SummaryTldD 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 β 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.
Copper trafficking proteins and copper-sensitive regulators are often found to be able to bind multiple Cu(I) ions in the form of Cu(I) clusters. We have determined the high-resolution X-ray crystal structure of an Atx1-like copper chaperone protein from Bacillus subtilis containing a novel tetranuclear Cu(I) cluster. The identities and oxidation states of the cluster ions were established unambiguously by refinement of X-ray energy-dependent anomalous scattering factors. The [Cu(4)(S-Cys)(4)(N-His)(2)] cluster geometry provides new structural insights into not only the binding of multiple cuprous ions by metallochaperones but also protein-associated tetranuclear Cu(I) clusters, including those found in eukaryotic copper-responsive transcription factors.
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.
Multinuclear Cu(I) clusters are common in nature, but little is known about their formation or transfer between proteins. CopZ and CopA from Bacillus subtilis, which are involved in a copper-efflux pathway, both readily accommodate multinuclear Cu(I) clusters. Using the luminescence properties of a multinuclear Cu(I)-bound form of the two N-terminal soluble domains of CopA (CopAab) we have investigated the thermodynamic and kinetic properties of cluster formation and loss. We demonstrate that Cu(I)-bound forms of dimeric CopZ containing more than one Cu(I) per CopZ monomer can transfer Cu(I) to apo-CopAab, leading to the formation of luminescent dimeric CopAab. Kinetic studies demonstrated that transfer is a first-order process and that the rate-determining steps for transfer from CopZ to CopAab and vice versa are different processes. The rate of formation of luminescent CopAab via transfer of Cu(I) from CopZ was more rapid than that observed when Cu(I) was added 'directly' from solution or in complex with a cysteine variant of CopZ, indicating that transfer occurs via a transient protein-protein complex. Such a complex would probably require the interaction of at least one domain of CopAab with the CopZ dimer. Insight into how such a complex might form is provided by the high resolution crystal structure of Cu3(CopZ)3, a thus far unique trimeric form of CopZ containing a trinuclear Cu(I) cluster. Modelling studies showed that one of the CopZ monomers can be substituted for either domain of CopAab, resulting in a heterotrimer, thus providing a model for a 'trapped' copper exchange complex.
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.
New structures of the N-terminal 43 kDa fragment of the E. coli DNA gyrase B subunit reveal two discrete monovalent cation-binding sites that could have functional roles.
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.
The discovery of new antibiotics with novel modes of action to combat antimicrobial resistance (AMR) is of vital importance. The natural product simocyclinone D8 (SD8) is a potent inhibitor of DNA gyrase. Its bi-functional structure and novel mode of action serve as an inspiring lead for antibiotic development. Herein we describe a proof of principle fragment-based approach towards the development of a new class of coumarin-quinolone hybrids. We demonstrate that the coumarin moiety is required for the observed inhibitory activity (IC50 ~3 M) of the hybrid compound, which is in part mediated through stabilisation of a cleaved-DNA intermediate
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