The Experts below are selected from a list of 144 Experts worldwide ranked by ideXlab platform
Gurdyal S Besra - One of the best experts on this subject based on the ideXlab platform.
-
The Mycobacterium tuberculosis {beta}-Ketoacyl-Acyl Carrier Protein Synthase III Activity Is Inhibited by Phosphorylation on a Single Threonine Residue
Journal of Biological Chemistry, 2009Co-Authors: Romain Veyron-churlet, Alistair K Brown, Gurdyal S Besra, Virginie Molle, Rebecca C Taylor, Isabelle Zanella-cléon, Klaus Fütterer, Laurent KremerAbstract:Mycolic acids are hallmark features of the Mycobacterium tuberculosis cell wall. They are synthesized by the condensation of two fatty acids, a C(56-64)-meromycolyl chain and a C(24-26)-fatty acyl chain. Meromycolates are produced via the combination of type I and type II fatty acid Synthases (FAS-I and FAS-II). The beta-ketoacyl-acyl carrier protein (ACP) Synthase III (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-ACP. Because mtFabH represents a potential regulatory key point of the mycolic acid pathway, we investigated the hypothesis that phosphorylation of mtFabH controls its activity. Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently emerged as a major physiological mechanism of regulation in prokaryotes. We demonstrate here that mtFabH was efficiently phosphorylated in vitro by several mycobacterial STPKs, particularly by PknF and PknA, as well as in vivo in mycobacteria. Analysis of the phosphoamino acid content indicated that mtFabH was phosphorylated exclusively on threonine residues. Mass spectrometry analyses using liquid chromatography-electrospray ionization/tandem mass spectrometry identified Thr(45) as the unique phosphoacceptor. This was further supported by complete loss of PknF- or PknA-dependent phosphorylation of a mtFabH mutant. Mapping Thr(45) on the crystal structure of mtFabH illustrates that this residue is located at the entrance of the substrate channel, suggesting that the phosphate group may alter accessibility of the substrate and thus affect mtFabH enzymatic activity. A T45D mutant of mtFabH, designed to mimic constitutive phosphorylation, exhibited markedly decreased transacylation, malonyl-AcpM decarboxylation, and condensing activities compared with the wild-type protein or the T45A mutant. Together, these findings not only represent the first demonstration of phosphorylation of a Beta-Ketoacyl-ACP Synthase III enzyme but also indicate that phosphorylation of mtFabH inhibits its enzymatic activity, which may have important consequences in regulating mycolic acid biosynthesis.
-
X-ray crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein Synthase II (mtKasB).
Journal of Molecular Biology, 2007Co-Authors: Sudharsan Sridharan, Laurent Kremer, Lynn G Dover, Alistair K Brown, Gurdyal S Besra, Lei Wang, James C SacchettiniAbstract:Mycolic acids are long chain alpha-alkyl branched, beta-hydroxy fatty acids that represent a characteristic component of the Mycobacterium tuberculosis cell wall. Through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. Mycobacteria possess two fatty acid Synthases (FAS); FAS-I carries out de novo synthesis of fatty acids while FAS-II is considered to elongate medium chain length fatty acyl primers to provide long chain (C(56)) precursors of mycolic acids. Here we report the crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein Synthase (ACP) II mtKasB, a mycobacterial elongation condensing enzyme involved in FAS-II. This enzyme, along with the M. tuberculosis beta-ketoacyl ACP Synthase I mtKasA, catalyzes the Claisen-type condensation reaction responsible for fatty acyl elongation in FAS-II and are potential targets for development of novel anti-tubercular drugs. The crystal structure refined to 2.4 A resolution revealed that, like other KAS-II enzymes, mtKasB adopts a thiolase fold but contains unique structural features in the capping region that may be crucial to its preference for longer fatty acyl chains than its counterparts from other bacteria. Modeling of mtKasA using the mtKasB structure as a template predicts the overall structures to be almost identical, but a larger entrance to the active site tunnel is envisaged that might contribute to the greater sensitivity of mtKasA to the inhibitor thiolactomycin (TLM). Modeling of TLM binding in mtKasB shows that the drug fits the active site poorly and results of enzyme inhibition assays using TLM analogues are wholly consistent with our structural observations. Consequently, the structure described here further highlights the potential of TLM as an anti-tubercular lead compound and will aid further exploration of the TLM scaffold towards the design of novel compounds, which inhibit mycobacterial KAS enzymes more effectively.
-
Biphenyl-based analogues of thiolactomycin, active against Mycobacterium tuberculosis mtFabH fatty acid condensing enzyme.
Bioorganic & medicinal chemistry letters, 2003Co-Authors: Suzanne J Senior, David E Minnikin, Petr A Illarionov, Sudagar S Gurcha, Ian B Campbell, Merrill L Schaeffer, Gurdyal S BesraAbstract:Analogues of the antibiotic thiolactomycin, with biphenyl-based 5-substituents, were found to have excellent in vitro inhibitory activity against the recombinant Mycobacterium tuberculosis Beta-Ketoacyl-ACP Synthase mtFabH condensing enzyme. In particular, 5-(4'-benzyloxy-biphen-4-ylmethyl)-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one exhibited approximately a 4-fold increased potency against this key condensing enzyme involved in M. tuberculosis mycolic acid biosynthesis, compared to thiolactomycin.
-
mycolic acid biosynthesis and enzymic characterization of the beta ketoacyl acp Synthase a condensing enzyme from mycobacterium tuberculosis
Biochemical Journal, 2002Co-Authors: Laurent Kremer, Lynn G Dover, Severine Carrere, Madhavan K Nampoothiri, Sarah Lesjean, Alistair K Brown, Patrick J Brennan, David E Minnikin, Camille Locht, Gurdyal S BesraAbstract:Mycolic acids consist of long-chain alpha-alkyl-beta-hydroxy fatty acids that are produced by successive rounds of elongation catalysed by a type II fatty acid Synthase (FAS-II). A key feature in the elongation process is the condensation of a two-carbon unit from malonyl-acyl-carrier protein (ACP) to a growing acyl-ACP chain catalysed by a Beta-Ketoacyl-ACP Synthase (Kas). In the present study, we provide evidence that kasA from Mycobacterium tuberculosis encodes an enzyme that elongates in vivo the meromycolate chain, in both Mycobacterium smegmatis and Mycobacterium chelonae. We demonstrate that KasA belongs to the FAS-II system, which utilizes primarily palmitoyl-ACP rather than short-chain acyl-ACP primers. Furthermore, in an in vitro condensing assay using purified recombinant KasA, palmitoyl-AcpM and malonyl-AcpM, KasA was found to express Kas activity. Also, mutated KasA proteins, with mutation of Cys(171), His(311), Lys(340) and His(345) to Ala abrogated the condensation activity of KasA in vitro completely. Finally, purified KasA was highly sensitive to cerulenin, a well-known inhibitor of Kas, which may lead to the development of novel anti-mycobacterial drugs targeting KasA.
Laurent Kremer - One of the best experts on this subject based on the ideXlab platform.
-
The Mycobacterium tuberculosis {beta}-Ketoacyl-Acyl Carrier Protein Synthase III Activity Is Inhibited by Phosphorylation on a Single Threonine Residue
Journal of Biological Chemistry, 2009Co-Authors: Romain Veyron-churlet, Alistair K Brown, Gurdyal S Besra, Virginie Molle, Rebecca C Taylor, Isabelle Zanella-cléon, Klaus Fütterer, Laurent KremerAbstract:Mycolic acids are hallmark features of the Mycobacterium tuberculosis cell wall. They are synthesized by the condensation of two fatty acids, a C(56-64)-meromycolyl chain and a C(24-26)-fatty acyl chain. Meromycolates are produced via the combination of type I and type II fatty acid Synthases (FAS-I and FAS-II). The beta-ketoacyl-acyl carrier protein (ACP) Synthase III (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-ACP. Because mtFabH represents a potential regulatory key point of the mycolic acid pathway, we investigated the hypothesis that phosphorylation of mtFabH controls its activity. Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently emerged as a major physiological mechanism of regulation in prokaryotes. We demonstrate here that mtFabH was efficiently phosphorylated in vitro by several mycobacterial STPKs, particularly by PknF and PknA, as well as in vivo in mycobacteria. Analysis of the phosphoamino acid content indicated that mtFabH was phosphorylated exclusively on threonine residues. Mass spectrometry analyses using liquid chromatography-electrospray ionization/tandem mass spectrometry identified Thr(45) as the unique phosphoacceptor. This was further supported by complete loss of PknF- or PknA-dependent phosphorylation of a mtFabH mutant. Mapping Thr(45) on the crystal structure of mtFabH illustrates that this residue is located at the entrance of the substrate channel, suggesting that the phosphate group may alter accessibility of the substrate and thus affect mtFabH enzymatic activity. A T45D mutant of mtFabH, designed to mimic constitutive phosphorylation, exhibited markedly decreased transacylation, malonyl-AcpM decarboxylation, and condensing activities compared with the wild-type protein or the T45A mutant. Together, these findings not only represent the first demonstration of phosphorylation of a Beta-Ketoacyl-ACP Synthase III enzyme but also indicate that phosphorylation of mtFabH inhibits its enzymatic activity, which may have important consequences in regulating mycolic acid biosynthesis.
-
X-ray crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein Synthase II (mtKasB).
Journal of Molecular Biology, 2007Co-Authors: Sudharsan Sridharan, Laurent Kremer, Lynn G Dover, Alistair K Brown, Gurdyal S Besra, Lei Wang, James C SacchettiniAbstract:Mycolic acids are long chain alpha-alkyl branched, beta-hydroxy fatty acids that represent a characteristic component of the Mycobacterium tuberculosis cell wall. Through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. Mycobacteria possess two fatty acid Synthases (FAS); FAS-I carries out de novo synthesis of fatty acids while FAS-II is considered to elongate medium chain length fatty acyl primers to provide long chain (C(56)) precursors of mycolic acids. Here we report the crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein Synthase (ACP) II mtKasB, a mycobacterial elongation condensing enzyme involved in FAS-II. This enzyme, along with the M. tuberculosis beta-ketoacyl ACP Synthase I mtKasA, catalyzes the Claisen-type condensation reaction responsible for fatty acyl elongation in FAS-II and are potential targets for development of novel anti-tubercular drugs. The crystal structure refined to 2.4 A resolution revealed that, like other KAS-II enzymes, mtKasB adopts a thiolase fold but contains unique structural features in the capping region that may be crucial to its preference for longer fatty acyl chains than its counterparts from other bacteria. Modeling of mtKasA using the mtKasB structure as a template predicts the overall structures to be almost identical, but a larger entrance to the active site tunnel is envisaged that might contribute to the greater sensitivity of mtKasA to the inhibitor thiolactomycin (TLM). Modeling of TLM binding in mtKasB shows that the drug fits the active site poorly and results of enzyme inhibition assays using TLM analogues are wholly consistent with our structural observations. Consequently, the structure described here further highlights the potential of TLM as an anti-tubercular lead compound and will aid further exploration of the TLM scaffold towards the design of novel compounds, which inhibit mycobacterial KAS enzymes more effectively.
-
mycolic acid biosynthesis and enzymic characterization of the beta ketoacyl acp Synthase a condensing enzyme from mycobacterium tuberculosis
Biochemical Journal, 2002Co-Authors: Laurent Kremer, Lynn G Dover, Severine Carrere, Madhavan K Nampoothiri, Sarah Lesjean, Alistair K Brown, Patrick J Brennan, David E Minnikin, Camille Locht, Gurdyal S BesraAbstract:Mycolic acids consist of long-chain alpha-alkyl-beta-hydroxy fatty acids that are produced by successive rounds of elongation catalysed by a type II fatty acid Synthase (FAS-II). A key feature in the elongation process is the condensation of a two-carbon unit from malonyl-acyl-carrier protein (ACP) to a growing acyl-ACP chain catalysed by a Beta-Ketoacyl-ACP Synthase (Kas). In the present study, we provide evidence that kasA from Mycobacterium tuberculosis encodes an enzyme that elongates in vivo the meromycolate chain, in both Mycobacterium smegmatis and Mycobacterium chelonae. We demonstrate that KasA belongs to the FAS-II system, which utilizes primarily palmitoyl-ACP rather than short-chain acyl-ACP primers. Furthermore, in an in vitro condensing assay using purified recombinant KasA, palmitoyl-AcpM and malonyl-AcpM, KasA was found to express Kas activity. Also, mutated KasA proteins, with mutation of Cys(171), His(311), Lys(340) and His(345) to Ala abrogated the condensation activity of KasA in vitro completely. Finally, purified KasA was highly sensitive to cerulenin, a well-known inhibitor of Kas, which may lead to the development of novel anti-mycobacterial drugs targeting KasA.
Alistair K Brown - One of the best experts on this subject based on the ideXlab platform.
-
The Mycobacterium tuberculosis {beta}-Ketoacyl-Acyl Carrier Protein Synthase III Activity Is Inhibited by Phosphorylation on a Single Threonine Residue
Journal of Biological Chemistry, 2009Co-Authors: Romain Veyron-churlet, Alistair K Brown, Gurdyal S Besra, Virginie Molle, Rebecca C Taylor, Isabelle Zanella-cléon, Klaus Fütterer, Laurent KremerAbstract:Mycolic acids are hallmark features of the Mycobacterium tuberculosis cell wall. They are synthesized by the condensation of two fatty acids, a C(56-64)-meromycolyl chain and a C(24-26)-fatty acyl chain. Meromycolates are produced via the combination of type I and type II fatty acid Synthases (FAS-I and FAS-II). The beta-ketoacyl-acyl carrier protein (ACP) Synthase III (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-ACP. Because mtFabH represents a potential regulatory key point of the mycolic acid pathway, we investigated the hypothesis that phosphorylation of mtFabH controls its activity. Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently emerged as a major physiological mechanism of regulation in prokaryotes. We demonstrate here that mtFabH was efficiently phosphorylated in vitro by several mycobacterial STPKs, particularly by PknF and PknA, as well as in vivo in mycobacteria. Analysis of the phosphoamino acid content indicated that mtFabH was phosphorylated exclusively on threonine residues. Mass spectrometry analyses using liquid chromatography-electrospray ionization/tandem mass spectrometry identified Thr(45) as the unique phosphoacceptor. This was further supported by complete loss of PknF- or PknA-dependent phosphorylation of a mtFabH mutant. Mapping Thr(45) on the crystal structure of mtFabH illustrates that this residue is located at the entrance of the substrate channel, suggesting that the phosphate group may alter accessibility of the substrate and thus affect mtFabH enzymatic activity. A T45D mutant of mtFabH, designed to mimic constitutive phosphorylation, exhibited markedly decreased transacylation, malonyl-AcpM decarboxylation, and condensing activities compared with the wild-type protein or the T45A mutant. Together, these findings not only represent the first demonstration of phosphorylation of a Beta-Ketoacyl-ACP Synthase III enzyme but also indicate that phosphorylation of mtFabH inhibits its enzymatic activity, which may have important consequences in regulating mycolic acid biosynthesis.
-
X-ray crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein Synthase II (mtKasB).
Journal of Molecular Biology, 2007Co-Authors: Sudharsan Sridharan, Laurent Kremer, Lynn G Dover, Alistair K Brown, Gurdyal S Besra, Lei Wang, James C SacchettiniAbstract:Mycolic acids are long chain alpha-alkyl branched, beta-hydroxy fatty acids that represent a characteristic component of the Mycobacterium tuberculosis cell wall. Through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. Mycobacteria possess two fatty acid Synthases (FAS); FAS-I carries out de novo synthesis of fatty acids while FAS-II is considered to elongate medium chain length fatty acyl primers to provide long chain (C(56)) precursors of mycolic acids. Here we report the crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein Synthase (ACP) II mtKasB, a mycobacterial elongation condensing enzyme involved in FAS-II. This enzyme, along with the M. tuberculosis beta-ketoacyl ACP Synthase I mtKasA, catalyzes the Claisen-type condensation reaction responsible for fatty acyl elongation in FAS-II and are potential targets for development of novel anti-tubercular drugs. The crystal structure refined to 2.4 A resolution revealed that, like other KAS-II enzymes, mtKasB adopts a thiolase fold but contains unique structural features in the capping region that may be crucial to its preference for longer fatty acyl chains than its counterparts from other bacteria. Modeling of mtKasA using the mtKasB structure as a template predicts the overall structures to be almost identical, but a larger entrance to the active site tunnel is envisaged that might contribute to the greater sensitivity of mtKasA to the inhibitor thiolactomycin (TLM). Modeling of TLM binding in mtKasB shows that the drug fits the active site poorly and results of enzyme inhibition assays using TLM analogues are wholly consistent with our structural observations. Consequently, the structure described here further highlights the potential of TLM as an anti-tubercular lead compound and will aid further exploration of the TLM scaffold towards the design of novel compounds, which inhibit mycobacterial KAS enzymes more effectively.
-
mycolic acid biosynthesis and enzymic characterization of the beta ketoacyl acp Synthase a condensing enzyme from mycobacterium tuberculosis
Biochemical Journal, 2002Co-Authors: Laurent Kremer, Lynn G Dover, Severine Carrere, Madhavan K Nampoothiri, Sarah Lesjean, Alistair K Brown, Patrick J Brennan, David E Minnikin, Camille Locht, Gurdyal S BesraAbstract:Mycolic acids consist of long-chain alpha-alkyl-beta-hydroxy fatty acids that are produced by successive rounds of elongation catalysed by a type II fatty acid Synthase (FAS-II). A key feature in the elongation process is the condensation of a two-carbon unit from malonyl-acyl-carrier protein (ACP) to a growing acyl-ACP chain catalysed by a Beta-Ketoacyl-ACP Synthase (Kas). In the present study, we provide evidence that kasA from Mycobacterium tuberculosis encodes an enzyme that elongates in vivo the meromycolate chain, in both Mycobacterium smegmatis and Mycobacterium chelonae. We demonstrate that KasA belongs to the FAS-II system, which utilizes primarily palmitoyl-ACP rather than short-chain acyl-ACP primers. Furthermore, in an in vitro condensing assay using purified recombinant KasA, palmitoyl-AcpM and malonyl-AcpM, KasA was found to express Kas activity. Also, mutated KasA proteins, with mutation of Cys(171), His(311), Lys(340) and His(345) to Ala abrogated the condensation activity of KasA in vitro completely. Finally, purified KasA was highly sensitive to cerulenin, a well-known inhibitor of Kas, which may lead to the development of novel anti-mycobacterial drugs targeting KasA.
John T. Lonsdale - One of the best experts on this subject based on the ideXlab platform.
-
Structure-activity relationships at the 5-position of thiolactomycin: an intact (5R)-isoprene unit is required for activity against the condensing enzymes from Mycobacterium tuberculosis and Escherichia coli
Journal of medicinal chemistry, 2006Co-Authors: Pilho Kim, Yong-mei Zhang, Gautham G. Shenoy, Quynh-anh Nguyen, Helena I. Boshoff, Ujjini H. Manjunatha, Michael B. Goodwin, John T. Lonsdale, Allen C. Price, Darcie J. MillerAbstract:Thiolactomycin inhibits bacterial cell growth through inhibition of the Beta-Ketoacyl-ACP Synthase activity of type II fatty acid Synthases. The effect of modifications of the 5-position isoprenoid side chain on both IC(50) and MIC were determined. Synthesis and screening of a structurally diverse set of 5-position analogues revealed very little tolerance for substitution in purified enzyme assays, but a few analogues retained MIC, presumably through another target. Even subtle modifications such as reducing one or both double bonds of the diene were not tolerated. The only permissible structural modifications were removal of the isoprene methyl group or addition of a methyl group to the terminus. Cocrystallization of these two inhibitors with the condensing enzyme from Escherichia coli revealed that they retained the TLM binding mode at the active site with reduced affinity. These results suggest a strict requirement for a conjugated, planar side chain inserting within the condensing enzyme active site.
-
Purification and biochemical characterization of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein Synthases KasA and KasB.
The Journal of biological chemistry, 2001Co-Authors: Merrill L Schaeffer, Patrick J Brennan, Craig Volker, Gautam Agnihotri, Howard Kallender, John T. LonsdaleAbstract:Mycolic acids are vital components of the Mycobacterium 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 Beta-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.
Merrill L Schaeffer - One of the best experts on this subject based on the ideXlab platform.
-
Biphenyl-based analogues of thiolactomycin, active against Mycobacterium tuberculosis mtFabH fatty acid condensing enzyme.
Bioorganic & medicinal chemistry letters, 2003Co-Authors: Suzanne J Senior, David E Minnikin, Petr A Illarionov, Sudagar S Gurcha, Ian B Campbell, Merrill L Schaeffer, Gurdyal S BesraAbstract:Analogues of the antibiotic thiolactomycin, with biphenyl-based 5-substituents, were found to have excellent in vitro inhibitory activity against the recombinant Mycobacterium tuberculosis Beta-Ketoacyl-ACP Synthase mtFabH condensing enzyme. In particular, 5-(4'-benzyloxy-biphen-4-ylmethyl)-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one exhibited approximately a 4-fold increased potency against this key condensing enzyme involved in M. tuberculosis mycolic acid biosynthesis, compared to thiolactomycin.
-
Purification and biochemical characterization of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein Synthases KasA and KasB.
The Journal of biological chemistry, 2001Co-Authors: Merrill L Schaeffer, Patrick J Brennan, Craig Volker, Gautam Agnihotri, Howard Kallender, John T. LonsdaleAbstract:Mycolic acids are vital components of the Mycobacterium 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 Beta-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.