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• W2058707711 abstract "Mycolic acids are vital components of theMycobacterium tuberculosis cell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performs de novo fatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two β-ketoacyl-ACP synthase (KAS) enzymes of the M. tuberculosis FASII system. KasA and KasB were expressed in E. coli and purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use of M. tuberculosis AcpM, suggesting that structural differences between AcpM and E. coli ACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery. Mycolic acids are vital components of theMycobacterium tuberculosis cell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performs de novo fatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two β-ketoacyl-ACP synthase (KAS) enzymes of the M. tuberculosis FASII system. KasA and KasB were expressed in E. coli and purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use of M. tuberculosis AcpM, suggesting that structural differences between AcpM and E. coli ACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery. isoniazid acyl carrier protein β-ketoacyl-ACP synthase thiolactomycin fatty acid synthase isopropyl β-d-thiogalactopyranoside dithiothreitol 3-[N-morpholino]propanesulfonic acid 2-[N-morpholino]ethanesulfonic acid cerulenin 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid The cell wall of Mycobacterium tuberculosis is a complex structure containing many components that contribute to the communication between the bacterial cell and its host (1Barry III, C.E. Trends Microbiol. 2001; 9: 237-241Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) as well as its structural integrity and characteristic impermeability (2Liu J. Barry III, C.E. Besra G.S. Nikaido H. J. Biol. Chem. 1996; 271: 29545-29551Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 3Liu J. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4011-4016Crossref PubMed Scopus (84) Google Scholar, 4Wang L. Slayden R.A. Barry III, C.E. Liu J. J. Biol. Chem. 2000; 275: 7224-7229Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In fact, the innate impermeability displayed by M. tuberculosisto many common broad spectrum antibiotics and other small hydrophilic molecules can be directly attributed to the hydrophobic nature of the cell envelope (4Wang L. Slayden R.A. Barry III, C.E. Liu J. J. Biol. Chem. 2000; 275: 7224-7229Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The unique core structure of the cell wall consists of a covalently linked complex of peptidoglycan, arabinogalactan, and mycolic acids (5Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1563) Google Scholar, 6Kremer L. Dover L.G. Morehouse C. Hitchin P. Everett M. Morris H.R. Dell A. Brennan P.J. McNeil M.R. Flaherty C. Duncan K. Besra G.S. J. Biol. Chem. 2001; 276: 26430-26440Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 7McNeil M. Daffé M. Brennan P.J. J. Biol. Chem. 1991; 266: 13217-13233Abstract Full Text PDF PubMed Google Scholar). The latter, which are high molecular weight, α-alkyl, β-hydroxy fatty acids, are the largest fatty acids in nature, ranging from 70 to 90 carbons (for reviews, see Refs. 5Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1563) Google Scholar and 8Barry III, C.E. Lee R.E. Mdluli K. Sampson A.E. Schroeder B.J. Slayden R.A. Yuan Y. Prog. Lipid Res. 1998; 37: 143-179Crossref PubMed Scopus (451) Google Scholar). In addition to their characteristically long acyl chains, M. tuberculosis mycolic acids also contain a variety of functionalities, including desaturations and cyclopropyl rings, α-methyl-branched methyl ethers, and α-methyl-branched ketones which define the α-, methoxy-, and ketomycolates, respectively (8Barry III, C.E. Lee R.E. Mdluli K. Sampson A.E. Schroeder B.J. Slayden R.A. Yuan Y. Prog. Lipid Res. 1998; 37: 143-179Crossref PubMed Scopus (451) Google Scholar,9Glickman M.S. Cahill S.M. Jacobs Jr., W.R. J. Biol. Chem. 2001; 276: 2228-2233Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The low permeability of the cell can be directly attributed to the nature of functional groups in mycolic acids and their effect on the fluidity of the cell wall (2Liu J. Barry III, C.E. Besra G.S. Nikaido H. J. Biol. Chem. 1996; 271: 29545-29551Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 5Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1563) Google Scholar, 10Jarlier V. Nikaido H. FEMS Microbiol. Lett. 1994; 123: 1-8Crossref Scopus (433) Google Scholar). Variations in functional group structure can also have effects on the ability of M. tuberculosis to grow in macrophages (11Yuan Y. Zhu Y. Crane D.D. Barry III, C.E. Mol. Microbiol. 1998; 29: 1449-1458Crossref PubMed Scopus (145) Google Scholar). In addition to the influence of mycolic acids on cell wall structure and function, cell wall components such as trehalose dimycolate (cord factor) have been shown to exhibit immunomodulatory activities that can enhance the pathogenicity of M. tuberculosis (12Bloch H. J. Exp. Med. 1950; 91: 197-217Crossref PubMed Scopus (163) Google Scholar). The importance of mycolic acids to bacterial survival and pathogenesis has generated much interest in the enzymes responsible for their biosynthesis, based on the hypothesis that inhibitors of these proteins will be potential antimycobacterial agents. Indeed, the cessation of mycolic acid synthesis is one of the primary effects of isoniazid (INH),1 a front-line anti-tuberculosis drug (13Takayama K. Wang L. David H.L. Antimicrob. Agents Chemother. 1972; 2: 29-35Crossref PubMed Scopus (230) Google Scholar). Various other compounds including ethionamide (14Quémard A. Laneele G. Lacave C. Antimicrob. Agents Chemother. 1992; 36: 1316-1321Crossref PubMed Scopus (49) Google Scholar, 15Winder F.G. Collins P.B. Whelan D. J. Gen. Microbiol. 1971; 66: 379-380Crossref PubMed Scopus (70) Google Scholar), isoxyl (15Winder F.G. Collins P.B. Whelan D. J. Gen. Microbiol. 1971; 66: 379-380Crossref PubMed Scopus (70) Google Scholar, 16Phetsuksiri B. Baulard A.R. Cooper A.M. Minnikin D.E. Douglas J.D. Besra G.S. Brennan P.J. Antimicrob. Agents Chemother. 1999; 43: 1042-1051Crossref PubMed Google Scholar), and thiolactomycin (TLM) (17Slayden 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), have also been shown to inhibit mycolic acid synthesis. Biosynthesis of mycolic acid precursors requires the interaction of two fatty acid synthase (FAS) systems, the multifunctional polypeptide, FASI, and the dissociated FASII system, the latter composed of monofunctional enzymes and a discrete acyl carrier protein (ACP) 2The abbreviation ACP is typically used to describe acyl carrier protein. When the source of ACP is relevant,i.e. from either Escherichia coli or M. tuberculosis, the abbreviations ACPEc and AcpM are used, respectively, to differentiate the two. (8Barry III, C.E. Lee R.E. Mdluli K. Sampson A.E. Schroeder B.J. Slayden R.A. Yuan Y. Prog. Lipid Res. 1998; 37: 143-179Crossref PubMed Scopus (451) Google Scholar) (Fig. 1). Although the specific details of mycolic acid synthesis are not completely understood, the mycobacterial FASI system appears to be responsible for the de novosynthesis of C16–26 fatty acyl primers (18Bloch K. Methods Enzymol. 1975; 35: 84-90Crossref PubMed Scopus (48) Google Scholar), which are then passed to the FASII system and elongated to produce intermediates of the long meromycolate chain (19Qureshi N. Sathyamoorthy N. Takayama K. J. Bacteriol. 1984; 157: 46-52Crossref PubMed Google Scholar). Such intermediates can be modified and condensed with α-branch fatty acids to form mature mycolic acids (8Barry III, C.E. Lee R.E. Mdluli K. Sampson A.E. Schroeder B.J. Slayden R.A. Yuan Y. Prog. Lipid Res. 1998; 37: 143-179Crossref PubMed Scopus (451) Google Scholar). The availability of the M. tuberculosis genome sequence has allowed the identification of putative genes encoding proteins homologous to other bacterial FASII enzymes (8Barry III, C.E. Lee R.E. Mdluli K. Sampson A.E. Schroeder B.J. Slayden R.A. Yuan Y. Prog. Lipid Res. 1998; 37: 143-179Crossref PubMed Scopus (451) Google Scholar, 20Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry III, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver S. Osborne J. Quail M.A. Rajandream M.A. Rogers J. Rutter S. Seeger K. Skelton S. Squares S. Sqares R. Sulston J.E. Taylor K. Whitehead S. Barrel B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6522) Google Scholar). In turn, this has facilitated the characterization of several components of the M. tuberculosis FASII system, including FabH (21Choi K-H. Kremer L. Besra G.S. Rock C.O. J. Biol. Chem. 2000; 275: 28201-28207Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 22Scarsdale J.N. Kazanina G. He X. Reynolds K.A. Wright H.T. J. Biol. Chem. 2001; 276: 20516-20522Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), FabD (23Kremer L. Nampoothiri K.M. Lesjean S. Dover L.G. Graham S. Betts J. Brennan P.J. Minnikin D.E. Locht C. Besra G.S. J. Biol. Chem. 2001; 276: 27967-27974Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), and AcpM (24Schaeffer M.L. Agnihotri G. Kallender H. Brennan P.J. Lonsdale J.T. Biochim. Biophys. Acta. 2001; 1532: 67-78Crossref PubMed Scopus (37) Google Scholar). Of particular interest in the FASII system are the roles played by three condensing enzymes, FabH, KasA, and KasB. Recent reports describe the isolation of the FASII initiation enzyme FabH (21Choi K-H. Kremer L. Besra G.S. Rock C.O. J. Biol. Chem. 2000; 275: 28201-28207Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 22Scarsdale J.N. Kazanina G. He X. Reynolds K.A. Wright H.T. J. Biol. Chem. 2001; 276: 20516-20522Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) and show that this enzyme is inhibited by TLM but not by cerulenin (CER) (21Choi K-H. Kremer L. Besra G.S. Rock C.O. J. Biol. Chem. 2000; 275: 28201-28207Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). TLM also inhibits KasA and KasB, both putatively involved in fatty acid elongation, based on overexpression of the kasA andkasB genes in Mycobacterium bovis BCG, which resulted in increased resistance to TLM in vitro andin vivo (25Kremer L. Douglas J.D. Baulard A.R. Morehouse C. Guy M.R. Alland D. Dover L.G. Lakey J.H. Jacobs Jr., W.R. Brennan P.J. Minnikin D.E. Besra G.S. J. Biol. Chem. 2000; 275: 16857-16864Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Distinct roles for KasA and KasB in mycolic acid synthesis were postulated based upon increases in the incorporation of radioactivity into fatty acids in various FAS assays after overexpression of either kasA or kasB (25Kremer L. Douglas J.D. Baulard A.R. Morehouse C. Guy M.R. Alland D. Dover L.G. Lakey J.H. Jacobs Jr., W.R. Brennan P.J. Minnikin D.E. Besra G.S. J. Biol. Chem. 2000; 275: 16857-16864Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The antimycobacterial activity of TLM suggests the essentiality of at least one of the three condensing enzymes, as is the case for orthologs in other bacterial species such as E. coli FabB (26Garwin J.L. Klages A.L. Cronan Jr., J.E. J. Biol. Chem. 1980; 255: 3263-3265Abstract Full Text PDF PubMed Google Scholar) andStreptococcus pneumoniae FabH (27Khandekar S.S. Gentry D.R. Van Aller G.S. Warren P. Xiang H. Silverman C. Doyle M.L. Chambers P.A. Konstantinidis A.K. Brandt M. Daines R.A. Lonsdale J.T. J. Biol. Chem. 2001; 276: 30024-30030Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and FabF, 3H. Kallender, manuscript in preparation. thus making them attractive targets for the development of screens to identify new lead compounds. Therefore, we purified and characterized KasA and KasB, and despite many similarities, these enzymes display differing enzymatic properties and may indeed play distinct roles in mycolic acid biosynthesis. Chemicals and reagents were purchased from Sigma unless otherwise noted. All cloning steps were performed in E. coli DH5α cells purchased from Life Technologies, Inc. E. coli BL21(DE3) cells and expression vector pET28a(+) were purchased from Novagen. M. tuberculosis H37Rv genomic DNA was obtained from Dr. John Belisle at Colorado State University. Q-Sepharose and HiTrap chelating columns were purchased from Amersham Pharmacia Biotech. Materials for polyacrylamide gel electrophoresis were purchased from Novex. An AKTAprime liquid chromatography system (Amersham Pharmacia Biotech) was utilized for protein purification. Mass spectra were obtained using a SELDI protein biology system purchased from Ciphergen Biosystems, Inc. Enzyme assays were performed on a SpectraMax Plus 384 plate reader (Molecular Devices). Representative sequences among selected classes of related fatty acid biosynthetic enzymes were aligned with the MACAW manual sequence alignment program (28Schuler G.D. Altschul S.F. Lipman D.J. Proteins. 1991; 9: 180-190Crossref PubMed Scopus (895) Google Scholar). The alignments of conserved sequence blocks were evaluated with the segment pair overlap and Gibbs sampler options in MACAW based on the BLOSUM62 similarity matrix. Preliminary sequence data forEnterococcus faecalis sequences was obtained from The Institute for Genomic Research (TIGR) web site at www.tigr.org. The aligned sequences included: actinomycete KAS proteins (M. tuberculosis KasA, GI:1706746; Mycobacterium lepraeKasA, GI:3150229; M. tuberculosis KasB, GI:1706747; M. leprae KasB, GI:3150228; Streptomyces coelicolor Kas1, GI:125235), FabF proteins (E. coli, GI:729460;Moraxella catarrhalis (GlaxoSmithKline); Pseudomonas aeruginosa FabF1, GI:7433751; P. aeruginosa FabF2, GI:11347289; Bacillus subtilis, GI:7433750; E. faecalis FabF1 (TIGR), E. faecalis FabF2 (TIGR),Staphylococcus aureus, GI:13700788; S. pneumoniae, GI:9789234; Homo sapiens, GI:8923559;Drosophila melanogaster, GI:7296703; Caenorhabditis elegans, GI:7498864), FabB proteins (E. coli, GI:119783; Hemophilus influenzae, GI:1169589; M. catarrhalis (GlaxoSmithKline), P. aeruginosa, GI:11347502), and multi-domain fatty acid synthase proteins from actinomycetes (M. tuberculosis, GI:7447225; M. leprae, GI:4539127; Corynebacterium ammoniagenes, GI:580746), fungi (Saccharomyces cerevisiae, GI:119832;Candida albicans, GI:1169645, Schizosaccharomyces pombe, GI:3023732; Emericella nidulans, GI:2492657), and animals (H. sapiens, GI:1345959; Gallus gallus, GI:1345958; D. melanogaster, GI:7295848;C. elegans, GI:3876624). Positions with gaps or ambiguous alignment were compared with E. coli FabF and FabB crystal structures (1B3N (29Moche M. Schneider G. Edwards P. Dehesh K. Lindqvist Y. J. Biol. Chem. 1999; 274: 6031-6034Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), 1FJ8 (30Price A.C. Choi K.H. Heath R.J. Li Z. White S.W. Rock C.O. J. Biol. Chem. 2001; 276: 6551-6559Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar)). In each case, such positions were found to be in predicted loop regions that suggested structural inequivalence among the aligned sequences in these regions. Thus, these regions, including the non-homologous domains of the multi-domain fatty acid synthase proteins, were removed from the alignment before phylogenetic analyses. In the final edited alignment, 395 of the 414 amino acids of M. tuberculosis KasA were maintained. Phylogenetic analyses were based on the total number of sites in edited multiple-sequence alignments. Trees were constructed by neighbor-joining and maximum-parsimony methods. Neighbor-joining trees were built from pairwise distances between amino acid sequences based on the Dayhoff PAM120 substitution matrix (31Dayhoff M.O. Schwartz M. Orcutt B.C. Dayhoff M.O. Atlas of Protein Sequence and Structure. 5th Ed. National Biomedical Research Foundation, Washington, D. C.1978: 345-352Google Scholar) using the programs NEIGHBOR and PROTDIST of the PHYLIP 3.572c package (at genetics.washington.edu/phylip.html (32Felsenstein J. PHYLIP (Phylogeny Inference Package) Version 3.57c. Department of Genetics, University of Washington, Seattle1993Google Scholar)). The programs SEQBOOT and CONSENSE were used to estimate the confidence limits of branch points from 1000 bootstrap replicates. Maximum parsimony construction was accomplished with the software package PAUP*, version 4.0 (33Swofford D.L. PAUP*: Phylogenetic Analysis Using Parsimony, Version 4.0. Sinauer Associates, Sunderland, Mass1999Google Scholar). The number and length of minimal trees were estimated with 100 replicate random heuristic searches. Confidence limits for branch points were estimated from 1000 bootstrap replicates. To generate clones for expressing N-terminal hexahistidine-tagged KasA and KasB, the M. tuberculosis kasA and kasB genes (Rv2245 and Rv2246, respectively, GenBankTM accession number Z70692) were PCR-amplified from M. tuberculosis genomic DNA using standard PCR strategies (34Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1991) Current Protocols in Molecular Biology, Vol. 2, pp. 15.0.1-15.8.8, Greene and Wiley, New YorkGoogle Scholar) with Pfu Turbo DNA polymerase (Stratagene) and primers designed for directional cloning intoNheI/HindIII (kasA)- orNdeI/SacI (kasB)-digested pET28a(+) (5′-CGAGGCTTGAGGCCGAGCTAGCGTGAGTCAGCCTTC-3′, forwardkasA primer with NheI site underlined, and 5′-CCCGCGATGTCAAGCTTCAGTAACG-3′, reverse kasAprimer with HindIII site underlined, and 5′-GACATCGCGGGTCGCGAGGCATATGGTGGGGGTC-3′, forwardkasB primer with NdeI site underlined, and 5′-CTGTCGCGTAGAGCTCGGGTTTAGTACCG-3′, reversekasB primer with SacI site underlined). PCR programs consisted of an initial 5-min denaturation step (94 °C) followed by 25 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min and then a final elongation step at 72 °C for 7 min. PCR products were digested withNheI/HindIII (kasA) orNdeI/SacI (kasB) and ligated with vector using T4 DNA ligase (Life Technologies, Inc.) to produce pETkasA and pETkasB. Positive clones were identified by PCR screening using the above primers, and the plasmid insert was sequenced to assure authenticity of the construct. The pETkasA and pETkasB plasmids were electroporated into E. coli BL21(DE3) cells, and single colony transformants were grown at 37 °C to mid-log phase (A 600 = 0.7) in LB broth containing kanamycin (50 μg/ml) and glucose (1%). For KasA, the cells were induced with 0.3 mm IPTG at 37 °C for 2.5 h. KasB cultures were equilibrated to 18 °C and induced with 0.3 mm IPTG for ∼24 h at 18 °C. Cells were harvested by centrifugation, resuspended in Buffer A (50 mm sodium phosphate, pH 7.5, 500 mm NaCl, 10 mm imidazole, 5 mmβ-mercaptoethanol (5 ml/g)), and lysed by the addition of 2 mg of lysozyme followed by 3 cycles of freezing and thawing. The KasA lysate was centrifuged at 20,000 × g for 20 min, the supernatant was discarded, and the pellet was dissolved in 20 ml of Buffer A containing 8 m urea (Buffer A′). The urea solution was centrifuged at 27,000 × g, and the supernatant containing denatured KasA was loaded onto a 5-ml nickel-chelating column. The column was washed with 5 column volumes of Buffer A′ and eluted using a 100-ml linear gradient of 10–500 mm imidazole in Buffer A′. Fractions containing KasA were identified by SDS-PAGE, pooled, and diluted to an approximate concentration of 0.2 mg/ml in Buffer A′. KasA was refolded during stepwise dialyses (6–8-h/step) against 2 liters of 50 mmsodium phosphate, pH 7.5, 0.5 m NaCl, 2 mm DTT containing 4, 2, and 1 m urea and then dialyzed against 4 liters of 50 mm sodium phosphate, pH 7.5, 0.5 mNaCl, 2 mm DTT. Finally, the sample was dialyzed overnight against 2 liters of 50 mm Tris-HCl, pH 9.5, 0.3m NaCl, 2 mm DTT, and 50% glycerol and filtered through 0.45 μm filter. Aliquots of KasA were snap-frozen in liquid nitrogen and stored at −80 °C. The KasB lysate was centrifuged at 27,000 × g, and the pellet was discarded. The soluble lysate was loaded onto a 5-ml nickel-chelating column, washed with 5 column volumes of Buffer A, and eluted with a 100-ml linear gradient of 10–500 mmimidazole in Buffer A. Fractions containing KasB were identified by SDS-PAGE, pooled, and dialyzed against 4 liters of 50 mmsodium phosphate, 0.3 m NaCl, 2 mm DTT, 5% glycerol, after which KasB was divided into 100-μl aliquots, snap-frozen in liquid nitrogen, and stored at −80 °C. Lauroyl (C12:0)-, palmitoyl (C16:0)-, and arachidoyl (C20:0)-ACPEc and C16:0-AcpM substrates were generated using holo-ACPEc (or holo-AcpM), lauric, palmitic, or arachidic acid and the E. coli Aas enzyme as previously described (24Schaeffer M.L. Agnihotri G. Kallender H. Brennan P.J. Lonsdale J.T. Biochim. Biophys. Acta. 2001; 1532: 67-78Crossref PubMed Scopus (37) Google Scholar, 35Rock C.O. Garwin J.L. Cronan Jr., J.E. Methods Enzymol. 1981; 72: 379-403Google Scholar, 36Edwards P. Nelsen J.S. Metz J.G. Dehesh K. FEBS Lett. 1997; 402: 62-66Crossref PubMed Scopus (74) Google Scholar). Newly synthesized substrates were purified by anion exchange chromatography on Q-Sepharose columns and characterized by SELDI-MS, and the acyl-ACPs were quantitated by amino acid analysis and enzymatically with KasA. Butyryl (C4:0)- and malonyl-ACPEc were provided by Martin Brandt (GlaxoSmithKline). Malonyl-AcpM was synthesized using apo-AcpM, malonyl-CoA, and E. coli AcpS and purified by anion exchange chromatography. A continuous assay format was used to monitor KasA and KasB activity by coupling the condensing activity of the KAS enzymes to a β-ketoacyl-ACP reductase, either M. tuberculosis MabA or S. pneumoniae FabG (obtained from Joshua West, GlaxoSmithKline). Both MabA and FabG reduce β-ketoacyl-ACP intermediates to the corresponding β-hydroxyacyl-ACPs, enabling the reaction course to be monitored spectrophotometrically by following the oxidation of NADPH to NADP+ at 340 nm. MabA was purified by expressing themabA gene as an N-terminal hexahistidine-tagged protein inE. coli followed by affinity chromatography of the soluble fraction of the whole-cell lysate. 4M. L. Schaeffer, unpublished results. Although various concentrations of enzyme and ACP substrates were utilized in the experiments described, the details of which are included in the figure legends and text, typical KasA assays (100-μl total volume) contained 96–240 nm KasA, 3 μmMabA or FabG, 26–54 μm malonyl-ACPEc (AcpM), 6–10 μm acyl-ACPEc (AcpM), 50 μm NADPH, and 0.01% CHAPS in 50 mm HEPES buffer, pH 6.8. KasB reactions (100 μl total volume) contained 460–920 nm KasB, 3 μm MabA or FabG, 26–54 μm malonyl-ACPEc, 6–10 μmacyl-ACPEc, 50 μm NADPH, and 0.01% CHAPS in 50 mm sodium phosphate buffer, pH 7.0. For KasB assays using AcpM, typical conditions were as described above with 140–185 nm KasB, 6–10 μm acyl-AcpM, and 18–26 μm malonyl-AcpM. Reactions were preincubated for 5 min at 37 °C before initiation and allowed to proceed for 20 min at 37 °C unless specified in half-area 96-well microtiter plates (Costar). Initial rates were determined by measuring the decrease inA 340 at either 10- or 30-s intervals throughout the course of the reaction and expressed as pmol/min. The presence of a bacterial FASII system in M. tuberculosis is well established, and sequencing of the M. tuberculosisgenome confirmed the presence of numerous genes postulated to comprise a single FASII system (8Barry III, C.E. Lee R.E. Mdluli K. Sampson A.E. Schroeder B.J. Slayden R.A. Yuan Y. Prog. Lipid Res. 1998; 37: 143-179Crossref PubMed Scopus (451) Google Scholar, 18Bloch K. Methods Enzymol. 1975; 35: 84-90Crossref PubMed Scopus (48) Google Scholar, 20Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry III, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver S. Osborne J. Quail M.A. Rajandream M.A. Rogers J. Rutter S. Seeger K. Skelton S. Squares S. Sqares R. Sulston J.E. Taylor K. Whitehead S. Barrel B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6522) Google Scholar). Located within a cluster of FASII-related genes including fabD and acpM are two genes, kasA and kasB, that have been predicted to encode KAS enzymes (20Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry III, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver S. Osborne J. Quail M.A. Rajandream M.A. Rogers J. Rutter S. Seeger K. Skelton S. Squares S. Sqares R. Sulston J.E. Taylor K. Whitehead S. Barrel B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6522) Google Scholar, 25Kremer L. Douglas J.D. Baulard A.R. Morehouse C. Guy M.R. Alland D. Dover L.G. Lakey J.H. Jacobs Jr., W.R. Brennan P.J. Minnikin D.E. Besra G.S. J. Biol. Chem. 2000; 275: 16857-16864Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Also, sequence alignments of KasA and KasB along with E. coli FabF and other related structures suggest that KasA and KasB are related to FabF (25Kremer L. Douglas J.D. Baulard A.R. Morehouse C. Guy M.R. Alland D. Dover L.G. Lakey J.H. Jacobs Jr., W.R. Brennan P.J. Minnikin D.E. Besra G.S. J. Biol. Chem. 2000; 275: 16857-16864Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). In the present context, phylogenetic analysis was applied to examine the relationship of KasA and KasB to selected classes of FAS enzymes (Fig. 2). M. tuberculosis andM. leprae KasA sequences share 93% identity and 99% similarity, and their KasB sequences share 92% identity and 98% similarity (Table I). M. tuberculosis KasA and KasB sequences are also closely related to one another, sharing 67% identity and 86% similarity (Table I). Neighbor-joining and maximum parsimony methods (Fig. 2) showed strong statistical support in terms of bootstrap values and minimal-length trees for placing these enzymes together in a clade distinct from the other classes of fatty acid biosynthetic enzymes. Additionally, these phylogenetic and sequence homology analyses indicate that KasA and KasB are more closely related to FabF and FabB than to any of the multi-subunit fatty acid synthases.Table I% similarities and % identities of selected homologs to M. tuberculosis KasA and KasB proteinsProtein% identity to KasA% identity to KasB% similarity to KasA% similarity to KasBKas proteins M. tuberculosis KasA1006710086 M. tuberculosis KasB6710086100 M. leprae KasA93679986 M. leprae KasB67928698FabF proteins E. coli41366665 M. catarrhalis37366667 P. aeruginosa 138366665 P. aeruginosa 242406967 B. subtilis39366968 E. faecalis 137366664 E. faecalis 236376661 S. aureus38386868 S. pneumoniae36346463 H. sapiens35356263FabB proteins E. coli33345759 H. influenzae35356163 M. catarrhalis34355961 P. aeruginosa31325860FASI proteins M. tuberculosis23245147 M. leprae24235148 S. cerevisiae20214646 H. sapiens22245050Pairwise similarities and identities were determined based on the length of the shorter sequence without gaps from the edited alignment. Similarities were calculated based on the mutational difference matrix (MDM78) of Schwartz and Dayhoff (48Schwartz R.M. Dayhoff M.O. Dayhoff M.O. Atlas of Protein Sequence and Structure. 5th Ed. National Biomedical Re" @default.
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