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ABBREVIATIONS |
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The abbreviations used are: ACP, acyl carrier protein; mtFabH, M. tuberculosis FabH; sgFabH, S. glaucescens FabH; ecFabH, E. coli FabH; SPA, scintillation proximity assay; PEG, polyethylene glycol.
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