New polyenic antibiotics active against Gram-positive and Gram-negative bacteria. IV. Structural elucidation of enacyloxin IIa.

Watanabe, Toshihiko; Sugiyama, Takeyoshi; Takahashi, Masahiro; Shima, Jun; Izaki, Kazuo; Furihata, Kazuo; Seto, Haruo

doi:10.7164/antibiotics.45.470

Cited by 27 publications

(17 citation statements)

References 34 publications

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“…Enacyloxin IIa is a linear polyenic acid similar to kirromycin (Appendix D1) isolated from Fratueria W-315 (i.e., not from the actinomycetes as all other EF-Tu-binding inhibitory antibiotics) and is active against Gram-positive and Gram-negative bacteria [91,92]. It is easy to envisage enacyloxin IIa operating through a similar mechanism to kirromycin, since enacyloxin is structurally similar and the mutations that give rise to enacyloxin resistance were originally identified as kirromycin-resistant mutations (indicated in Figs.…”

Section: The Kirromycins Trap Ef-tu On the Ribosomementioning

confidence: 99%

Antibiotics and the Inhibition of Ribosome Function

Wilson

2004

Protein Synthesis and Ribosome Structure

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Section: The Kirromycins Trap Ef-tu On the Ribosomementioning

confidence: 99%

Antibiotics and the Inhibition of Ribosome Function

Wilson

2004

Protein Synthesis and Ribosome Structure

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“…2A) is active against Gram-positive and Gram-negative bacteria, slightly active against fungi, but inactive against yeasts (17)(18)(19). It stabilizes the EF-Tu⅐GTP complex (7) and enables EF-Tu⅐GDP to interact with aa-tRNA and ribosomes (8).…”

Section: Enacyloxin Iia Binding Site On Ef-tu-enacyloxinmentioning

confidence: 99%

Enacyloxin IIa Pinpoints a Binding Pocket of Elongation Factor Tu for Development of Novel Antibiotics

Parmeggiani

Krab

Watanabe

et al. 2006

Journal of Biological Chemistry

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Elongation factor (EF-) Tu⅐GTP is the carrier of aminoacyl-tRNA to the programmed ribosome. Enacyloxin IIa inhibits bacterial protein synthesis by hindering the release of EF-Tu⅐GDP from the ribosome. The crystal structure of the Escherichia coli EF-Tu⅐guanylyl iminodiphosphate (GDPNP)⅐enacyloxin IIa complex at 2.3 Å resolution presented here reveals the location of the antibiotic at the interface of domains 1 and 3. The binding site overlaps that of kirromycin, an antibiotic with a structure that is unrelated to enacyloxin IIa but that also inhibits EF-Tu⅐GDP release. As one of the major differences, the enacyloxin IIa tail borders a hydrophobic pocket that is occupied by the longer tail of kirromycin, explaining the higher binding affinity of the latter. EF-Tu⅐GDPNP⅐enacyloxin IIa shows a disordered effector region that in the PhetRNA Phe ⅐EF-Tu (Thermus aquaticus)⅐GDPNP⅐enacyloxin IIa complex, solved at 3.1 Å resolution, is stabilized by the interaction with tRNA. This work clarifies the structural background of the action of enacyloxin IIa and compares its properties with those of kirromycin, opening new perspectives for structure-guided design of novel antibiotics. Elongation factor (EF)5 Tu is a GTPase that, as all members of this superfamily, cycles between the active GTP-bound and the inactive GDP-bound state. In bacterial protein synthesis EF-Tu is the carrier of aa-tRNA to the mRNA-programmed ribosome (for review see Refs. 1 and 2). The codon-anticodon interaction triggers the hydrolysis of the bound GTP, inducing the release of EF-Tu⅐GDP, the positioning of aa-tRNA into the ribosomal A-site, and peptide bond formation with the P-site-bound peptidyl-tRNA. EF-Tu folds in three distinct domains: the nucleotide-binding domain 1 (residues 1-199 in Escherichia coli (Ec) EF-Tu⅐GDP) shows the classical GTPase mixed ␣/␤ Rossman fold, whereas domains 2 and 3 (residues 209 -299 and 300 -393, respectively) are ␤-barrels. The association of the domains in EF-Tu⅐GDP with reduced interfaces and a central hole becomes more compact in EF-Tu⅐GTP with extensive interfaces and no hole (3-5). EF-Tu is the target of four structurally unrelated families of antibiotic inhibitors of protein synthesis (for review see Ref.2), of which kirromycin and enacyloxin IIa hinder the release of EF-Tu⅐GDP from the ribosome after GTP hydrolysis, thus inhibiting its recycling and peptide bond formation. The high-resolution crystal structure of Thermus thermophilus (Tt) EF-Tu⅐GDP in complex with methylkirromycin (also called aurodox) showed that the antibiotic binds in the interface of domains 1 and 3, inducing a unique, compact EF-Tu-GTP-like conformation, which is the reason for its inhibition of the EF-Tu⅐GDP release (6). These conclusions have been further confirmed in studies of the Phe-tRNA Phe ⅐ EF-Tu⅐GDPNP⅐kirromycin. 6 Specific differences between the enacyloxin IIa and kirromycin complexes with EF-Tu concern various aspects such as the electrophoretic migration on native gel, binding affinity, GTPase activity, different effects of...

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“…These compounds were previously isolated from Frateuria sp. and Burkholderia ambifaria . To prove that the induced products are identical with enacyloxins, we isolated the derivatives from a large‐scale culture by a combination of different chromatographic techniques and elucidated their structures by 1D and 2D NMR analyses.…”

Section: Resultsmentioning

confidence: 99%

Biosynthesis of antifungal and antibacterial polyketides by Burkholderia gladioli in coculture with Rhizopus microsporus

Ross

Opel

Scherlach

et al. 2014

Mycoses

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SummaryFungi-bacteria interactions can impact the course of fungal infection and biotechnological use. The mucoralean fungus Rhizopus microsporus, traditionally used in food fermentations (tempe and sufu), is frequently accompanied by Burkholderia gladioli pv. cocovenenans. When producing tempe bongkrek, the bacterial contamination can lead to lethal food-related intoxications caused by the respiratory toxin bongkrekic acid. To unveil the metabolic potential of the fungus-associated bacterium, we sequenced its genome, assigned secondary metabolite biosynthesis gene clusters and monitored the metabolic profile under various growth conditions. In addition to the bongkrekic acid biosynthesis gene cluster we found gene clusters coding for the biosynthesis of toxoflavin and a complex polyketide. The orphan polyketide synthase gene cluster was activated under conditions that emulate tempe production, which enabled isolation and structure elucidation of four members of the enacyloxin family of antibiotics, out of which one is new. Moreover, we found that the fungus positively influences the growth of the bacteria and dramatically increases bongkrekic acid production in stationary culture, which inhibits the growth of the fungus. These results showcase the context-dependent formation of antifungal and antibacterial agents at the fungal-bacterial interface, which may also serve as a model for scenarios observed in mixed infections.

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New polyenic antibiotics active against Gram-positive and Gram-negative bacteria. IV. Structural elucidation of enacyloxin IIa.

Cited by 27 publications

References 34 publications

Antibiotics and the Inhibition of Ribosome Function

Antibiotics and the Inhibition of Ribosome Function

Enacyloxin IIa Pinpoints a Binding Pocket of Elongation Factor Tu for Development of Novel Antibiotics

Biosynthesis of antifungal and antibacterial polyketides by Burkholderia gladioli in coculture with Rhizopus microsporus

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