Inhibition of β-Ketoacyl-Acyl Carrier Protein Synthases by Thiolactomycin and Cerulenin
2001; Elsevier BV; Volume: 276; Issue: 9 Linguagem: Inglês
10.1074/jbc.m007101200
ISSN1083-351X
AutoresAllen C. Price, Keum-Hwa Choi, Richard J. Heath, Zhenmei Li, Stephen W. White, Charles O. Rock,
Tópico(s)Microbial Natural Products and Biosynthesis
ResumoThe β-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 π 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. The β-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 π 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. acyl carrier protein β-ketoacyl-ACP synthase I β-ketoacyl-ACP synthase II β-ketoacyl-ACP synthase III thiolactomycin, [(4S)(2E,5E)]-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene-4-thiolide (2R,3S)-2,3-epoxy-4-oxo-7,10-dodecandienolyamide 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 (1Smith S. FASEB J. 1994; 8: 1248-1259Crossref PubMed Scopus (513) Google Scholar). 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 (2Rock C.O. Cronan Jr., J.E. Biochim. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (289) Google Scholar, 3Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtis R. Gross C.A. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W. Riley M. Schaechter M. Umbarger H.E. Biosynthesis of Membrane Lipids. American Society for Microbiology, Washington, D. C.1996: 612-636Google Scholar). Triclosan (4McMurray L.M. Oethinger M. Levy S. Nature. 1998; 394: 531-532Crossref PubMed Scopus (824) Google Scholar, 5Heath R.J., Yu, Y.-T. Shapiro M.A. Olson E. Rock C.O. J. Biol. Chem. 1998; 273: 30316-30321Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar) and isoniazid (6Dessen A. Quémard A. Blanchard J.S. Jacobs Jr., W.R. Sacchettini J.C. Science. 1995; 267: 1638-1641Crossref PubMed Scopus (395) Google Scholar) are two commonly used antibacterial agents that target fatty acid synthesis. The type II system has been most extensively studied inEscherichia coli where the three β-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 β-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 (7Rosenfeld I.S. D'Agnolo G. Vagelos P.R. J. Biol. Chem. 1973; 248: 2452-2460Abstract Full Text PDF PubMed Google Scholar, 8D'Agnolo G. Rosenfeld I.S. Vagelos P.R. J. Biol. Chem. 1975; 250: 5289-5294Abstract Full Text PDF PubMed Google Scholar). Synthase II (FabF) controls the temperature-dependent regulation of fatty acid composition (9Garwin J.L. Klages A.L. Cronan Jr., J.E. J. Biol. Chem. 1980; 255: 3263-3265Abstract Full Text PDF PubMed Google Scholar, 10de Mendoza D. Cronan Jr., J.E. Trends Biochem. Sci. 1983; 8: 49-52Abstract Full Text PDF Scopus (136) Google Scholar). Mutants lacking synthase II (fabF) are deficient in the elongation of palmitoleate to cis-vaccenate but grow normally under standard culture conditions (9Garwin J.L. Klages A.L. Cronan Jr., J.E. J. Biol. Chem. 1980; 255: 3263-3265Abstract Full Text PDF PubMed Google Scholar, 11Garwin J.L. Klages A.L. Cronan Jr., J.E. J. Biol. Chem. 1980; 255: 11949-11951Abstract Full Text PDF PubMed Google Scholar, 12Gelmann E.P. Cronan Jr., J.E. J. Bacteriol. 1972; 112: 381-387Crossref PubMed Google Scholar). 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 (13Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 1833-1836Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 14Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 10996-11000Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 15Choi K.-H. Heath R.J. Rock C.O. J. Bacteriol. 2000; 182: 365-370Crossref PubMed Scopus (202) Google Scholar, 16Choi K.-H. Kremer L. Besra G.S. Rock C.O. J. Biol. Chem. 2000; 275: 28201-28207Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Unlike FabB and FabF, FabH enzymes use an acyl-CoA rather than an acyl-ACP as the primer (15Choi K.-H. Heath R.J. Rock C.O. J. Bacteriol. 2000; 182: 365-370Crossref PubMed Scopus (202) Google Scholar, 16Choi K.-H. Kremer L. Besra G.S. Rock C.O. J. Biol. Chem. 2000; 275: 28201-28207Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 17Jackowski S. Rock C.O. J. Biol. Chem. 1987; 262: 7927-7931Abstract Full Text PDF PubMed Google Scholar, 18Tsay J.-T. Oh W. Larson T.J. Jackowski S. Rock C.O. J. Biol. Chem. 1992; 267: 6807-6814Abstract Full Text PDF PubMed Google Scholar). FabH is further distinguished by a His-Asn-Cys (19Qiu X. Janson C.A. Konstantinidis A.K. Nwagwu S. Silverman C. Smith W.W. Khandekar S. Lonsdale J. Abdel-Meguid S.S. J. Biol. Chem. 1999; 274: 36465-36471Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 20Davies C. Heath R.J. White S.W. Rock C.O. Structure. 2000; 8: 185-195Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) catalytic triad in contrast to the His-His-Cys triad in the FabB (21Olsen J.G. Kadziola A. Wettstein-Knowles P. Siggaard-Andersen M. Lindquist Y. Larsen S. FEBS Lett. 1999; 460: 46-52Crossref PubMed Scopus (102) Google Scholar) and FabF (22Huang W. Jia J. Edwards P. Dehesh K. Schneider G. Lindqvist Y. EMBO J. 1998; 17: 1183-1191Crossref PubMed Scopus (178) Google Scholar) enzymes. The crystal structures of all three condensing enzymes from E. coli (FabB, FabF, and FabH) have now been determined (19Qiu X. Janson C.A. Konstantinidis A.K. Nwagwu S. Silverman C. Smith W.W. Khandekar S. Lonsdale J. Abdel-Meguid S.S. J. Biol. Chem. 1999; 274: 36465-36471Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 20Davies C. Heath R.J. White S.W. Rock C.O. Structure. 2000; 8: 185-195Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 21Olsen J.G. Kadziola A. Wettstein-Knowles P. Siggaard-Andersen M. Lindquist Y. Larsen S. FEBS Lett. 1999; 460: 46-52Crossref PubMed Scopus (102) Google Scholar, 22Huang W. Jia J. Edwards P. Dehesh K. Schneider G. Lindqvist Y. EMBO J. 1998; 17: 1183-1191Crossref PubMed Scopus (178) Google Scholar). 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 α-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 (20Davies C. Heath R.J. White S.W. Rock C.O. Structure. 2000; 8: 185-195Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The initial transacylation half-reaction, which attaches the acyl primer to the active site cysteine, is facilitated by an α-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 (20Davies C. Heath R.J. White S.W. Rock C.O. Structure. 2000; 8: 185-195Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) and differs somewhat from the mechanisms proposed by others (19Qiu X. Janson C.A. Konstantinidis A.K. Nwagwu S. Silverman C. Smith W.W. Khandekar S. Lonsdale J. Abdel-Meguid S.S. J. Biol. Chem. 1999; 274: 36465-36471Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 22Huang W. Jia J. Edwards P. Dehesh K. Schneider G. Lindqvist Y. EMBO J. 1998; 17: 1183-1191Crossref PubMed Scopus (178) Google Scholar). Two natural products inhibit type II fatty acid synthesis by blocking the activity of one or more of the β-ketoacyl-ACP synthases. Cerulenin is an irreversible inhibitor of β-ketoacyl-ACP synthases I and II (23Vance D.E. Goldberg I. Mitsuhashi O. Bloch K. Omura S. Nomura S. Biochem. Biophys. Res. Commun. 1972; 48: 649-656Crossref PubMed Scopus (213) Google Scholar, 24D'Agnolo G. Rosenfeld I.S. Awaya J. Omura S. Vagelos P.R. Biochim. Biophys. Acta. 1973; 326: 155-166Crossref PubMed Scopus (183) Google Scholar, 25Kawaguchi A. Tomoda H. Nozoe S. Omura S. Okuda S. J. Biochem. (Tokyo). 1982; 92: 7-12Crossref PubMed Scopus (60) Google Scholar) and forms a covalent adduct with the active site cysteine (26Kauppinen S. Siggaard-Anderson M. van Wettstein-Knowles P. Carlsburg. Res. Commun. 1988; 53: 357-370Crossref PubMed Scopus (120) Google Scholar). 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 (27Omura S. Microbiol. Rev. 1976; 40: 681-697Google Scholar, 28Omura S. Methods Enzymol. 1981; 72: 520-532Crossref PubMed Scopus (102) Google Scholar). However, cerulenin and related compounds have antineoplastic activity (29Kuhajda F.P. Pizer E.S. Li J.N. Mani N.S. Frehywot G.L. Townsend C.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3450-3454Crossref PubMed Scopus (507) Google Scholar) and reduce food intake and body weight in mice (30Loftus T.M. Jaworsky D.E. Frehywot G.L. Townsend C.A. Ronnett G.V. Lane M.D. Kuhajda F.P. Science. 2000; 288: 2379-2381Crossref PubMed Scopus (825) Google Scholar). TLM is a unique thiolactone molecule that reversibly inhibits type II, but not type I, fatty-acid synthases (31Hayashi T. Yamamoto O. Sasaki H. Okazaki H. J. Antibiot. (Tokyo). 1984; 37: 1456-1461Crossref PubMed Scopus (43) Google Scholar, 32Hayashi T. Yamamoto O. Sasaki H. Kawaguchi A. Okazaki H. Biochem. Biophys. Res. Commun. 1983; 115: 1108-1113Crossref PubMed Scopus (82) Google Scholar) and is effective against many pathogens. The antibiotic is not toxic to mice and affords significant protection against urinary tract and intraperitoneal bacterial infections (33Miyakawa S. Suzuki K. Noto T. Harada Y. Okazaki H. J. Antibiot. (Tokyo). 1982; 35: 411-419Crossref PubMed Scopus (120) Google Scholar). TLM is active against Gram-negative anaerobes associated with periodontal disease (34Hamada S. Fujiwara T. Shimauchi H. Ogawa T. Nishihara T. Koga T. Neheshi T. Matsuno T. Oral Microbiol. Immunol. 1990; 5: 340-345Crossref PubMed Scopus (17) Google Scholar) and exhibits antimycobacterial action by virtue of its inhibition of mycolic acid synthesis (35Slayden R.A. Lee R.E. Armour J.W. Cooper A.M. Orme I.M. Brennan P.J. Besra G.S. Antimicrob. Agents Chemother. 1996; 40: 2813-2819Crossref PubMed Google Scholar). TLM also has activity against malaria (36Waller R.F. Keeling P.J. Donald R.G.K. Striepen B. Handman E. Kang-Unnasch N. Cowman A.F. Besra G.S. Roos D. McFadden G.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12352-12357Crossref PubMed Scopus (636) Google Scholar) and trypanosomes (37Morita Y.S. Paul K.S. Englund P.T. Science. 2000; 288: 140-143Crossref PubMed Scopus (108) Google Scholar), 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. 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 (38Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214435) Google Scholar) unless otherwise indicated. Acyl-ACP was prepared using an established acyl-ACP synthetase method (14Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 10996-11000Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 39Rock C.O. Garwin J.L. Cronan Jr., J.E. Methods Enzymol. 1981; 72: 397-403Crossref PubMed Scopus (44) Google Scholar, 40Rock C.O. Garwin J.L. J. Biol. Chem. 1979; 254: 7123-7128Abstract Full Text PDF PubMed Google Scholar). All other supplies were reagent grade or better. The three condensing enzymes of E. coli and malonyl-CoA:ACP transacylase (FadD) were expressed and purified to homogeneity as described previously (13Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 1833-1836Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 14Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 10996-11000Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 41Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 42Heath R.J. Rock C.O. J. Biol. Chem. 1995; 270: 26538-26542Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Purified enzymes were then dialyzed against 20 mm Tris-HCl, pH 7.6, 1 mmdithiothreitol, 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 (15Choi K.-H. Heath R.J. Rock C.O. J. Bacteriol. 2000; 182: 365-370Crossref PubMed Scopus (202) Google Scholar, 18Tsay J.-T. Oh W. Larson T.J. Jackowski S. Rock C.O. J. Biol. Chem. 1992; 267: 6807-6814Abstract Full Text PDF PubMed Google Scholar). The assays contained 50 μm ACP, 1 mmβ−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. (11Garwin J.L. Klages A.L. Cronan Jr., J.E. J. Biol. Chem. 1980; 255: 11949-11951Abstract Full Text PDF PubMed Google Scholar) using myristoyl-ACP as the substrate. The assays contained 100 μm ACP, 0.3 mm dithiothreitol, 1 mm EDTA, 0.1 mpotassium 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 (0.1m 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. Kd values for TLM and the condensing enzymes were measured by fluorescence spectroscopy on a SLM-Amicon 8100 spectrofluorimeter (43Anderson K.S. Sikorski J.A. Johnson K.A. Biochemistry. 1988; 27: 1604-1610Crossref PubMed Scopus (115) Google Scholar). 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 0.1m 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(43Anderson K.S. Sikorski J.A. Johnson K.A. Biochemistry. 1988; 27: 1604-1610Crossref PubMed Scopus (115) Google Scholar), FC=Fo+(ΔF/2[E])(Kd+[E]+[TLM])−((Kd+[E]+[TLM])2−4[E][TLM])Equation 1 where Fo is the initial fluorescence; ΔF 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. A portion of thefabB 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 aBamHI 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. Both structures were solved using electron density difference maps. Pure FabB protein was dialyzed (10 mmTris-HCl, pH 8.0, 1 mm dithiothreitol, 1 mmEDTA) 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 (21Olsen J.G. Kadziola A. Wettstein-Knowles P. Siggaard-Andersen M. Lindquist Y. Larsen S. FEBS Lett. 1999; 460: 46-52Crossref PubMed Scopus (102) Google Scholar). 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 (44Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38445) Google Scholar). 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).Table IStatistics of data collection and refinement for the FabB-TLM binary complexData collectionParameterSpace groupP212121Cell dimensions (Å)a, 59.1,b, 139.0, c, 211.9Resolution range (Å)19.96–2.35 (2.40–2.35)1-aParameters in parentheses refer to the outer resolution shell.Multiplicity4.1 (3.7)Rsym1-bRsym = ΣΣ‖Ii −Im‖/ΣΣIi, whereIi is the intensity of the measured reflection, andIm is the mean intensity of all symmetry-related reflections.14.8 (56.6)I/ς8.3 (2.0)Completeness (%)95.5 (92.1)Reflections626,204Unique reflections75,201RefinementResolution range of data included (Å)19.96–2.35Number of reflections in working set (Rwork)69,481Number of reflections in test set (Rfree)5720Number of protein atoms in asymmetric unit11,824Number of TLM atoms in asymmetric unit56Number of water molecules in asymmetric unit306Rwork (%)19.7Rfree (%)25.3Root mean square deviations from ideal stereochemistryBond lengths (Å)0.007Bond angles (°)1.35Dihedrals (°)26.3Impropers (°)0.70Mean B factor (main chain) (Å2)19.2Root mean square deviation in main chain B factor (Å2)5.3Mean B factor (side chains and waters) (Å2)19.3Root mean square deviation in side chain B factors (Å2)7.9Ramachandran plot:Residues in most favored region (%)89.1Residues in additionally allowed region (%)9.4Residues in generously allowed regions (%)1.2Residues in disallowed regions (%)0.3 (1 residue)1-a Parameters in parentheses refer to the outer resolution shell.1-b Rsym = ΣΣ‖Ii −Im‖/ΣΣIi, whereIi is the intensity of the measured reflection, andIm is the mean intensity of all symmetry-related reflections. Open table in a new tab Table IIStatistics for data collection and refinement of the FabB-cerulenin binary complexData collectionParametersSpace groupP212121Cell dimensions (Å)a, 59.2, b, 139.6, c, 212.2Resolution range (Å)19.88–2.27 (2.31–2.27) 2-aParameters in parentheses refer to the outer resolution shell.Multiplicity4.1 (3.6)Rsym2-bRsym = ΣΣ‖Ii −Im‖/ΣΣIi, whereIi is the intensity of the measured reflection, andIm is the mean intensity of all symmetry-related reflections.9.2 (33.9)I/ς17.0 (3.7)Completeness (%)97.3 (91.8)Reflections741083Unique reflections79821RefinementResolution range of data included (Å)19.88–2.27Number of reflections in working set (Rwork)71,817Number of reflections in test set (Rfree)8004Number of protein atoms in asymmetric unit11,824Number of CER atoms in asymmetric unit64Number of water molecules in asymmetric unit295Rwork (%)23.0Rfree (%)26.5Root mean square deviations from ideal stereochemistry:Bond lengths (Å)0.008Bond angles (°)1.39Dihedrals (°)26.3Impropers (°)0.74Mean B factor (main chain) (Å2)20.4Root mean square deviation in main chain B factor (Å2)5.9Mean B factor (side chains and waters) (Å2)19.7Root mean square deviation in side chain B factors (Å2)8.2Ramachandran plot:Residues in most favored region (%)87.6Residues in additionally allowed region (%)11.5Residues in generously allowed regions (%)0.6Residues in disallowed regions (%)0.3 (1 residue)2-a Parameters in parentheses refer to the outer resolution shell.2-b Rsym = ΣΣ‖Ii −Im‖/ΣΣIi, whereIi is the intensity of the measured reflection, andIm is the mean intensity of all symmetry-related reflections. Open table in a new tab All refinements of models against our data were carried out using XPLOR (version 3.851) (45Brünger A.T. X-PLOR, A System for X-ray Crystallography and NMR, version 3.851. Yale University Press, New Haven, CT1992Google Scholar). First, the native FabB structure (21Olsen J.G. Kadziola A. Wettstein-Knowles P. Siggaard-Andersen M. Lindquist Y. Larsen S. FEBS Lett. 1999; 460: 46-52Crossref PubMed Scopus (102) Google Scholar) was refined against our data, and then 2 mFo −DFc maps were calculated using CCP4 programs (46Collaborative Computation Project, N. 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19730) Google Scholar). To optimize the maps, the program DM (47Cowtan K. Joint CCP4 and ESF-EACBM Newslett. on Protein Crystallogr. 1994; 31: 34-38Google Scholar) 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 (48Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13006) Google Scholar) 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. 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. 1 A). 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 (49Tsay J.-T. Rock C.O. Jackowski S. J. Bacteriol. 1992; 174: 508-513Crossref PubMed Google Scholar). Increased expression of FabF blocks growth (50Subrahmanyam S. Cronan Jr., J.E. J. Biol. Chem. 1998; 180: 4596-4602Google Scholar) precluding a similar experiment with this condensing enzyme. However, since FabF is not essential for the growth of E. coli (11Garwin J.L. Klages A.L. Cronan Jr., J.E. J. Biol. Chem. 1980; 255: 11949-11951Abstract Full Text PDF PubMed Google Scholar), FabB is the physiologically relevant TLM target in this bacterium. As expected, both FabB and FabF were inhibited by cerulenin, with FabB being the most sensitive enzyme under our assay conditions (Fig.1 B). Since cerulenin forms an irreversible covalent complex with the FabB and FabF, the differences noted in Fig. 1 Bref
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