Inhibition of beta -Ketoacyl-Acyl Carrier Protein Synthases by Thiolactomycin and Cerulenin

STRUCTURE AND MECHANISM*

Allen C. PriceDagger , Keum-Hwa Choi§, Richard J. Heath§, Zhenmei LiDagger , Stephen W. WhiteDagger , and Charles O. Rock§||

From the Departments of Dagger  Structural Biology and § Biochemistry, St. Jude Children's Research Hospital and the  Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38105

Received for publication, August 6, 2000, and in revised form, October 9, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The beta -ketoacyl-acyl carrier protein (ACP) synthases are key regulators of type II fatty acid synthesis and are the targets for two natural products, thiolactomycin (TLM) and cerulenin. The high resolution structures of the FabB-TLM and FabB-cerulenin binary complexes were determined. TLM mimics malonyl-ACP in the FabB active site. It forms strong hydrogen bond interactions with the two catalytic histidines, and the unsaturated alkyl side chain interaction with a small hydrophobic pocket is stabilized by pi  stacking interactions. Cerulenin binding mimics the condensation transition state. The subtle differences between the FabB-cerulenin and FabF-cerulenin (Moche, M., Schneider, G., Edwards, P., Dehesh, K., and Lindqvist, Y. (1999) J. Biol. Chem. 244, 6031-6034) structures explain the differences in the sensitivity of the two enzymes to the antibiotic and may reflect the distinct substrate specificities that differentiate the two enzymes. The FabB[H333N] protein was prepared to convert the FabB His-His-Cys active site triad into the FabH His-Asn-Cys configuration to test the importance of the two His residues in TLM and cerulenin binding. FabB[H333N] was significantly more resistant to both antibiotics than FabB and had an affinity for TLM an order of magnitude less than the wild-type enzyme, illustrating that the two-histidine active site architecture is critical to protein-antibiotic interaction. These data provide a structural framework for understanding antibiotic sensitivity within this group of enzymes.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Drug resistance in infectious organisms has become a serious medical problem, and fatty acid synthesis has emerged as a promising target for the development of novel therapeutic agents. Lipid synthesis is not only essential to cell viability but specificity for bacteria and other infectious organisms can be achieved by taking advantage of the organizational and structural differences that exist in the fatty acid synthetic systems of different organisms. There are two major types. The associated, or type I, systems exist in higher organisms such as mammals and compose a single, multifunctional polypeptide (1). The dissociated, or type II, fatty-acid synthases exist in bacteria and plants and are composed of a collection of discrete enzymes that each carry out an individual step in the cycles of chain elongation (2, 3). Triclosan (4, 5) and isoniazid (6) are two commonly used antibacterial agents that target fatty acid synthesis.

The type II system has been most extensively studied in Escherichia coli where the three beta -ketoacyl-ACP1 synthases have emerged as important regulators of the initiation and elongation steps in the pathway. These enzymes catalyze the Claisen condensation reaction, transferring an acyl primer to malonyl-ACP and thereby creating a beta -ketoacyl-ACP that has been lengthened by two carbon units. Two of these synthases are elongation condensing enzymes. Synthase I (FabB) is required for a critical step in the elongation of unsaturated fatty acids. Mutants (fabB) lacking synthase I activity require supplementation with exogenous unsaturated fatty acids to support growth (7, 8). Synthase II (FabF) controls the temperature-dependent regulation of fatty acid composition (9, 10). Mutants lacking synthase II (fabF) are deficient in the elongation of palmitoleate to cis-vaccenate but grow normally under standard culture conditions (9, 11, 12). The third synthase functions as the initiation condensing enzyme. Synthase III (FabH) catalyzes the first condensation step in the pathway and is thus ideally situated to govern the rate of fatty acid synthesis (13-16). Unlike FabB and FabF, FabH enzymes use an acyl-CoA rather than an acyl-ACP as the primer (15-18). FabH is further distinguished by a His-Asn-Cys (19, 20) catalytic triad in contrast to the His-His-Cys triad in the FabB (21) and FabF (22) enzymes.

The crystal structures of all three condensing enzymes from E. coli (FabB, FabF, and FabH) have now been determined (19-22). Their primary structures are clearly related, and these translate into similar dimeric structures and active site architectures. The structures of the monomers compose an internally duplicated helix-sheet-helix motif, and the active site is located at the convergence of the pseudo dyad-related alpha -helices at the center of the molecule. The buried active site is accessed by a tunnel that accommodates the 4'-phosphopantetheine prosthetic group of ACP (and also CoA in the case of FabH). The active site is functionally and architecturally divided into halves, and each half is associated with one of the duplicated motifs (20). The initial transacylation half-reaction, which attaches the acyl primer to the active site cysteine, is facilitated by an alpha -helix dipole and an oxyanion hole. The decarboxylation half-reaction, which transfers the acyl primer to malonyl-ACP, is accelerated by the formation of two adjacent hydrogen bonds to the thioester carbonyl of the incoming malonyl-ACP. The hydrogen bond donors are two histidines in the FabB/FabF class and a histidine and an asparagine in the FabH class. Also, the side chain of a conserved phenylalanine promotes the decarboxylation step in both types of enzymes. This scheme is supported by mutagenesis studies of FabH (20) and differs somewhat from the mechanisms proposed by others (19, 22).

Two natural products inhibit type II fatty acid synthesis by blocking the activity of one or more of the beta -ketoacyl-ACP synthases. Cerulenin is an irreversible inhibitor of beta -ketoacyl-ACP synthases I and II (23-25) and forms a covalent adduct with the active site cysteine (26). Cerulenin is not a selective antibacterial because it is also a potent inhibitor of the condensation reaction catalyzed by the mammalian multifunctional (type I) fatty-acid synthase (27, 28). However, cerulenin and related compounds have antineoplastic activity (29) and reduce food intake and body weight in mice (30). TLM is a unique thiolactone molecule that reversibly inhibits type II, but not type I, fatty-acid synthases (31, 32) and is effective against many pathogens. The antibiotic is not toxic to mice and affords significant protection against urinary tract and intraperitoneal bacterial infections (33). TLM is active against Gram-negative anaerobes associated with periodontal disease (34) and exhibits antimycobacterial action by virtue of its inhibition of mycolic acid synthesis (35). TLM also has activity against malaria (36) and trypanosomes (37), extending the potential for using this template as a platform to develop more antimicrobials.

We report the structures of the FabB-TLM and FabB-cerulenin complexes, and we identify structural features that define the differences in the biochemical mode of action and target selectivity of the two antibiotics. We have further validated our understanding of the mechanisms of antibiotic binding through the mutagenesis of a key residue involved in the protein-drug interaction, and the subsequent assay of the mutant. This work contributes not only to the development of new antibacterials that target the condensation step in type II fatty acid synthesis but also to the understanding of the condensation reaction mechanism.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Sources of supplies are as follows: [14C]malonyl-CoA (specific activity, 55.0 Ci/mol) and [14C]acetyl-CoA (specific activity, 52.0 Ci/mol) from Amersham Pharmacia Biotech; microbiological media from Difco; molecular reagents from Promega; cerulenin and ACP from Sigma; Ni2+-agarose resin from Qiagen; pET vector and expression strains from Novagen; and pCR2.1 vector from Invitrogen. Proteins were quantitated by the Bradford method (38) unless otherwise indicated. Acyl-ACP was prepared using an established acyl-ACP synthetase method (14, 39, 40). All other supplies were reagent grade or better.

Purification and Assay of Condensing Enzymes-- The three condensing enzymes of E. coli and malonyl-CoA:ACP transacylase (FadD) were expressed and purified to homogeneity as described previously (13, 14, 41, 42). Purified enzymes were then dialyzed against 20 mM Tris-HCl, pH 7.6, 1 mM dithiothreitol, concentrated with an Amicon stirred cell, and stored in 50% glycerol at -20 °C.

A filter disc assay was used to assay FabH activity with [1-14C]acetyl-CoA as described previously (15, 18). The assays contained 50 µM ACP, 1 mM beta -mercaptoethanol, 45 µM [14C]acetyl-CoA (specific activity, 52.0 Ci/mol), and 50 µM malonyl-CoA, E. coli FabD (0.3 µg) and 0.1 M sodium phosphate buffer, pH 7.0, in a final volume of 40 µl. The reaction was initiated by the addition of FabH, and the mixture was incubated at 37 °C for 12 min. A 35-µl aliquot was removed and deposited on a Whatman 3MM filter disc. The discs were washed with three changes (20 ml/disc for 20 min) of ice-cold trichloroacetic acid. The concentration of the trichloroacetic acid was reduced from 10 to 5 to 1% in each successive wash. The filters were dried and counted in 3 ml of scintillation mixture.

FabB and FabF radiochemical assay was performed using the scheme devised by Garwin et al. (11) using myristoyl-ACP as the substrate. The assays contained 100 µM ACP, 0.3 mM dithiothreitol, 1 mM EDTA, 0.1 M potassium phosphate buffer, pH 6.8, 50 µM [14C]malonyl-CoA (specific activity 55 Ci/mol), 100 µM myristoyl-ACP, FabD (0.3 µg of protein), in a final volume of 20 µl. A mixture of ACP, 0.3 mM dithiothreitol, 1 mM EDTA, and the buffer was incubated at 37 °C for 30 min to ensure complete reduction ACP, and then the remaining components (except the condensing enzyme) were added. The mixture was then aliquoted into the assay tubes, and the reaction was initiated by the addition of FabB or FabF. The reaction mixture was incubated at 37 °C for 20 min, and then 400 µl of reducing agent (0.1 M K2HPO4, 0.4 M KCl, 30% tetrahydrofuran, and 5 mg/ml sodium borohydride) was added into the reaction tubes and incubated for 40 min. Finally, 400 µl of toluene was added and vigorously mixed, and 300 µl of upper phase solution was counted in 3 ml of scintillation mixture.

Assay for TLM Binding-- Kd values for TLM and the condensing enzymes were measured by fluorescence spectroscopy on a SLM-Amicon 8100 spectrofluorimeter (43). Quenching of the intrinsic protein fluorescence was measured with excitation at 280 nm and emission at 337 (FabB), 332 (FabF), or 321 nm (FabH). The concentration of each enzyme was 1 µM in 0.1 M sodium phosphate buffer, pH 7.5, and TLM was titrated in 2-µl aliquots from stock solutions in water. Each curve was corrected for the nominal absorption by TLM at both excitation and emission wavelengths and normalized. Data was then fitted to Equation 1 (43),


F<SUB>C</SUB>=F<SUB>o</SUB>+(&Dgr;F/2[E]) <FENCE>(K<SUB>d</SUB>+[E]+[<UP>TLM</UP>])−<RAD><RCD>((K<SUB>d</SUB>+[E]+[<UP>TLM</UP>])<SUP>2</SUP>−4[E][<UP>TLM</UP>])</RCD></RAD></FENCE> (Eq. 1)

where Fo is the initial fluorescence; Delta F is the change in fluorescence; [E] is the enzyme concentration; and [TLM] is the drug concentration. Each experiment was repeated several times and with the same results.

Construction of the FabB[H333N] Mutant-- A portion of the fabB gene was amplified using the polymerase chain reaction with two specific primers. The first primer introduced the desired mutation and extended over a unique AgeI site (5'-AAAGCCATGACCGGTAACTCTC-3'). The second primer created a BamHI site downstream of the stop codon (5'-GCAGGATCCGGCGATTGTCAATGATG-3'). The resulting fragment was sequenced to confirm that the mutation had been correctly introduced and then was digested with AgeI and BamHI and cloned into the pET-15b-FabB expression vector that had been digested with the same enzymes. The FabB[H333N] protein was expressed and purified as described above.

Structure Determination of the FabB-Cerulenin and FabB-TLM Complexes-- Both structures were solved using electron density difference maps. Pure FabB protein was dialyzed (10 mM Tris-HCl, pH 8.0, 1 mM dithiothreitol, 1 mM EDTA) and concentrated to 15 mg/ml. The inhibitors (TLM and cerulenin) were added directly to separate aliquots of the protein solution and were gently agitated for 1 h. The ratio of inhibitor molecules to FabB monomers was about 10:1. The FabB-antibiotic complexes were crystallized by the same procedure as followed for the native crystals (21). Crystals measuring 0.1 × 0.3 × 1.0 mm grew in 1-2 weeks. The crystals were mounted on standard nylon loops, passed through a cryoprotectant of 50% paratone-N, 50% mineral oil and were frozen directly in liquid nitrogen. Data were collected at 100 K using a Nonius FR591 x-ray generator and DIP 2030H detector system. All diffraction data were integrated using the HKL software package (44). Integrated data were merged and scaled using SCALEPACK. Crystals of both complexes have space group P212121 and cell dimensions similar to the native crystals (Tables I and II).

All refinements of models against our data were carried out using XPLOR (version 3.851) (45). First, the native FabB structure (21) was refined against our data, and then 2 mFo - DFc maps were calculated using CCP4 programs (46). To optimize the maps, the program DM (47) was used to perform histogram matching, solvent flattening, and 4-fold NCS averaging (there are 2 dimers in the asymmetric unit). Maps were examined using the program O (48) and were determined to be of good quality. Both maps clearly showed the presence of an antibiotic in the active site. The three-dimensional structures of the inhibitors were fit by hand into the electron density of one monomer and then extended by NCS operators into the other sites. For both antibiotics, all four sites showed a good fit between the map and the hand-fit molecule. Waters were picked using XPLOR and were visually inspected for good electron density and for sensible H-bonding geometry. Incorrectly assigned waters were rejected, and some additional waters were added by hand. Full scale refinements using NCS restraints were then performed. The residues to include in NCS restraints were chosen to be those residues not involved in crystal contacts, as determined by the XPLOR script "geomanal" and by a visual inspection of crystal packing. Two to three cycles of refinement followed by manual rebuilding of each model completed the structure determinations. The statistics of the final models are shown in Tables I and II.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Inhibition of Condensing Enzymes by TLM and Cerulenin-- The relative sensitivities of the condensing enzymes to TLM and cerulenin are known from the inhibition of the pathway in crude cell extracts and from analyses of growth inhibition in genetically modified E. coli strains. However, the activities of the purified enzymes have not been compared using natural substrates. Therefore, we determined the IC50 values for all three condensing enzymes for both TLM and cerulenin (Fig. 1). Each of the three condensing enzymes was inhibited by TLM (Fig. 1A). Under the in vitro assay conditions employed, FabF was the most sensitive enzyme (IC50 = 6 µM) followed by FabB (IC50 = 25 µM) and FabH, which was considerably less sensitive (IC50 = 110 µM). These data are consistent with genetic experiments that show that overexpression of FabB confers TLM resistance, whereas FabH overexpression does not (49). Increased expression of FabF blocks growth (50) precluding a similar experiment with this condensing enzyme. However, since FabF is not essential for the growth of E. coli (11), FabB is the physiologically relevant TLM target in this bacterium.



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Fig. 1.   Inhibition of E. coli condensing enzymes by TLM and cerulenin. A, the inhibition of FabF (), FabB (open circle ), and FabH (black-square) by TLM. The IC50 values are as follows: FabF, 6 µM; FabB, 25 µM; and FabH, 110 µM. B, the inhibition of FabF (), FabB (open circle ), and FabH (black-square) by cerulenin. The IC50 values are as follows: FabF, 20 µM; FabB, 3 µM; and FabH, >700 µM. The activities of the three condensing enzymes were compared using the radiochemical assays described under "Experimental Procedures."

As expected, both FabB and FabF were inhibited by cerulenin, with FabB being the most sensitive enzyme under our assay conditions (Fig. 1B). Since cerulenin forms an irreversible covalent complex with the FabB and FabF, the differences noted in Fig. 1B reflect the rate of complex formation under our assay conditions, and eventually both FabB and FabF will be completely inactivated by cerulenin. However, our experiments are consistent with previous work that examined the effect of cerulenin on fatty acid production in either normal E. coli strains, strains lacking FabF activity, or strains that overexpressed FabB, which all indicated that FabB was the more sensitive of the two enzymes in vivo (51, 52). FabH was not effectively inhibited by cerulenin, as reported previously for cell extracts (17).

TLM Is a Reversible Inhibitor-- The thiolactone structure in TLM suggested that it may form a covalent adduct with the condensing enzyme via a thioester exchange reaction. We examined this hypothesis by comparing the kinetics of TLM inhibition to that of cerulenin (Fig. 2), which is known to form a covalent complex with the active site cysteine of the condensing enzymes. The kinetics of cerulenin inhibition exhibited the hallmarks of a slow binding, irreversible inhibitor (53). When the reaction was initiated with enzyme, there was an initial burst of product formation, but the FabB reaction rate rapidly decreased and ceased by 20 min (Fig. 2). When cerulenin was preincubated with FabB and the reaction was initiated by the addition of the substrates, there was no discernible product formation. This indicates the formation of the irreversible FabB-cerulenin binary complex. The same two types of time course experiments were performed with TLM (Fig. 2), and there was no evidence for the formation of an irreversible or slow-binding FabB-TLM complex. To verify that TLM was a reversible inhibitor, we performed a dilution experiment in which FabB was first exposed to 100 µM TLM, which inhibits the enzyme about 95%, and then the TLM concentration was diluted to 1 µM, and the enzyme activity was recovered to the same value as when FabB was continually exposed to 1 µM TLM. These data verify the reversibility of TLM inhibition that was shown in previous experiments with cell extracts (32).



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Fig. 2.   FabB is reversibly inhibited by TLM and irreversibly inhibited by cerulenin. The time course for the inhibition of FabB activity under five experimental conditions was determined to address the reversibility of the antibiotics. Time course for the FabB reaction in the absence of inhibitors (black-triangle) and in the presence of 20 µM TLM ( or 3 µM cerulenin (open circle ) is shown. FabB was preincubated with either 20 µM TLM (black-square) or 3 µM cerulenin () for 30 min prior to initiation of the reaction by the addition of the other substrates. FabB activity was measured using the radiochemical assay with myristoyl-ACP as the substrate as described under "Experimental Procedures."

Direct Measurement of TLM Affinities-- The kinetic experiments reported in Fig. 1 illustrate the relative effectiveness of TLM as an inhibitor under standard assay conditions for the three condensing enzymes. However, the in vitro assays are different as are the substrate specificities of the enzymes, which precludes using the IC50 values as a direct comparison of the affinities of the enzymes for TLM. Therefore, we used fluorescence spectroscopy to measure directly the binding of TLM to the enzymes (Fig. 3). FabB exhibited the highest affinity (26 µM), FabF intermediate affinity (60 µM), and FabH (158 µM) the lowest affinity. Whereas the actual affinities of TLM for FabB and FabH were consistent with the kinetic experiments, the TLM affinity for FabF was lower than would be extrapolated from the IC50 determination.



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Fig. 3.   Direct measurement of TLM binding to the condensing enzymes. Binding of TLM to the condensing enzymes was measured by quenching of the intrinsic tryptophan fluorescence of the proteins as described under "Experimental Procedures." Data are presented as the observed minus initial fluorescence, normalized to the total change. The calculated Kd values are as follows: FabB (open circle ), 26 µM; FabB[H333N] (), 256 µM; FabF (), 60 µM; and FabH (triangle ), 158 µM.

The Structure of the FabB-TLM Binary Complex-- The final refined electron density of the complex clearly shows the conformation of the entire TLM molecule and all of its important interactions. The statistics of the final model are good (Table I). TLM makes a number of specific but noncovalent interactions within the FabB active site (Fig. 4). The C-9 and C-10 methyl groups are nestled within two hydrophobic pockets comprising phenylalanines 229 and 392 and Pro-272 and Phe-390, respectively. The isoprenoid moiety is wedged between two peptide bonds, 391-392 "below" and 271-272 "above," and this intercalated stacking interaction between three delocalized systems is clearly an important element of specificity. The ring of Pro-272 participates in this molecular sandwich by van der Waals interactions. The O-1 and O-2 exocyclic oxygens are both involved in hydrogen bonding interactions. O-1 interacts with the Nepsilon -2 nitrogens of the two active site histidines 298 and 333, and O-2 bonds to the carbonyl oxygen of residue 270 and the amide nitrogen of residue 305 through a lattice of water molecules. These water molecules are at the base of the active site tunnel, and the O-2 is appropriately oriented toward the tunnel. Finally, the thiolactone sulfur does not make any obvious specific interactions, but it is adjacent to the active site cysteine 163. 


                              
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Table I
Statistics of data collection and refinement for the FabB-TLM binary complex



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Fig. 4.   The structure of the FabB-TLM binary complex. A, stereo diagram of the complex. TLM is shown with magenta bonds. Secondary structural elements are shown in orange, as are all alpha  carbons. The atoms in TLM, the atoms in the active site residues (His-298, His-333, Cys-163, Phe-392), and important protein backbone atoms are color-coded as follows: carbon, black; nitrogen, blue; oxygen, red; and sulfur, yellow. Hydrophobic residues are shown green. Water molecules are shown as cyan. Hydrogen bonds are shown as dotted red lines. The native, unbound conformations of the active site residues are shown in purple. TLM forms hydrogen bonds with the two active site histidines, His-298 and His-333, and to a network of waters which is held in place by the carbonyl oxygen of Val-270 and by the amine group of Gly-305. TLM is further stabilized by the intercalation of its isoprenoid tail into the space between Pro-272 and its associated peptide bond and the peptide bond between Gly-391 and Phe-392. B, electron density of bound TLM. The electron density is contoured at the one sigma  level.

The exquisite fit of TLM into the FabB active site is reflected in the minimal distortion it causes to the native FabB structure. The movements that do occur involve local changes in the FabB main chain positions, but these do not extend beyond the immediate vicinity of the active site (Fig. 4). The side chain of Cys-163 shifts by 2.1 Å to avoid a clash with the TLM sulfur, and His-298 moves by 2 Å to improve the hydrogen bonding geometry to the O-1 of TLM. Finally, phenylalanines 390 and 392, and the associated loop comprising residues 388-394, all move by about 1.0 Å, and the side chain of Phe-392 also rotates ~40°. These latter movements correlate with the new location of His-298. A hydrogen bonding interaction between the Nepsilon -2 nitrogen and the carbonyl oxygen of residue 390 fixes the orientation of the histidine. When the histidine moves, the loop also moves to maintain this important interaction.

The Structure of the FabB-Cerulenin Complex-- In the final, refined electron density map, the conformation of the attached end of the cerulenin molecule is unambiguous (Fig. 5B). The electron density corresponding to the acyl chain of the inhibitor is weaker. This may be due to the conformation flexibility of the chain. The final model has good statistics (Table II). In contrast to TLM, cerulenin forms a covalent complex with FabB (Fig. 5). Also, with one major exception, it primarily occupies a different region of the active site than TLM. The covalent bond is formed between the central C-2 of cerulenin and the active site Cys-163. The O-2 and O-3s are crucial specificity determinants that form important hydrogen bonds. O-2 interacts with the Nepsilon -2s of the active site histidines 298 and 333 and is in the same position as the O-1 of TLM. O-3 is hydrogen-bonded to the amide nitrogens of residues 163 and 392. The O-1 and N-1 do not form hydrogen bonds but likely interact with the pi  electrons of the adjacent Phe-292 side chain. Finally, the extended acyl chain of cerulenin is located within a deep hydrophobic pocket at the dimer interface comprising Gly-107, Pro-110, Val-134' (prime refers to the other monomer), alanines 137' and 162, methionines 138' and 197, Phe-201, and Leu-335. No electron density was observed for C-12, indicating that the end of the cerulenin chain may be flexible.



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Fig. 5.   The structure of the FabB-cerulenin covalent complex. A, stereo diagram of the complex. Cerulenin is shown with yellow bonds. The rest of the coloring scheme in this figure is the same as in Fig. 3. Cerulenin forms a covalent bond with the active site cysteine (Cys-163). Its O-2 forms hydrogen bonds with the two active site histidines, His-298 and His-333. The O-3 sits in the oxyanion hole, forming hydrogen bonds to the amide of Phe-392 and the amide of Cys-163. The tail of cerulenin occupies a long hydrophobic cavity, which normally contains the growing acyl chain of the natural substrate. B, electron density of bound cerulenin. The electron density is contoured at the one sigma  level.


                              
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Table II
Statistics for data collection and refinement of the FabB-cerulenin binary complex

Like TLM, cerulenin occupies the active site with minimal distortion of the surrounding FabB structure, and the slight movements that do occur are similar to those observed in the TLM complex. Thus, Cys-163 moves to form the covalent bond; histidines 298 and 333 move to optimize the hydrogen bond interactions with the O-2, and phenylalanines 390 and 392 and their associated loop shift by ~1.5 Å in concert with the movement of His-298. Notably, residues in the deep hydrophobic pocket of FabB do not move upon cerulenin binding, whereas Ile-108 must rotate to accommodate the cerulenin chain in the FabF-cerulenin structure (54).

Importance of His-His Active Site Configuration in TLM Inhibition-- The structural information indicates that the strong hydrogen bond interactions between the two active site histidines and the O-1 of TLM are important determinants of high affinity TLM binding. The FabH condensing enzyme has a His-Asn configuration and was much less sensitive to TLM (Figs. 1 and 3). The importance of the two histidines was directly addressed by creating the FabB[H333N] mutant that converts the FabB active site into a FabH configuration. The specific activity of FabB[H333N] was reduced compared with FabB, but it still retained significant condensation activity (Fig. 6A). However, FabB[H333N] was significantly less sensitive to inhibition by both TLM (Fig. 6B) and cerulenin (Fig. 6C) in the standard biochemical assay. Accordingly, the affinity of FabB[H333N] for TLM was an order of magnitude lower than FabB (Fig. 3) supporting the importance of the two-histidine motif in TLM binding. These data indicate that the two-histidine active site architecture is an important determinant of the reactivity of condensing enzymes toward both TLM and cerulenin and that it contributes to the observed resistance of the FabH class of enzymes to these two antibiotics (Fig. 1).



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Fig. 6.   FabB[H333N] is resistant to TLM. A, the specific activities of FabB and FabB[H333N]. The specific activity of FabB (open circle ) was 425 ± 6 pmol/min/µg, whereas FabB[H333N] () had 1.6% of the condensation activity of the wild-type protein (6.6 ± 0.4 pmol/min/µg). B, a comparison of the inhibition of FabB[H333N] () and FabB (open circle ) by TLM. The IC50 for FabB was 25 µM compared with the higher IC50 of FabB[H333N] (350 µM). C, a comparison of the inhibition of FabB[H333N] () and FabB (open circle ) by cerulenin. The IC50 for FabB was 3 µM compared with the higher IC50 of FabB[H333N] (30 µM). The assays were performed using 14:0-ACP as the primer for the radiochemical assay described under "Experimental Procedures."



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The structural and biochemical analyses of the binding of TLM and cerulenin to FabB provide the framework for understanding the specificity of these antibiotics and provide clues for the development of more potent compounds that target type II fatty acid synthesis. Although the condensation enzymes have similar active sites, subtle structural variations define their differential response to these molecules. The sensitivity of the condensing enzymes to TLM inhibition is FabF > FabB FabH. The interactions that account for the slight difference in TLM binding to FabF and FabB is not clear. In contrast, there are two reasons why TLM should be a poor inhibitor of FabH. First, histidines 298 and 333 in FabB are replaced by His-244 and Asn-274 in FabH, and our FabB-TLM structure reveals that the two histidines form strong hydrogen bonds with the antibiotic (Fig. 4). To test the importance of this interaction, we converted the FabB active site into a FabH active site by constructing the FabB[H333N] mutant, and we showed that the absence of the histidine dyad decreases the sensitivity to TLM (Figs. 3 and 6). However, the exact mechanism for the lower TLM affinity is not entirely clear since the asparagine is capable of promoting the condensation reaction and could presumably donate a hydrogen bond to O-1 of TLM. Second, the peptide bond "sandwich" that binds the isoprenoid group may not be able to form in FabH. Specifically, there is no equivalent in FabH to the loop containing Pro-272 in FabB, and the sandwich cannot form.

The isoprenoid moiety in TLM takes advantage of a specific hydrophobic crevice that is present in the active sites of both FabB and FabF. However, these hydrophobic pockets extend further back into the interior of the proteins and are not optimally filled by the TLM side chain. This would explain the results of inhibition studies against plant (55) and mycobacterial (56) fatty-acid synthase systems that used TLM analogs in which the isoprenoid was replaced by various acyl chains. Analogs with longer, more flexible chains showed increased activity against FAS II in these organisms, and these longer chains may more completely fill the available space. Also, TLM analogs with shorter chains lacking the double bond are less active. It is worth noting that the analog studies assumed that the isoprenoid chain binds in the natural substrate pocket, and our structural studies clearly show that this is not the case. This structural insight has important implications for the design of more potent inhibitors against these enzymes.

In the case of cerulenin, the order of inhibition is FabB > FabF FabH. One reason cerulenin is a poor inhibitor of FabH is the fact that FabH lacks the substrate hydrophobic pocket to accommodate the acyl chain of the drug. This pocket is not required in E. coli FabH where the substrate acyl group is simply the initiating acetyl moiety. However, long chain acyl groups are accommodated by Mycobacterium tuberculosis FabH, and this enzyme is still resistant to cerulenin (16). This supports our finding that the His-His active site, as opposed to the FabH His-Asn active site configuration, is crucial for optimal cerulenin inhibition (Fig. 6). The reason why FabB is more susceptible than FabF is understood by comparing the FabB-cerulenin complex (Fig. 5) with the FabF-cerulenin complex (54). In the FabB-cerulenin complex, Gly-107 and Met-197 face each other in the substrate hydrophobic pocket and direct the cerulenin acyl chain toward strand beta 4 (to the back in Fig. 5). However, in the FabF-cerulenin complex, the steric configuration is reversed, and Ile-108 and Gly-198 direct the cerulenin tail away from strand beta 4 (to the front in Fig. 5). Furthermore, Ile-108 must swing around to accommodate the acyl chain in FabF, but Met-197 does not move in FabB. This required movement in FabF probably explains why cerulenin is a better inhibitor of FabB than of FabF. It is also possible that this structural difference between FabF and FabB relates to the differences in their substrate specificities and physiological functions. When the cerulenin structures of FabB and FabF are superimposed, they are identical except for the acyl chain that adopts these different positions. FabB catalyzes reactions in the elongation of short chain unsaturated fatty acid intermediates, and its active site must accommodate acyl chains with the characteristic kink imposed by the cis double bond. In contrast, FabF does not accept these intermediates. Thus, these cerulenin-bound structures provide a framework for modifying the substrate binding pocket by site-directed mutagenesis to define the important differences in the elongation condensing enzymes that define their physiological functions (57).

It is clear that FabB is the physiologically important TLM target in E. coli since FabF is not an essential enzyme (11), and elevated expression of the fabB gene confers TLM resistance whereas increased levels of FabH do not (49). In contrast, FabF-like proteins are the only elongation condensing enzymes expressed in many pathogens, such as Streptococcus pneumoniae and Staphylococcus aureus, and it is likely that FabF is the relevant TLM target in these organisms. Bacillus subtilis stands out as an organism that is uniquely resistant to TLM (58, 59). Accordingly, TLM is a very weak inhibitor of B. subtilis FabF in vitro.2 The nature of the subtle differences among these enzymes that determine TLM sensitivity are under investigation.

The structures of the FabB-TLM and FabB-cerulenin complexes support the division of the common active site of the enzymes into a transacylation half and a decarboxylation half based on our site-directed mutagenesis work (20). The location of the two antibiotics with respect to the two sides of the FabB active site is clearly seen in an overlay of the FabB-TLM and FabB-cerulenin structures (Fig. 7). The tail of cerulenin occupies the hydrophobic cavity that accommodates the fatty acid chain of the acyl-enzyme intermediate. On the other hand, the ring of TLM takes the place of the incoming malonyl group that participates in the decarboxylation reaction. The O-1 of TLM and O-2 of cerulenin are the only two atoms that occupy the same space in the two structures. In both cases, these carbonyl oxygens form strong hydrogen bonds with the active site histidine dyad, an important component of antibiotic interactions with the enzyme (Fig. 7). It is interesting to note that the movements in the FabB active site caused by TLM and cerulenin are very similar. The fact that these movements directly relate to the hydrogen bonding interactions of the inhibitors with the histidine dyad suggests that they also occur during substrate binding and are part of the active site mechanism. One possibility is that they represent communication between the two half-sites that promotes the ping-pong mechanism.



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Fig. 7.   Overlay of TLM and cerulenin in the FabB active site. The FabB-TLM and FabB-cerulenin structures were superimposed to illustrate the differences in the binding of the antibiotics in the active site. The coloring scheme in this figure is the same as in Figs. 3 and 4. TLM binds on the malonyl-ACP side and cerulenin occupies the acyl-enzyme intermediate half. The O-1 of TLM and O-2 of cerulenin are the only portions of the antibiotics that overlap in the structure, and they form hydrogen bonds with the His-His dyad in the active site. Note that the protein structure shown is that of the FabB-TLM complex. Binding of the two antibiotics results in essentially identical changes in the conformations of the active site residues.

Cerulenin mimics the condensation transition state (Fig. 8, lower panel). The C-2 forms a covalent bond with the active site cysteine; the O-3 mimics the substrate oxygen in the oxyanion hole formed by the amide nitrogens of residues 163 and 392, and the O-2 represents the carbonyl oxygen of the incoming malonyl group. The acyl chain mimics the location of the acyl-enzyme intermediate, and its location identifies the hydrophobic substrate-binding pocket.



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Fig. 8.   Schematic diagrams illustrating how cerulenin and TLM mimic substrates in the active site of FabB. Upper panel, the thiolactone ring of TLM mimics the bent conformation of the thiomalonate, and this is emphasized by the shaded atoms. The O-1s form hydrogen bonds with His-298 and His-333, and the C-1, C-2, and C-3s of malonate are mimicked by the C-1, C-2, and C-9s of TLM. The O-2 of TLM points out the active site tunnel that would be occupied by the pantetheine arm of the malonyl-ACP substrate. Lower panel, cerulenin mimics the condensation transition state and spans the two halves of the active site. The O-3 of cerulenin lies in the oxyanion hole formed by the amides of Cys-163 and Phe-392 enclosed by the phenyl side chain of Phe-392. This structure mimics the postulated location of the oxyanion of the tetrahedral transition state. The side chain of Cys-163 rotates in the cerulenin structure to form a covalent bond with C-2, but in the transition state, it is postulated to reside in the location observed in the native enzyme. The acyl chain of cerulenin feeds into the hydrophobic groove that accommodates the long chain acyl-enzyme intermediate.

In contrast, TLM mimics the noncovalently bound thiomalonate that enters the active site after the formation of the acyl-enzyme intermediate to participate in the decarboxylation/condensation half-step of the reaction (Fig. 8, upper panel). During the decarboxylation reaction, we proposed (20) that the thiomalonate is oriented such that the thioester carbonyl oxygen is hydrogen-bonded to the two acceptors (histidines 298 and 333 in the case of FabB), and the terminal carboxyl group is adjacent to a conserved phenylalanine (Phe-229 in the case of FabB). This promotes the movement of electrons away from the carboxyl group during decarboxylation and the formation of a carbanion at C-2 of malonate. This scheme requires that the thiomalonate be bent within the active site, and the TLM thiolactone ring mimics this bent structure. Thus, we propose that the ring sulfur, the O-1, and the C-9 of TLM corresponds to the positions of the thiol, the carbonyl oxygen, and the carboxylate carbon of the malonyl-ACP substrate, respectively. The isoprenoid TLM side chain nestles into a hydrophobic side pocket in the active site tunnel and does not represent a structural feature of the malonyl-ACP substrate. The function, if any, of the hydrophobic pocket and pi  stacking interaction exploited by TLM for high affinity binding in the condensing enzyme reaction cycle remains enigmatic.


    ACKNOWLEDGEMENT

We thank C. Hornsby for technical assistance.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM34496 (to C. O. R.) and GM44973 (to S. W. W.), Cancer Center (CORE) Support Grant CA 21765, and the American Lebanese Syrian Associated Charities.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1FJ4 and 1FJ8) 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: Dept. of Biochemistry, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. Tel.: 901-495-3491; Fax: 901-525-8025; E-mail: charles.rock@stjude.org.

Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M007101200

2 K.-C. Choi and C. O. Rock, unpublished observations.


    ABBREVIATIONS

The abbreviations used are: ACP, acyl carrier protein; FabB, beta -ketoacyl-ACP synthase I; FabF, beta -ketoacyl-ACP synthase II; FabH, beta -ketoacyl-ACP synthase III; TLM, thiolactomycin, [(4S)(2E,5E)]-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene-4-thiolide; cerulenin, (2R,3S)-2,3-epoxy-4-oxo-7,10-dodecandienolyamide.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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