title: Purification and biochemical characterization of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthases KasA and KasB.
abstract: 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.
authors.affiliation: GlaxoSmithKline, 1250 S. Collegeville Rd., Collegeville, PA 19426, USA. Merrill_L_Schaeffer@gsk.com
authors.forename: M L
authors.initials: ML
authors.lastname: Schaeffer
authors.forename: G
authors.initials: G
authors.lastname: Agnihotri
authors.forename: C
authors.initials: C
authors.lastname: Volker
authors.forename: H
authors.initials: H
authors.lastname: Kallender
authors.forename: P J
authors.initials: PJ
authors.lastname: Brennan
authors.forename: J T
authors.initials: JT
authors.lastname: Lonsdale
mesh_terms: D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D000123:Acetyltransferases; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology; D001426:Bacterial Proteins; D002244:Carbon / Q000378:metabolism; D002569:Cerulenin / Q000494:pharmacology; D002846:Chromatography, Affinity; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D054889:Fatty Acid Synthase, Type II; D005227:Fatty Acids / Q000096:biosynthesis; D017353:Gene Deletion; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008956:Models, Chemical; D009097:Multienzyme Complexes; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009171:Mycolic Acids / Q000378:metabolism; D010802:Phylogeny; D011485:Protein Binding; D013379:Substrate Specificity; D013876:Thiophenes / Q000494:pharmacology; D013997:Time Factors
chemical_list: D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D005227:Fatty Acids; D009097:Multienzyme Complexes; D009171:Mycolic Acids; D013876:Thiophenes; D002569:Cerulenin; D002244:Carbon; C035068:thiolactomycin; D000123:Acetyltransferases; C444656:KasB protein, Mycobacterium tuberculosis; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D054889:Fatty Acid Synthase, Type II