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§1§ Crystal Structure of the Mycobacterium tuberculosisβ-Ketoacyl-Acyl
Carrier Protein Synthase III[15]* §1§
1. [16]J. Neel Scarsdale[17]‡,
2. [18]Galina Kazanina[19]‡,
3. [20]Xin He[21]§,
4. [22]Kevin A. Reynolds[23]§ and
5. [24]H. Tonie Wright[25]‡[26]¶
1.
From the Departments of ^‡Biochemistry and ^§Medicinal Chemistry,
Institute for Structural Biology and Drug Discovery, Virginia
Commonwealth University, Richmond, Virginia 23219
[27]Next Section
§2§ Abstract §2§
Mycolic acids (α-alkyl-β-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
(C[14]–C[26]) generated by a type I fatty-acid synthase can be used
directly for the α-branch of mycolic acid or can be extended by a type
II fatty-acid synthase to make the meromycolic acid
(C[50]–C[56]))-derived component. An unusual Mycobacterium
tuberculosisβ-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 (C[14]) 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 α-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.
An estimated annual incidence rate of 8 million people and an annual
mortality rate of 3 million (1992) continue to make infection
byMycobacterium tuberculosis a serious worldwide health problem
([28]1). The appearance of drug-resistant strains of M. tuberculosis
and the human immunodeficiency virus pandemic have exacerbated this
situation ([29]2, [30]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
([31]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
([32]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 ([33]8).
Mycobacteria are unusual in that they possess both a type I and a type
II fatty-acid synthase (Fig. [34]1) ([35]9, [36]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 ([37]11). These acids are high molecular mass
α-alkyl-β-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
([38]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)
([39]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
([40]4, [41]15). A crystal structure has not been reported for KasA,
but a hypothetical structure has been presented as an aid in drug
design ([42]4).
[43]Figure 1
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Figure 1
Proposed role of mtFabH in initiation of mycolate biosynthesis in M.
tuberculosis. Carbon chain lengths are as follows: R [1] = 13–15
(saturated), R [2] = 48–54 (containing double bonds and cyclopropyl
groups), and R [3] = 22–24 (saturated). The acyl-CoA substrate for the
initiation of β-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 β-ketoacyl
synthase III (FabH, ketoacyl-ACP synthase III), which catalyzes a
decarboxylative condensation between an acetyl-CoA or similar substrate
and malonyl-ACP ([47]16, [48]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.
[49]Previous Section[50]Next Section
§2§ EXPERIMENTAL PROCEDURES §2§
§5§ Materials §5§
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 ([51]Novagen);
Ni^2+-agarose resin (QIAGEN Inc.); and crystal screen kits 1 and 2 and
polyethylene glycol (PEG) 4000 (Hampton Research).
§5§ Expression Plasmid of the M. tuberculosis fabH Gene in E. coli §5§
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 andBamHI, and ligated intoNdeI/BamHI-digested pET15b
to create pXH8. The insert coding sequence of FabH was verified by DNA
sequence analysis.
§5§ Purification of His-tagged mtFabH §5§
The pXH8 plasmid was used to transform E. coli BL21(DE3) pLysS cells
([52]Novagen), and transformants were grown in LB medium to an
absorbance at 600 nm of 0.35–0.4, induced with 0.5
mmisopropyl-β-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 Ni^2+-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 β-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 β-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.
§5§ Molecular Mass Determination §5§
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.
§5§ mtFabH Assays §5§
Radioactive FabH assays using an SPA format and biotinylated
malonyl-ACP were conducted essentially as described previously ([53]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 μmacetyl-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 ([54]19).
§5§ Enzyme Crystallization and Characterization §5§
Crystallization trials were performed by the vapor diffusion method,
initially using Hampton crystal screen kits ([55]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 CaCl[2]. These
crystals grew as very thin rhomboid plates (0.1 × 0.1 × 0.01 mm) in 3–5
days.
§5§ X-ray Intensity Data Collection §5§
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 ([56]21). Merged intensity data were converted to structure
factor amplitudes using the program Truncate ([57]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 theE. 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 P2[1] with unit cell dimensionsa = 64.1, b =
54.8, andc = 89.2 Å and β = 90.3^o. The Matthews coefficient was
consistent with a dimer of FabH in the asymmetric unit.
§5§ Structure Determination §5§
A polyalanine chain based on residues 1–317 [58]2 of a monomer of the
refined structure of ecFabH (,[59]6, [60]23) (Protein Data Bank code
[61]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 ([62]24) as
implemented in CNS Version 1.0 ([63]25). The solutions corresponding to
the 15 highest peaks from the cross-rotation search were used as input
in a translation search ([64]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.
§5§ Model Building and Refinement §5§
All model building was done using O Version 7.0 ([65]27). An initial
2.8-Å SIGMAA weighted 2mF [o] − dF [c] ([66]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 ([67]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
([68]30) using CNS Version 1.0. Each refinement cycle was followed by
manual rebuilding into mF [o] − dF [c] SIGMAA weighted cross-validated
maps and mF [o] −dF [c] SIGMAA weighted cross-validated composite omit
maps ([69]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
([70]32) and WHATCHECK ([71]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 ≥3ς 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 3ς 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 ([72]34). Refinement statistics are summarized in
Table [73]I.
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Table I
Data collection and refinement statistics for mtFabH
[76]Previous Section[77]Next Section
§2§ RESULTS §2§
§5§ Catalytic Activity of mtFabH with Long Chain Acyl-CoA Substrates §5§
The translated open reading frame (Rv05366) of theM. tuberculosis
genome ([78]35) was identified as having high sequence similarity to
the identified FabH proteins of E. coli (ecFabH) and S. glaucescens
FabH (sgFabH) (Fig.[79]2) ([80]16, [81]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 (Phe^87) 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
([82]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
([83]17), indicating that mtFabH might similarly be able to utilize
longer acyl-CoA substrates.
[84]Figure 2
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Figure 2
Sequence alignment of FabH from E. coli, S. glaucescens, and M.
tuberculosis. Positions of inserts in the latter two sequences are
based on comparison of tertiary structures, not on sequence similarity,
and are numbered with extensions to the residue number just preceding
the insert. Boxed residues are α-helix present in mtFabH and missing in
ecFabH and the putative specificity determinant at residue 87.
Asterisks denote active-site residues.
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. [88]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.[89]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 (C[2]–C[4]). 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 (C[6]–C[16]) 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 ([90]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.
[91]Figure 3
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Figure 3
Time course for mtFabH - and sgFabH-catalyzed condensation of
[9,10-^3H]myristoyl-CoA with biotinylated malonyl-ACP using an SPA.
Reactions were performed as described under “Experimental Procedures.”
○, mtFabH; ●, sgFabH.
[95]Figure 4
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Figure 4
Analysis of acyl-CoA substrate length range for mtFabH. Assays were
conducted as described under “Experimental Procedures.” No significant
loss of malonyl-ACP substrate (lane 2) was observed upon incubation
with mtFabH alone (lane 1) or with mtFabH and acetyl-CoA (lane 10),
propionyl-CoA (lane 9), or butyryl-CoA (lane 8). The addition of
hexanoyl-CoA (lane 7), octanoyl-CoA (lane 6), lauroyl-CoA (lane 5),
myristoyl-CoA (lane 4), or palmitoyl-CoA (lane 3) to the incubation led
to loss of malonyl-ACP and formation of the appropriate 3-ketoacyl-ACP
and related degradation products.
§5§ Structure of mtFabH §5§
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.[99]5). Excluding the two interior sequence insertions at residues
202 and 263 and the amino- and carboxyl-terminal extensions of mtFabH
(Fig. [100]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 Cys^112, His^244, and Asn^274 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 Arg^144 is spatially replaced by the side
chain of Lys^141 in mtFabH, thereby maintaining stabilization of the
loops defining the channel.
[101]Figure 5
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Figure 5
Comparison of backbone folds for ecFabH (blue) and mtFabH (bronze)
dimers. The insert at position 202 in mtFabH relative to ecFabH and the
contiguous α-helix (cylinder) immediately preceding this insertion in
mtFabH are colored red. Thearrow indicates the non-crystallographic
2-fold axis defining the functional mtFabH dimer. This and subsequent
figures were created with the RIBBONS program ([105]37).
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. [106]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: Thr^− ^9–Gln^237, Arg^316–Asn^− ^5, and
Arg^316–Ile^174 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 Leu^298, Val^314, Val^233, and Ala^231.
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. [107]23), consisting of
residues 191–204, by the 4-residue insertion at position 202 has the
effect of inducing a new α-helix at positions 194–202 in mtFabH (Fig.
[108]5). This new α-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. [109]6). Differences in sequence between
ecFabH and mtFabH upstream of the insertion site are consistent with
the existence of an α-helix at this position in the latter, but not in
the former. In ecFabH, Asn^193, Asn^198, and Pro^199 would inhibit
helix formation, whereas in mtFabH, these residues are Ile, Phe, and
Ala, respectively.
[110]Figure 6
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Figure 6
Backbone structure of mtFabH with modeled lauric acid in binding
channel 1 and myristoyl in binding channel 2 in each of the monomers A
and B. The lauric acid (L; green) was built into unidentified electron
density of the mtFabH map; myristoyl (M;lavender) was modeled in the
site corresponding to the acetyl group site in the ecFabH structure.
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. [114]6). At their nexus, they create a
small hydrophobic core about the 2-fold symmetry axis consisting of
Phe^198, Ile^196, and Trp^195 from each monomer, with the two Trp^195
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: Asn^198 → Phe, Arg^196 → Ile, and Asp^195 →
Trp. These changes, plus the extra interactions at the amino termini of
the mtFabH dimer, result in an additional 1384 Å^2 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 β-turn, which results in two less
hydrogen bonds relative to ecFabH: Asn^264(N^δ ^2) to the peptide
oxygen of Asp^239 and Asp^239 to the peptide nitrogen of Leu^298. These
differences are compensated by formation of an ion pair between Arg^261
and the carboxylate of Asp^239. 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. [115]7). We have modeled this density
as a lauric acid group extending from the active-site Cys^112 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 Cys^112
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.
[116]Figure 7
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Figure 7
Electron density in the pantotheinyl-binding site (binding channel 1)
of mtFabH , with the unidentified ligand modeled as a lauroyl group.
N274A, Asn^274A;C112A, Cys^112A; H224A, His^224A.
Electron density in the other binding channel (binding channel 2) for
long chain fatty acid, inferred by analogy to the Cys^112-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 Phe^87B (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 ([120]6). In mtFabH, residue 87B is a
threonine, whose smaller size permits binding of longer chain fatty
acids (Fig.[121]8). The -OγH of the Thr^87Bside 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 Arg^196B and Leu^191A(where A is monomer A), which in
mtFabH are isoleucine and glutamine, respectively, and by Ile^203A and
Leu^205A, which are displaced in mtFabH by the changes around the
insertion at position 202.
[122]Figure 8
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Figure 8
Modeled position of the myristoyl group in binding channel 2 showing
the position of residue 87B, which is a threonine in mtFabH (blue) and
a phenylalanine in ecFabH (green).Residue 87 is proposed to contribute
to fatty acid chain length specificity for these two enzymes. T87B,
Thr^87B;F87B, Phe^87B.
The end of the putative acyl-binding channel (channel 2) distal to the
active-site Cys^112 in mtFabH is capped by the α-helix at positions
194–202 induced just before the 4-residue insertion (Fig.[126]9). The
Arg^2024A side chain, which is hydrogen-bonded to the peptide carbonyl
oxygen of Pro^144A, blocks the end of this substrate channel, as do, to
a lesser extent, the side chains of Gln^191A, Ile^196B, Phe^198A,
Ala^199B, and Gln^200B. 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.
[127]Figure 9
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Figure 9
Magnified view of the distal end of the myristoyl-binding site in
mtFabH at the junction of the inserts of each monomer. The insert and
preceding α-helix are shown inred, and the modeled myristoyl group (Ma
andMb in each subunit) is shown in lavender. Note the space in the FabH
binding site for two more carbons on the end of myristoyl. R2024A,
Arg^2024A; F198A; Phe^198A; A199B, Ala^199B;Q200B, Gln^200B; I196B,
Ile^196B.
[131]Previous Section[132]Next Section
§2§ DISCUSSION §2§
Initiation of fatty acid biosynthesis in all type II systems studied to
date requires the action of a specialized condensing enzyme, FabH
([133]7, [134]17, [135]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 ([136]6, [137]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. [138]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 ([139]4,[140]14, [141]15). mtFabH therefore offers a
unique opportunity to develop new therapeutic agents that could be
effective against drug-resistantM. 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 ([142]6, [143]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. [144]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 ([145]36).
As described above, the type I fatty-acid synthase in mycobacteria
produces a bimodal (C[14:0]–C[16:0] and C[24:0]–C[26:0]) 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 (C[50]–C[56])-derived component of
mycolates by the type II fatty-acid synthase ([146]36). The specificity
of mtFabH and the crystal structure now indicate that only the shorter
acyl-CoA products (C[14:0]–C[16:0]) obtained from the type I fatty-acid
synthase are elongated (Fig. [147]1). The longer chain acyl-CoA
products (C[24:0]–C[26:0]) 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 α-alkyl chain of the mycolates
(Fig. [148]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 ([149]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.
[150]Previous Section[151]Next Section
§2§ ACKNOWLEDGEMENT §2§
We thank Clifton Barry III (National Institutes Health) for providing
the M. tuberculosis (H37Rv) chromosomal DNA and for helpful
discussions.
[152]Previous Section[153]Next Section
§2§ Footnotes §2§
* [154]↵* 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 ) have been
deposited in the Protein Data Bank, Research Collaboratory for
Structural Bioinformatics, Rutgers University, New Brunswick, NJ
([155]http://www.rcsb.org/).
* [156]↵¶ 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
* [157]↵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.
* Abbreviations:
ACP
acyl carrier protein
mtFabH
M. tuberculosis FabH
sgFabH
S. glaucescens FabH
ecFabH
E. coli FabH
SPA
scintillation proximity assay
PEG
polyethylene glycol
*
+ Received November 29, 2000.
+ Revision received January 30, 2001.
* The American Society for Biochemistry and Molecular Biology, Inc.
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