RESUMO
BACKGROUND: Antimicrobial resistance is a persistent problem regarding infection treatment and calls for developing new antimicrobial agents. Inhibition of bacterial ß-ketoacyl acyl carrier protein synthase III (FabH), which catalyzes the condensation reaction between a CoAattached acetyl group and an ACP-attached malonyl group in bacteria is an interesting strategy to find new antibacterial agents. OBJECTIVE: The aim of this work was to design and synthesize arylsulfonylhydrazones potentially FabH inhibitors and evaluate their antimicrobial activity. METHODS: MIC50 values of sulfonylhydrazones against E. coli and S. aureus were determined. Antioxidant activity was evaluated by DPPH (1-1'-diphenyl-2-picrylhydrazyl) assay and cytotoxicity against LL24 lung fibroblast cells was verified by MTT method. Principal component analysis (PCA) was performed in order to suggest a structure-activity relationship. Molecular docking allowed to propose sulfonylhydrazones interactions with FabH. RESULTS: The most active compound showed activity against S. aureus and E. coli, with MIC50 = 0.21 and 0.44 µM, respectively. PCA studies correlated better activity to lipophilicity and molecular docking indicated that sulfonylhydrazone moiety is important to hydrogen-bond with FabH while methylcatechol ring performs π-π stacking interaction. The DPPH assay revealed that some sulfonylhydrazones derived from the methylcatechol series had antioxidant activity. None of the evaluated compounds was cytotoxic to human lung fibroblast cells, suggesting that the compounds might be considered safe at the tested concentration. CONCLUSION: Arylsufonylhydrazones is a promising scaffold to be explored for the design of new antimicrobial agents.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/antagonistas & inibidores , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Hidrazonas/farmacologia , Sulfonamidas/farmacologia , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/química , Acetiltransferases/metabolismo , Antibacterianos/síntese química , Antibacterianos/metabolismo , Domínio Catalítico , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintase Tipo II/antagonistas & inibidores , Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo II/metabolismo , Hidrazonas/síntese química , Hidrazonas/metabolismo , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Análise de Componente Principal , Ligação Proteica , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/metabolismoRESUMO
FabA and FabZ are the two dehydratase enzymes in Escherichia coli that catalyze the dehydration of acyl intermediates in the biosynthesis of fatty acids. Both enzymes form obligate dimers in which the active site contains key amino acids from both subunits. While FabA is a soluble protein that has been relatively straightforward to express and to purify from cultured E. coli, FabZ has shown to be mostly insoluble and only partially active. In an effort to increase the solubility and activity of both dehydratases, we made constructs consisting of two identical subunits of FabA or FabZ fused with a naturally occurring peptide linker, so as to force their dimerization. The fused dimer of FabZ (FabZ-FabZ) was expressed as a soluble enzyme with an ninefold higher activity in vitro than the unfused FabZ. This construct exemplifies a strategy for the improvement of enzymes from the fatty acid biosynthesis pathways, many of which function as dimers, catalyzing critical steps for the production of fatty acids.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Ácido Graxo Sintase Tipo II/metabolismo , Hidroliases/metabolismo , Biocatálise , Desidratação , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo II/isolamento & purificação , Ácidos Graxos/biossíntese , Ácidos Graxos/química , Hidroliases/química , Hidroliases/isolamento & purificação , Modelos Moleculares , Multimerização Proteica , SolubilidadeRESUMO
Aleurites moluccana L. is grown as a roadside tree in southern China and the oil content of its seed is higher than other oil plants, such as Jatropha curcas and Camellia oleifera. A. moluccana is considered a promising energy plant because its seed oil could be used to produce biodiesel and bio-jet fuel. In addition, the bark, leaves, and kernels of A. moluccana have various medical and commercial uses. Here, a novel gene coding the biotin carboxyl carrier protein subunit (BCCP) was cloned from A. moluccana L. using the homology cloning method combined with rapid amplification of cDNA end (RACE) technology. The isolated full-length cDNA sequence (designated AM-accB) was 1188 bp, containing a 795-bp open reading frame coding for 265 amino acids. The deduced amino acid sequence of AM-accB contained a biotinylated domain located between amino acids 190 and 263. A. moluccana BCCP shows high identity at the amino acid level to its homologues in other higher plants, such as Vernicia fordii, J. curcas, and Ricinus communis (86, 77, and 70%, respectively), which all contain conserved domains for ACCase activity. The expression of the AM-accB gene during the middle stage of development and maturation in A. moluccana seeds was higher than that in early and later stages. The expression pattern of the AM-accB gene is very similar to that of the oil accumulation rate.
Assuntos
Acetil-CoA Carboxilase/genética , Aleurites/genética , Clonagem Molecular , Expressão Gênica , Subunidades Proteicas/genética , Acetil-CoA Carboxilase/química , Aleurites/metabolismo , Sequência de Aminoácidos , DNA Complementar/genética , Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo II/genética , Dados de Sequência Molecular , Óleos de Plantas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de TempoRESUMO
UNLABELLED: Cerulenin is a fungal toxin that inhibits both eukaryotic and prokaryotic ketoacyl-acyl carrier protein synthases or condensing enzymes. It has been used experimentally to treat cancer and obesity, and is a potent inhibitor of bacterial growth. Understanding the molecular mechanisms of resistance to cerulenin and similar compounds is thus highly relevant for human health. We have previously described a Bacillus subtilis cerulenin-resistant strain, expressing a point-mutated condensing enzyme FabF (FabF[I108F]) (i.e. FabF with isoleucine 108 substituted by phenylalanine). We now report the crystal structures of wild-type FabF from B. subtilis, both alone and in complex with cerulenin, as well as of the FabF[I108F] mutant protein. The three-dimensional structure of FabF[I108F] constitutes the first atomic model of a condensing enzyme that remains active in the presence of the inhibitor. Soaking the mycotoxin into preformed wild-type FabF crystals allowed for noncovalent binding into its specific pocket within the FabF core. Interestingly, only co-crystallization experiments allowed us to trap the covalent complex. Our structure shows that the covalent bond between Cys163 and cerulenin, in contrast to that previously proposed, implicates carbon C3 of the inhibitor. The similarities between Escherichia coli and B. subtilis FabF structures did not explain the reported inability of ecFabF[I108F] (i.e. FabF from Escherichia coli with isoleucine 108 substituted by phenylalanine) to elongate medium and long-chain acyl-ACPs. We now demonstrate that the E. coli modified enzyme efficiently catalyzes the synthesis of medium and long-chain ketoacyl-ACPs. We also characterized another cerulenin-insensitive form of FabF, conferring a different phenotype in B. subtilis. The structural, biochemical and physiological data presented, shed light on the mechanisms of FabF catalysis and resistance to cerulenin. DATABASE: Crystallographic data (including atomic coordinates and structure factors) have been deposited in the Protein Data Bank under accession codes 4LS5, 4LS6, 4LS7 and 4LS8.
Assuntos
Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cerulenina/farmacologia , Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo II/metabolismo , Acetiltransferases/química , Acetiltransferases/genética , Acetiltransferases/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Domínio Catalítico/genética , Cristalografia por Raios X , Farmacorresistência Bacteriana/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintase Tipo II/genética , Inibidores da Síntese de Ácidos Graxos/farmacologia , Genes Bacterianos , Humanos , Modelos Moleculares , Micotoxinas/farmacologia , Mutação Puntual , Estrutura Quaternária de Proteína , Eletricidade EstáticaRESUMO
All organisms that produce fatty acids do so via a repeated cycle of reactions. In mammals and other animals, these reactions are catalyzed by a type I fatty acid synthase (FAS), a large multifunctional protein to which the growing chain is covalently attached. In contrast, most bacteria (and plants) contain a type II system in which each reaction is catalyzed by a discrete protein. The pathway of fatty acid biosynthesis in Escherichia coli is well established and has provided a foundation for elucidating the type II FAS pathways in other bacteria (White et al., 2005). However, fatty acid biosynthesis is more diverse in the phylum Actinobacteria: Mycobacterium, possess both FAS systems while Streptomyces species have only the multienzyme FAS II system and Corynebacterium species exclusively FAS I. In this review, we present an overview of the genome organization, biochemical properties and physiological relevance of the two FAS systems in the three genera of actinomycetes mentioned above. We also address in detail the biochemical and structural properties of the acyl-CoA carboxylases (ACCases) that catalyzes the first committed step of fatty acid synthesis in actinomycetes, and discuss the molecular bases of their substrate specificity and the structure-based identification of new ACCase inhibitors with antimycobacterial properties.