RESUMO
In a recent study of polyketide biosynthetic gene clusters cloned directly from soil, we isolated two antibiotics, fasamycins A and B, which showed activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. To identify the target of the fasamycins, mutants with elevated fasamycin A minimum inhibitory concentrations were selected from a wild-type culture of E. faecalis OG1RF. Next-generation sequencing of these mutants, in conjunction with in vitro biochemical assays, showed that the fasamycins inhibit FabF of type II fatty acid biosynthesis (FASII). Candidate gene overexpression studies also showed that fasamycin resistance is conferred by fabF overexpression. On the basis of comparisons with known FASII inhibitors and in silico docking studies, the chloro-gem-dimethyl-anthracenone substructure seen in the fasamycins is predicted to represent a naturally occurring FabF-specific antibiotic pharmacophore. Optimization of this pharmacophore should yield FabF-specific antibiotics with increased potencies and differing spectra of activity. This study demonstrates that culture-independent antibiotic discovery methods have the potential to provide access to novel metabolites with modes of action that differ from those of antibiotics currently in clinical use.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/efeitos dos fármacos , Antibacterianos/química , Proteínas de Bactérias/efeitos dos fármacos , Compostos de Bifenilo/síntese química , Química Farmacêutica/métodos , DNA/química , Enterococcus faecalis/metabolismo , Ácidos Graxos/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/síntese química , Sequência de Bases , Bioquímica/métodos , Cromatografia/métodos , Clonagem Molecular , Primers do DNA/genética , Biblioteca Gênica , Humanos , Concentração Inibidora 50 , Modelos Químicos , Dados de Sequência Molecular , Família Multigênica , MutaçãoRESUMO
Bacteria stringently regulate the synthesis of their membrane phospholipids, but the responsible regulatory mechanisms are incompletely understood. Bacillus subtilis FabF, the target of the mycotoxin cerulenin, catalyses the condensation of malonyl-ACP with acyl-ACP to extend the growing acyl chain by two carbons. Here we show that B. subtilis strains containing the fabF1 allele, which codes for the cerulenin-insensitive protein FabF[I108F], overexpressed several genes involved in fatty acid and phospholipid biosynthesis (the fap regulon) and had significantly elevated levels of malonyl-CoA. These results pinpointed FabF[I108F] as responsible for the increased malonyl-CoA production, which in turn acts as an inducer of the fap regulon by impairing the binding of the FapR repressor to its DNA targets. Synthesis of acyl-ACPs by a cell-free fatty acid system prepared from fabF1 cells showed the accumulation of short- and medium-chain acyl-ACPs. These results indicate that the acyl-ACP chain length acceptance of FabF[I108F] is biased towards shorter acyl-ACPs. We also provide evidence that upregulation of FabF[I108F] is essential for survival and for resistance to cerulenin of fabF1 cells. These findings indicate that malonyl-CoA is a key molecule to monitor lipid metabolism functioning and trigger appropriate genetic and biochemical adjustments to relieve dysfunctions of this essential metabolic pathway.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Bacillus subtilis/enzimologia , Regulação Bacteriana da Expressão Gênica , Metabolismo dos Lipídeos/genética , Malonil Coenzima A/genética , Proteínas Repressoras/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/efeitos dos fármacos , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Bacillus subtilis/genética , Cerulenina/farmacologia , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Malonil Coenzima A/metabolismo , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Regulon , Proteínas Repressoras/genéticaRESUMO
The emergence of drug resistant strains of important human pathogens has made urgent the necessity of finding new targets and novel antimicrobial agents. One of the most promising targets is FabH. In this review we summarize the progress made in the design of FabH inhibitors and the role played by the 3D-structure of the enzyme in the drug design process.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/efeitos dos fármacos , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Desenho de Fármacos , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Modelos BiológicosRESUMO
In studies of the outstanding salt tolerance of the unicellular green alga Dunaliella salina, we isolated a cDNA for a salt-inducible mRNA encoding a protein homologous to plant beta-ketoacyl-coenzyme A (CoA) synthases (Kcs). These microsomal enzymes catalyze the condensation of malonyl-CoA with acyl-CoA, the first and rate-limiting step in fatty acid elongation. Kcs activity, localized to a D. salina microsomal fraction, increased in cells transferred from 0.5 to 3.5 M NaCl, as did the level of the kcs mRNA. The function of the kcs gene product was directly demonstrated by the condensing activity exhibited by Escherichia coli cells expressing the kcs cDNA. The effect of salinity on kcs expression in D. salina suggested the possibility that salt adaptation entailed modifications in the fatty acid composition of algal membranes. Lipid analyses indicated that microsomes, but not plasma membranes or thylakoids, from cells grown in 3.5 M NaCl contained a considerably higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty acids compared with cells grown in 0.5 M salt. Thus, the salt-inducible Kcs, jointly with fatty acid desaturases, may play a role in adapting intracellular membrane compartments to function in the high internal glycerol concentrations balancing the external osmotic pressure.
