Tuberculosis (TB) is the leading cause of infectious disease mortality in the world by a bacterial pathogen. We previously demonstrated that a bisubstrate inhibitor of the adenylation enzyme MbtA, which is responsible for the second step of mycobactin biosynthesis, exhibited potent antitubercular activity. Here we systematically investigate the structure activity relationships of the bisubstrate inhibitor glycosyl domain resulting in the identification of a carbocyclic analogue that possesses a K I app value of 2.3 nM and MIC 99 values of 1.56 μM against M. tuberculosis H37Rv. The SAR data suggest the intriguing possibility that the bisubstrate inhibitors utilize a transporter for entry across the mycobacterial cell-envelope. Additionally, we report improved conditions for the expression of MbtA and biochemical analysis demonstrating that MbtA follows a random sequential enzyme mechanism for the adenylation half-reaction.

This manuscript calls for an international effort to generate a comprehensive catalog from genome sequences of all the archaeal and bacterial type strains.

The pikromycin biosynthetic gene cluster contains the pikAV gene encoding a type II thioesterase (TEII). TEII is not responsible for polyketide termination and cyclization, and its biosynthetic role has been unclear. During polyketide biosynthesis, extender units such as methylmalonyl acyl carrier protein (ACP) may prematurely decarboxylate to generate the corresponding acyl-ACP, which cannot be used as a substrate in the condensing reaction by the corresponding ketosynthase domain, rendering the polyketide synthase module inactive. It has been proposed that TEII may serve as an "editing" enzyme and reactivate these modules by removing acyl moieties attached to ACP domains. Using a purified recombinant TEII we have tested this hypothesis by using in vitro enzyme assays and a range of acyl-ACP, malonyl-ACP, and methylmalonyl-ACP substrates derived from either PikAIII or the loading didomain of DEBS1 (6- Polyketides are a large and structurally diverse class of natural products that possess a wide range of biological activities (1). These compounds are used throughout medicinal and agricultural fields as antimicrobials, immunosuppressants, antiparasitics, and anticancer agents. Despite their structural diversity, polyketides are assembled by a common mechanism of decarboxylative condensations of simple malonate derivatives by polyketide synthases (PKSs) 1 in a manner very similar to fatty acid biosynthesis (2, 3). Type I PKSs are a family of PKSs that are analogous to vertebrate fatty acid synthase that catalyze the biosynthesis of the polyketide moieties of various secondary metabolites in Streptomyces (4 -6). They are gigantic multifunctional modular proteins. Each module is responsible for one cycle of polyketide chain elongation and contains a set of discrete catalytic domains of ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP). Ketoreductase, dehydratase (DH), and enoyl reductase domains may also be present, allowing structural variation in the level of processing of the ␤-ketoacyl chain (4, 6). The fully extended polyketide chain bound to the PKS as an acyl-ACP thioester is often released and cyclized by a thioesterase domain (TEI) covalently linked to the last extending module of the PKS (7).In many cases, additional genes encoding a TE have been found within a polyketide biosynthetic gene cluster, for example, the tylosin PKS of Streptomyces fradiae (8), pikromycin PKS of Streptomyces venezuelae (5), rifamycin PKS of Amycolatopsis mediterranei (9), and the erythromycin PKS (DEBS) of Saccharopolyspora erythraea (10). These genes encoding a discrete protein were named as TEII to differentiate from the chain releasing TEI domains in modular polyketide synthases. Discrete TEII enzymes are also associated with bacterial nonribosomal peptide synthases, responsible for the production of macrocyclic peptide compounds (11), and animal fatty acid synthases (12). Sequence analysis has revealed that these thioesterases are probably structurally and evolutionarily related (13). They have a common...