J Biol Chem, Vol. 274, Issue 51, 36465-36471, December 17, 1999

Crystal Structure of -Ketoacyl-Acyl Carrier Protein Synthase III
A KEY CONDENSING ENZYME IN BACTERIAL FATTY ACID BIOSYNTHESIS^*

Xiayang Qiu, Cheryl A. Janson, Alex K. Konstantinidis, Silas Nwagwu, Carol Silverman, Ward W. Smith, Sanjay Khandekar, John Lonsdale, and Sherin S. Abdel-Meguid

From SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

-Ketoacyl-acyl carrier protein synthase III (FabH), the most divergent member of the family of condensing enzymes, is a key catalyst in bacterial fatty acid biosynthesis and a promising target for novel antibiotics. We report here the crystal structures of FabH determined in the presence and absence of acetyl-CoA. These structures display a fold that is common for condensing enzymes. The observed acetylation of Cys¹¹² proves its catalytic role and clearly defines the primer binding pocket. Modeling based on a bound CoA molecule suggests catalytic roles for His²⁴⁴ and Asn²⁷⁴. The structures provide the molecular basis for FabH substrate specificity and reaction mechanism and are important for structure-based design of novel antibiotics.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Condensing enzymes catalyze carbon-carbon bond formation by condensing an acyl primer with an elongating carbon source often attached to a holo-acyl carrier protein (ACP).¹ These enzymes act either as single polypeptides or domains of multienzyme complexes. They are responsible for the biosynthesis of a diverse set of natural products, including fatty acids (1, 2) and polyketides (3), and have recently been used to catalyze the synthesis of "unnatural" compounds (4, 5). Three condensing enzymes are involved in bacterial fatty acid biosynthesis: FabB, FabF, and FabH (-ketoacyl-ACP synthase I, II, and III, respectively). Each catalyzes a distinct biochemical reaction (1, 2). FabH plays a central role in fatty acid synthesis because it catalyzes the initiating condensation reaction and is responsible for the feedback regulation of the pathway via product (palmitoyl-ACP) inhibition (6-9). FabH catalyzes the condensation of acetyl-CoA and malonyl-ACP to yield acetoacetyl-ACP. The reaction is believed to occur in three steps: the acetyl transfer from acetyl-CoA to a catalytic cysteine, the decarboxylation of malonyl-ACP to form a carbanion, and the condensation of the acetyl group and carbanion (1, 2). FabH most likely acts as a homodimer of ~70 kDa (10, 11). In a similar manner to other components of the dissociated (type II) fatty acid synthase system, FabH exists as an isolated enzyme. In vertebrates, however, this enzymatic activity is encoded in the N terminus of a large multi-functional Type I fatty acid synthase, which displays no overall sequence homology to the bacterial enzyme. Emerging resistance to currently used antibacterial agents has generated an urgent need for antibiotics acting via novel mechanisms (12, 13). FabH exists ubiquitously in bacteria and is essential for viability,² suggesting it is a potential broad-spectrum antibacterial target. Moreover, thiolactomycin has been shown to be an effective inhibitor of bacterial condensing enzymes; it also possesses antibacterial activity (1, 2). The significant differences between bacterial and human fatty acid synthase systems suggest great potential for specific and selective inhibition. Most condensing enzymes contain a consensus sequence of 45 amino acids, but FabH is the most divergent member of the family and matches few of the consensus residues (14). The antibiotic cerulenin inhibits almost all condensing enzymes but not FabH (15). Thus, FabH could have a fold and evolutionary origin different from other condensing enzymes. Here we report the crystal structure of Escherichia coli FabH in the presence and absence of ligand and show that the enzyme contains a quasi-2-fold symmetry, most likely resulting from gene duplication. The structures suggest a conserved fold for all condensing enzymes, provide the structural basis for the catalytic mechanism, and will serve as molecular templates for structure-based drug design.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein Purification and Crystallization-- Native E. coli FabH protein was overexpressed in E. coli DH10B cells using the pET29 (Novagen) vector and purified to homogeneity in three chromatographic steps (Q-Sepharose, MonoQ, and hydroxyapatite) at 4 °C. The selenomethionine-substituted protein was expressed in E. coli BL21 cells and purified in a similar way. Apo FabH crystals were obtained with the native protein using 20% polyethylene glycol 8000 at neutral pH, whereas the acetyl-CoA complex crystals were obtained with the selenomethionine protein using saturating acetyl-CoA and 14% polyethylene glycol 6000 at neutral pH. Crystals were ~0.3 mm in size and were frozen before data collection.

Data Collection and Multiple-wavelength Anomalous Dispersion Phasing-- Diffraction data from the apo crystal were measured to 2.0 Å resolution at the Industrial Macromolecular Crystallography Association 17-ID beam line at the Advanced Photon Source (Table I). The crystal was of the orthorhombic P2₁2₁2₁ space group with cell dimensions a = 63.1, b = 65.1, and c = 166.5 Å, with one FabH dimer per asymmetric unit. The selenomethionine FabH·acetyl-CoA complex crystal belongs to the tetragonal space group P4₁2₁2, with a = b = 72.4 and c = 102.8 Å, and contains a monomer per asymmetric unit. The data (Table I), obtained at the X12C beam line of the Brookhaven National Laboratory, were measured at three wavelengths optimal for Se multiple-wavelength anomalous dispersion phasing experiments. Although the crystal mosaicity was as high as 1.4^o (compared with that of 0.7^o for the apo crystal) and the data were only 80% complete, the data quality was improved by its high redundancy. The acetyl-CoA complex structure was determined to 1.9-Å resolution using the multiple-wavelength anomalous dispersion phasing technique with the program SOLVE (16). There are 8 methionines in E. coli FabH; all were correctly located by SOLVE and refined to reasonable occupancies (0.62-1.00) and temperature factors (23 -41 Å²). The overall figure of merit was 0.6 from 30 to 1.9 Å resolutions, and the overall Z score was 148. The resulting electron density map was of very high quality (Fig. 1).

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Experimentally (MAD) phased electron density map at Tyr125 in a cross-eye stereo view. The map is contoured at 1.5 , and Tyr125 is from the refined model. The extra electron density near the main chain atoms of Tyr125 belongs to the backbone atoms of the neighboring residues.

Model Building and Refinement-- The whole FabH protein (317 residues), the bound acetyl and CoA, as well as 98 solvent molecules were built in the initial model. The data set collected at the inflection point wavelength (0.9785) (I > 2[I]) were used for structural refinement with XPLOR (17) because its completion is the highest among the three. The apo FabH structure (P2₁2₁2₁) was solved using the molecular replacement program AMORE (18) with the protein atoms from the acetyl-CoA complex structure as the search model. The two monomers were identified, and the solution gave an R-factor of 33% (8.0-3.5 Å). The model was initially built using the 2-fold averaged electron density map and refined to the final model using XPLOR. Both models (Table I) have good geometry with no Ramachandran outliers. The apo structure is clearly of good quality, as indicated by the low R-factors; the acetyl-CoA complex structure is also valid for the discussions in this report. This is because the high-quality electron density map (Fig. 1) is calculated from experimental phases; the complex structure has been used successfully to solve the apo structure; and the two structures are practically the same at -carbon level (r.m.s., 0.3 Å). We have not included the Se atoms and their anomalous signal in the current stage of refinement, resulting in the slightly higher R-factors of the complex structure. The figures were created with the computer programs XTALVIEW (19), MOLSCRIPT (20), and GRASP (21).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Overall Structure: E. coli-- FabH displays a five-layered core structure, ----, where each  comprises two -helices and each  is made of a five-stranded, mixed -sheet (Fig. 2A). Despite the lack of overall sequence homology, the FabH core structure is similar to that of FabF, the other condensing enzyme of known structure (22, 23). This fold was first identified in thiolase I, which catalyzes the degradation of ketoacyl-CoA structure (24, 25). The superposition of the FabH and FabF cores results in a r.m.s. difference of 1.7 Å for 160 pairs of -carbon atoms. In the comparison, Na2, Ca2, Cb3, and part of Ca3 were excluded because of large differences. The structures beyond the cores fold into a domain for substrate recognition (Fig. 2A) and intermolecular interactions, and this domain is completely unrelated to that of FabF or thiolase. The overall sequence identity is only 14% between FabH and FabF, but the 160 pairs of structurally matched residues have a higher sequence identity of 21%, indicating that FabH is evolutionally related to FabF. Because FabF fits well with the consensus sequence (14), our results suggest a common ancestor for all condensing enzymes. Their sequences and peripheral structures have diverged significantly throughout evolution to accommodate the differences in substrates. The FabH monomer appears to have been generated through gene duplication. The two halves, residues 1-170 and 171-317, have only 11% overall sequence identity. However, their structures are similar except for the insertion and loop regions (Fig. 2B). The best matched structural elements of the two FabH halves are strands b1, b2, b4, and b5. Within these strands, the 33 pairs of -carbons can be superimposed with a r.m.s. difference of only 1.0 Å, and the sequence identity between them is as high as 24%. A search of sequence data bases revealed no homologous proteins half the size of FabH, except for a few hypothetical proteins deduced from DNA sequences.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Overall structure of E. coli FabH. A, ribbon diagram of the FabH monomer. The N-terminal (1-170) and C-terminal (171-317) halves have a similar fold. The core secondary structural elements are labeled as b1-b5 and a1-a3, respectively and referred in the text with a prefix of N or C to indicate the domain they belong to. These -sheet and -helices are drawn in magenta (N, b1-b5), cyan (N, a1-a3), red (C, b1-b5), and blue (C, a1-a3). N is the N terminus, which precedes N, b1. C is the C terminus, which comes off C, b5. Secondary elements of insertion regions are drawn in yellow and orange for N- and C-terminal domains, respectively. The catalytic Cys112 is shown in a red ball-and-stick drawing, and the CoA molecule (green) was taken from the acetyl-CoA complex structure to orient the view. B, stereo view of the superposition between the N-terminal (red) and C-terminal (blue) domains of FabH. Core -strands are labeled; the overlay was generated by matching the 33 -carbon pairs from four of the strands.

Structure of the Dimer-- The crystal structure of FabH revealed the presence of a tight dimer (Fig. 3), consistent with our biochemical data³ and similar to the structures of FabF (22, 23) and thiolase I (24, 25). The dimer interface mainly involves Nb3, Na3, and Na2 and buries ~2670 Ų of accessible surface area. Strand Nb3 of one monomer interacts with that of the other monomer to form a continuous 10-stranded -sheet in the dimer. Helix Na3, with the catalytic Cys¹¹² near its N terminus (Gly¹¹⁴), uses its C-terminal half to interact with its counterpart from the other monomer. This suggests that the dimer interface is important for positioning the catalytic apparatus of FabH. The two active sites, facing opposite sides in the dimer, are formed mostly by residues within each monomer. However, Phe87 (in a loop preceding helix Na2) of one monomer projects into the active site of the other monomer. These structural features suggest that the observed dimer is the biologically relevant structure, and that its formation is essential for the proper functioning of the FabH enzyme.

View larger version (71K): [in this window] [in a new window]   Fig. 3.   Ribbon diagram of the FabH dimer. The dimer is viewed perpendicular to the two-fold axis. One monomer is shown in yellow and green; the other is shown in cyan and magenta. Phe87 and Cys112 (red) are shown as ball-and-stick models.

Catalytic Residues-- Each FabH active site, located at the center of the monomer, is formed at the interface of the N- and C-terminal domains (Fig. 2A). The catalytic residues in thiolase I were proposed to be Cys¹²⁵, His³⁷⁵, and Cys⁴⁰³ (24, 25), whereas those in FabF were predicted to be Cys¹⁶³, His³⁰³, and His³⁴⁰ (22, 23). The corresponding residues in FabH were found by structural alignment to be Cys¹¹², His²⁴⁴, and Asn²⁷⁴. Although FabH Cys¹¹² aligns well with Cys¹⁶³ of FabF and Cys¹²⁵ of thiolase I, the structural superposition revealed significant differences among other catalytic residues (Fig. 4). The lack of a Cys⁴⁰³-like residue in FabH implies a role for that residue that is relevant only to the thiolase reaction. Asn²⁷⁴ superimposes well with the histidines in FabF (His³⁴⁰) and thiolase I (His³⁷⁵), but His²⁴⁴ is far from its counterpart (His³⁰³) in FabF, suggesting different substrate specificity. A previously unsuspected thiolase residue, Asn³⁴³, is found near His²⁴⁴ of FabH. If Asn³⁴³ plays a role in catalysis, it is possible that the His-Asn pairs of FabH and thiolase are inverted structurally to match the reversed reactions they catalyze.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Superposition of potential catalytic residues in FabH (red), FabF (blue), and thiolase I (yellow). FabH residues are labeled. The corresponding residues in FabF are Cys163, His303, and His340, and those in thiolase are Cys125, Asn343, His375, and Cys403. Thiolase Asn343 and Cys403 are labeled in parentheses.

Substrate or Ligand Binding-- Both FabF and thiolase I structures were determined in the absence of a substrate; thus, the structural basis for catalysis and substrate specificity has not been established. To further investigate these important questions, we co-crystallized FabH with its first substrate, acetyl-CoA. The overall structure of FabH in the acetyl-CoA complex agrees well with the apo structure, with a r.m.s. difference of 0.3 Å for all 317 -carbons. The electron density reveals a covalent attachment of the acetyl moiety to Cys¹¹² (bond distance, 1.8 Å), indicating that the substrate was turned over by the enzyme to yield an initial product complex. This is direct evidence that Cys¹¹² is the catalytic nucleophile. The acetyl oxygen makes hydrogen bonds to the backbone nitrogens of Gly³⁰⁶ (2.8 Å) and Cys¹¹² (3.0 Å). These backbone nitrogens are ideal for stabilizing the oxyanion formed during the transition state of acetyl transfer. The same role can be suggested for the backbone nitrogens of Phe⁴⁰⁰ and Cys¹⁶³ of FabF by structural analogy. FabH Ala¹¹³ has a slightly unfavorable torsion angle, which may be required to make Cys¹¹² N available for the interaction. The acetyl methyl group occupies a hydrophobic pocket in the active site, formed by the side chains of Leu¹⁴², Phe¹⁵⁷, Leu¹⁸⁹, Leu²⁰⁵, and Phe⁸⁷', which belongs to the other monomer (Fig. 5A). The van der Waals interactions between the acetyl methyl and Phe⁸⁷' suggest that the dimer interface plays a critical role in determining the primer specificity of FabH.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Substrate binding and structure of the active site. A, stereo view of the acetyl binding pocket in the active site. Atom colors are as defined as follows: carbon, yellow; nitrogen, blue; oxygen, red; and sulfur, green. The acetyl group is shown with red bonds, whereas the dashed lines indicate hydrogen bonds. Phe87 of the other monomer in the FabH dimer is labeled as Phe87' and is shown in magenta. For clarity, only the mercaptoethylamine group of CoA is shown, which is drawn with green bonds. B, electron density of the bound CoA molecule. Atom colors are as defined before, with the three phosphorous atoms in blue as well. The map is contoured at 1. C, adenine binding site. Protein residues are in yellow bonds, and the CoA molecule is drawn in purple bonds. Carbon atoms are drawn in yellow, nitrogens in blue, oxygens in red, and phosphors in purple. The dashed lines denote hydrogen bonds.

Surprisingly, we found a CoA molecule bound in the acetyl-CoA complex, although the electron density is weak for parts of the molecule (Fig. 5B). Observing CoA in the crystal suggests that the reaction product has a notable FabH affinity. The bound CoA allows us to define the active site cavity, which is long (~15 Å) and narrow, ideal for fitting the pantetheine group of substrates in a more or less extended conformation. The adenine ring of CoA stacks between the side chains of Trp³² and Arg¹⁵¹ (Fig. 5C), a classical adenine binding mode. The Arg¹⁵¹ side chain also interacts with the ribose hydroxyl (3.4 Å) and phosphate (3.2 Å), whereas the N-cap dipole of helix Cb1 and Arg³⁶ are involved in stabilizing the diphosphate. The pantetheine group interacts with the hydrophobic wall of the active site cavity, comprised of residues such as Ile¹⁵⁶, Met²⁰⁷, Val²¹², Phe²¹³, and Ile²⁵⁰. The pantothenate also forms a couple of hydrogen bonds (O9 to Asn²⁴⁷ ND2, 2.9 Å; N8 to Gly²⁰⁹ O, 3.1 Å) with the enzyme, but the -mercaptoethylamine tail of CoA is flexible, as indicated by the weak electron density and the lack of specific protein interactions. Cys¹¹² is at the bottom of this cavity, with the acetyl carbon 3.9 Å away from the CoA sulfur atom (Fig. 5A).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It has been shown that FabH can accept primers such as acetyl-, propionyl-, and butyryl-CoA, but not long chain acyl-CoA (7, 8). This is supported by our FabH structure, in which the primer binding pocket is indeed quite small (Fig. 5A). The acetyl methyl group takes up most of the space in this pocket and is within reasonable van der Waals distances to Leu¹⁴², Phe¹⁵⁷, and Phe⁸⁷'. It is possible to model an additional carbon pointing between the side chains of Leu¹⁸⁹ and Phe¹⁵⁷, suggesting a propionyl group can also fit. Adding yet another carbon appears to require side chain rearrangements, consistent with a much reduced efficiency for butyryl-CoA. This is in contrast to the FabF primer binding pocket that is much more extended and is the locus of binding of cerulenin (23). A helical segment of FabF (Phe¹³³-Ile¹³⁸) is found by structural analogy to play a role similar to FabH Phe⁸⁷', suggesting the use of dimer interface as a conserved feature in condensing enzymes to modulate primer specificity. It is clear from structural inspection that cerulenin or a long acyl chain would not bind the FabH enzyme because the pocket is too small.

Although our acetyl-CoA structure turns out to be a product complex, the observed CoA provides a basis for modeling either of the FabH substrates. As indicated by the nature of the interactions, the mode of CoA binding should approximate that of the acetyl-CoA from adenine to most of the pantothenate. The observed flexible CoA tail is probably not an accurate representation of acetyl-CoA, but the substrate binding mode can be modeled with only a few free bond rotations. In such a model, this tail is probably in a more extended conformation. The hydrogen bonds between the acetyl carbonyl oxygen and the backbone nitrogens of Gly³⁰⁶ and Cys¹¹² may guide the acetyl group into the active site, positioning it for nucleophilic attack by Cys¹¹² (Fig. 6A). His²⁴⁴, 3.4 Å from Cys¹¹², is in position to act as a general base and extract a proton from Cys¹¹². Asn²⁷⁴ is too far (4.2 Å from Cys¹¹²) to assume this role. The side chain of Ser²⁷⁶ is 3.8 Å from Cys¹¹², but its nature prohibits it from accepting a proton. The reaction likely goes through a tetrahedral intermediate, with the carbonyl group forming an oxyanion. The backbone nitrogens of Gly³⁰⁶ and Cys¹¹² can stabilize this oxyanion. After the acetyl transfer to Cys¹¹², His²⁴⁴ could replenish the CoA sulfur with a proton to produce a neutral CoA (Fig. 6A). In well-known examples such as serine proteases, catalytic residues can be superimposed regardless of large differences in the overall protein folding (26). However, His²⁴⁴ and its analogous residue in FabF (His³⁰³) are in different positions with respect to the catalytic cysteines (Fig. 4). This is most likely attributable to differences in the substrates, suggesting that the histidine may interact directly with substrates or intermediates in addition to its role as the catalytic base. The positional differences in the catalytic apparatus suggest that condensing enzymes are under stringent evolutionary pressure to recognize and process different substrates, another reason for their sequence divergence.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Proposed mechanism of the FabH reactions. A, schematic diagram of the acetylation reaction mechanism. Dotted lines indicate hydrogen bonds; thick arrows indicate attack or electron transfer directions. His244 extracts a proton from Cys112, which may then attack the carbonyl of acetyl-CoA. The tetrahedral intermediate is stabilized by backbone nitrogens of Gly306 and Cys112. His244 may donate a proton at the end of acetylation to form a neutral CoA. B, FabH surface charge distribution viewed toward the active site cavity. The figure was calculated with the acetyl-CoA complex structure after removing the bound CoA. Only the monomer was used, because the dimer interface is not in the area of interest. Red indicates negative potential, and blue indicates positive potential. The CoA molecule is shown to occupy the active site, with white for carbon atoms, blue for nitrogens, red for oxygens, yellow for phosphors, and green for the sulfur. C, proposed structural basis for the decarboxylation and condensation mechanisms. According to the mechanism (14), the carboxylic acid moiety probably binds His244, whereas the carbonyl group interacts with Asn274. The decarboxylation produces a carbanion, which resonates with an enol intermediate that is stabilized by Asn274. The nucleophilic attack from the carbanion to the enzyme-bound acetyl group leads to a tetrahedral intermediate and finally yields the product acetoacetyl-ACP.

The observed active site cavity appears to be the only access to Cys¹¹² and, therefore, the possible binding cavity for the second substrate malonyl-ACP and the feedback inhibitor palmitoyl-ACP. Malonyl-ACP is a common substrate for many condensing enzymes. The phosphopantetheine group is linked to a serine of the ACP, which is an acidic protein of 78 residues (27). A region above the FabH cavity is positively charged and seems to be suitable for ACP binding (Fig. 6B). This region mainly includes two helices (Ca1 and Ca2) and contains several positively charged residues (Arg³⁶, Arg⁴⁰, Lys²¹⁴, His²²², Arg²³⁵, Arg²⁴⁹, Lys²⁵⁶, and Lys²⁵⁷). Binding of the prosthetic group of malonyl-ACP may be modeled based on the bound CoA. This is reasonable given that the acetylated FabH is known to react with ACP to produce acetyl-ACP (6), which is the reverse of the acetyl transfer reaction and a good indication that CoA and ACP can bind FabH similarly in the pantothenate region.

The acetyl transfer reaction is clearly a distinct step in the FabH reaction. A stepwise mechanism has also been proposed for decarboxylation and condensation (14, 28). FabH can catalyze the production of acetyl-ACP from butyryl-CoA and malonyl-ACP (8), further suggesting that decarboxylation need not occur in concert with condensation. The active sites are very similar between the apo and acetylated structures; only His²⁴⁴ shifts marginally (0.5 Å). However, it is known that malonyl-ACP only binds acetylated FabH but not the apo form of the enzyme (7, 8). This suggests that either the acetyl group interacts directly with the malonyl moiety, or the change of Cys¹¹² electrostatic potential upon acetylation is important for the binding of this substrate. The former is unlikely because iodoacetamide can also induce the binding of the malonyl group (29). The latter is probably true because His²⁴⁴ is counterbalanced by Cys¹¹² in apo FabH (Fig. 6A) but is available in acetylated FabH for a charged interaction with the malonyl carboxylic acid (Fig. 6C). His²⁴⁴ is the only potentially positively charged residue in the active site, which provides another reason for the proposed His²⁴⁴-carboxylic acid interaction during decarboxylation. In addition, His²⁴⁴ may stabilize the carbanion for the nucleophilic attack and donate a proton to Cys¹¹² after condensation. Modeling clearly suggests that Asn²⁷⁴ forms a hydrogen bond with the malonyl carbonyl oxygen. Therefore, Asn²⁷⁴ may promote decarboxylation by general acid catalysis via stabilizing an enol intermediate (Fig. 6C) and hold the carbanion for nucleophilic attack in condensation. Other condensing enzymes use a histidine in that position, and the histidine is suitable to play these same roles of Asn²⁷⁴. Based on our structures, mutagenesis and biochemical experiments can be devised to test these models and their application in other condensing enzymes. The structure will also facilitate the structure-based design of novel antibiotic drugs against this important target.

	ACKNOWLEDGEMENTS

We thank the Advanced Photon Source Industrial Macromolecular Crystallography Association-Collaborative Access Team staff and the National Synchrotron Light Source X12C staff for assistance in data collection, Kathleen Maley for help in E. coli transformation, Dean McNulty for N-terminal sequence analysis, Dr. David Tew for insightful discussions, and Drs. Robert Daines, Bill Kingsbury, Marti Head, John Elliot, John Gleason, Christine Debouck, Jim Kane, and Glen Van Aller for encouragement.

	FOOTNOTES

^* 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 1D9B) 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. Tel.: 610-270-4589; Fax: 610-270-4091; E-mail: xiayang_qiu-1@sbphrd.com.

² J. Lonsdale, unpublished data.

³ A. Konstandinidis, unpublished data.

	ABBREVIATIONS

The abbreviations used are: ACP, acyl-carrier protein; FabH, -ketoacyl-ACP synthase III; FabF, -ketoacyl-ACP synthase II; r.m.s., root mean square.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES