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     * [14]F1000 papers in the JBC

 §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-Ã… 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

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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. [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 Ã…^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