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1.
Structure ; 25(10): 1549-1561.e5, 2017 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-28943336

RESUMO

TldD and TldE proteins are involved in the biosynthesis of microcin B17 (MccB17), an Escherichia coli thiazole/oxazole-modified peptide toxin targeting DNA gyrase. Using a combination of biochemical and crystallographic methods we show that E. coli TldD and TldE interact to form a heterodimeric metalloprotease. TldD/E cleaves the N-terminal leader sequence from the modified MccB17 precursor peptide, to yield mature antibiotic, while it has no effect on the unmodified peptide. Both proteins are essential for the activity; however, only the TldD subunit forms a novel metal-containing active site within the hollow core of the heterodimer. Peptide substrates are bound in a sequence-independent manner through ß sheet interactions with TldD and are likely cleaved via a thermolysin-type mechanism. We suggest that TldD/E acts as a "molecular pencil sharpener": unfolded polypeptides are fed through a narrow channel into the active site and processively truncated through the cleavage of short peptides from the N-terminal end.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Bacteriocinas/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/química , Modelos Moleculares , Peptídeos/metabolismo , Conformação Proteica , Especificidade por Substrato
2.
Sci Rep ; 6: 27792, 2016 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-27283217

RESUMO

There is an urgent need to identify new treatments for tuberculosis (TB), a major infectious disease caused by Mycobacterium tuberculosis (Mtb), which results in 1.5 million deaths each year. We have targeted two essential enzymes in this organism that are promising for antibacterial therapy and reported to be inhibited by naphthoquinones. ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the human enzyme. DNA gyrase is a DNA topoisomerase present in bacteria and plants but not animals. The current study set out to understand the structure-activity relationships of these targets in Mtb using a combination of cheminformatics and in vitro screening. Here, we report the identification of new Mtb ThyX inhibitors, 2-chloro-3-(4-methanesulfonylpiperazin-1-yl)-1,4-dihydronaphthalene-1,4-dione) and idebenone, which show modest whole-cell activity and appear to act, at least in part, by targeting ThyX in Mtb.


Assuntos
Proteínas de Bactérias/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Timidilato Sintase/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Teorema de Bayes , DNA Girase/metabolismo , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Aprendizado de Máquina , Naftoquinonas/química , Naftoquinonas/farmacologia , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/química , Ubiquinona/análogos & derivados , Ubiquinona/química , Ubiquinona/farmacologia , Interface Usuário-Computador
3.
J Mol Biol ; 427(12): 2192-204, 2015 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-25861759

RESUMO

Simocyclinone D8 (SD8) is a potent DNA gyrase inhibitor produced by Streptomyces antibioticus Tü6040. The simocyclinone (sim) biosynthetic gene cluster has been sequenced and a hypothetical biosynthetic pathway has been proposed. The tetraene linker in SD8 was suggested to be the product of a modular type I polyketide synthase working in trans with two monofunctional enzymes. One of these monofunctional enzymes, SimC7, was proposed to supply a dehydratase activity missing from two modules of the polyketide synthase. In this study, we report the function of SimC7. We isolated the entire ~72-kb sim cluster on a single phage artificial chromosome clone and produced simocyclinone heterologously in a Streptomyces coelicolor strain engineered for improved antibiotic production. Deletion of simC7 resulted in the production of a novel simocyclinone, 7-oxo-SD8, which unexpectedly carried a normal tetraene linker but was altered in the angucyclinone moiety. We demonstrate that SimC7 is an NAD(P)H-dependent ketoreductase that catalyzes the conversion of 7-oxo-SD8 into SD8. 7-oxo-SD8 was essentially inactive as a DNA gyrase inhibitor, and the reduction of the keto group by SimC7 was shown to be crucial for high-affinity binding to the enzyme. Thus, SimC7 is an angucyclinone ketoreductase that is essential for the biological activity of simocyclinone.


Assuntos
Oxirredutases do Álcool/metabolismo , Antibacterianos/farmacologia , DNA Girase/metabolismo , NAD/metabolismo , Streptomyces antibioticus/enzimologia , Oxirredutases do Álcool/genética , Vias Biossintéticas/genética , Biotransformação , Cumarínicos/farmacologia , Inibidores Enzimáticos/farmacologia , Deleção de Genes , Glicosídeos/farmacologia , Família Multigênica , Oxirredução , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces antibioticus/genética , Streptomyces coelicolor/enzimologia , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo
4.
J Mol Biol ; 426(10): 2023-33, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24594357

RESUMO

Simocyclinone D8 (SD8) is an antibiotic produced by Streptomyces antibioticus that targets DNA gyrase. A previous structure of SD8 complexed with the N-terminal domain of the DNA gyrase A protein (GyrA) suggested that four SD8 molecules stabilized a tetramer of the protein; subsequent mass spectrometry experiments suggested that a protein dimer with two symmetry-related SD8s was more likely. This work describes the structures of a further truncated form of the GyrA N-terminal domain fragment with and without SD8 bound. The structure with SD8 has the two SD8 molecules bound within the same GyrA dimer. This new structure is entirely consistent with the mutations in GyrA that confer SD8 resistance and, by comparison with a new apo structure of the GyrA N-terminal domain, reveals the likely conformation changes that occur upon SD8 binding and the detailed mechanism of SD8 inhibition of gyrase. Isothermal titration calorimetry experiments are consistent with the crystallography results and further suggest that a previously observed complex between SD8 and GyrB is ~1000-fold weaker than the interaction with GyrA.


Assuntos
Antibacterianos/química , DNA Girase/química , Inibidores da Topoisomerase II/química , Substituição de Aminoácidos , Antibacterianos/farmacologia , Cumarínicos/química , Cumarínicos/farmacologia , Cristalografia por Raios X , DNA Girase/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Glicosídeos/química , Glicosídeos/farmacologia , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Inibidores da Topoisomerase II/farmacologia
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