Enacyloxin IIa Research Articles

Native ESI-MS and Collision-Induced Unfolding (CIU) of the Complex between Bacterial Elongation Factor-Tu and the Antibiotic Enacyloxin IIa.

Collision-induced unfolding (CIU) of protein ions, monitored by ion mobility-mass spectrometry, can be used to assess the stability of their compact gas-phase fold and hence provide structural information. The bacterial elongation factor EF-Tu, a key protein for mRNA translation in prokaryotes and hence a promising antibiotic target, has been studied by CIU. The major [M + 12H]12+ ion of EF-Tu unfolded in collision with Ar atoms between 40 and 50 V, corresponding to an Elab energy of 480-500 eV. Binding of the cofactor analogue GDPNP and the antibiotic enacyloxin IIa stabilized the compact fold of EF-Tu, although dissociation of the latter from the complex diminished its stabilizing effect at higher collision energies. Molecular dynamics simulations of the [M + 12H]12+ EF-Tu ion showed similar qualitative behavior to the experimental results.

P211 Evolutionary adaptation of Burkholderia multivorans to enacyloxin IIa leads to alterations in antimicrobial resistance and phenotype

P211 Evolutionary adaptation of Burkholderia multivorans to enacyloxin IIa leads to alterations in antimicrobial resistance and phenotype

Screening inhibitors against the Ef-Tu of Fusobacterium nucleatum: a docking, ADMET and PBPK assessment study.

The oral pathogen Fusobacterium nucleatum has recently been associated with an elevated risk of colorectal cancer (CRC), endometrial metastasis, chemoresistance, inflammation, metastasis, and DNA damage, along with several other diseases. This study aimed to explore the disruption of protein machinery of F. nucleatum via inhibition of elongation factor thermo unstable (Ef-Tu) protein, through natural products. No study on Ef-Tu inhibition by natural products or in Fusobacterium spp. exists till todate. Ef-Tu is an abundant specialized drug target in bacteria that varies from human Ef-Tu. Elfamycins target Ef-Tu and hence, Enacyloxin IIa was used to generate pharmacophore for virtual screening of three natural product libraries, Natural Product Activity and Species Source (NPASS) (n = 30000 molecules), Tibetan medicinal plant database (n = 54 molecules) and African medicinal plant database (n > 6000 molecules). Peptaibol Septocylindrin B (NPC141050), Hirtusneanoside, and ZINC95486259 were prioritized from these libraries as potential therapeutic candidates. ADMET profiling was done for safety assessment, physiological-based pharmacokinetic modeling in human and mouse for getting insight into drug interaction with body tissues and molecular dynamics was used to assess stability of the best hit NPC141050 (Septocylindrin B). Based on the promising results, we propose further in vitro, in vivo and pharmacokinetic testing on the lead Septocylindrin B, for possible translation into therapeutic interventions.

Antimicrobial activity of enacyloxin IIa and gladiolin against the urogenital pathogens Neisseria gonorrhoeae and Ureaplasma spp.

To determine the antimicrobial activity of enacyloxin IIa and gladiolin against Neisseria gonorrhoeae and Ureaplasma spp. The Burkholderia polyketide antibiotics enacyloxin IIa and gladiolin were tested against 14 N. gonorrhoeae and 10 Ureaplasma spp. isolates including multidrug-resistant N. gonorrhoeae isolates WHO V, WHO X and WHO Z as well as macrolide, tetracycline and ciprofloxacin-resistant ureaplasmas. Susceptibility testing of N. gonorrhoeae was carried out by agar dilution, whereas broth micro-dilution and growth kinetic assays were used for Ureaplasma spp. The MIC range for enacyloxin IIa and gladiolin against N. gonorrhoeae was 0·015-0·06mgl-1 and 1-2mgl-1 respectively. The presence of resistance to front line antibiotics had no effect on MIC values. The MIC range for enacyloxin IIa against Ureaplasma spp. was 4-32mgl-1 with a clear dose-dependent effect when observed using a growth kinetic assay. Gladiolin had no antimicrobial activity on Ureaplasma spp. at 32mgl-1 and limited impact on growth kinetics. Enacyloxin IIa and gladiolin antibiotics have antimicrobial activity against a range of antibiotic susceptible and resistant N. gonorrhoeae and Ureaplasma isolates. This study highlights the potential for a new class of antimicrobial against pathogens in which limited antibiotics are available. Development of these compounds warrants further investigation in the face of emerging extensively drug-resistant strains.

A rapid screening method for the detection of specialised metabolites from bacteria: Induction and suppression of metabolites from Burkholderia species

Screening microbial cultures for specialised metabolites is essential for the discovery of new biologically active compounds. A novel, cost-effective and rapid screening method is described for extracting specialised metabolites from bacteria grown on agar plates, coupled with HPLC for basic identification of known and potentially novel metabolites. The method allows the screening of culture collections to identify optimal production strains and metabolite induction conditions. The protocol was optimised on two Burkholderia species known to produce the antibiotics, enacyloxin IIa (B. ambifaria) and gladiolin (B. gladioli), respectively; it was then applied to strains of each species to identify high antibiotic producers. B. ambifaria AMMD and B. gladioli BCC0238 produced the highest concentrations of the respective antibiotic under the conditions tested. To induce expression of silent biosynthetic gene clusters, the addition of low concentrations of antibiotics to growth media was evaluated as known elicitors of Burkholderia specialised metabolites. Subinhibitory concentrations of trimethoprim and other clinically therapeutic antibiotics were evaluated and screened against a panel of B. gladioli and B. ambifaria. To enhance rapid strain screening with more antibiotic elicitors, antimicrobial susceptibility testing discs were included within the induction medium. Low concentrations of trimethoprim suppressed the production of specialised metabolites in B. gladioli, including the toxins, toxoflavin and bongkrekic acid. However, the addition of trimethoprim significantly improved enacylocin IIa concentrations in B. ambifaria AMMD. Rifampicin and ceftazidime significantly improved the yield of gladiolin and caryoynencin by B. gladioli BCC0238, respectively, and cepacin increased 2-fold with tobramycin in B. ambifaria BCC0191. Potentially novel metabolites were also induced by subinhibitory concentrations of tobramycin and chloramphenicol in B. ambifaria. In contrast to previous findings that low concentrations of antibiotic elicit Burkholderia metabolite production, we found they acted as both inducers or suppressors dependent on the metabolite and the strains producing them. In conclusion, the screening protocol enabled rapid characterization of Burkholderia metabolites, the identification of suitable producer strains, potentially novel natural products and an understanding of metabolite regulation in the presence of inducing or suppressing conditions.

Towards improved understanding of intersubunit interactions in modular polyketide biosynthesis: Docking in the enacyloxin IIa polyketide synthase

Modular polyketide synthases (PKSs) are molecular-scale assembly lines comprising multiple gigantic polypeptide subunits. Faithful ordering of the subunits is mediated by extreme C- and N-terminal regions called docking domains (DDs). Decrypting how specificity is achieved by these elements is important both for understanding PKS function and modifying it to generate useful polyketide analogues for biological evaluation. Here we report the biophysical and structural characterisation of all six PKS/PKS interfaces in the unusual, chimaeric cis-AT/trans-AT PKS pathway responsible for biosynthesis of the antibiotic enacyloxin IIa in Burkholderia ambifaria. Taken together with previous work, our data reveal that specificity is achieved in the enacyloxin PKS by deploying at least three functionally orthogonal classes of DDs. We also demonstrate for the first time that cis-AT PKS subunits incorporate DDs with intrinsically disordered character, reinforcing the utility of such regions for achieving both medium affinity and high specificity at PKS intersubunit junctions. Overall, this work substantially increases the number of orthogonal DDs available for creating novel, highly-specific interfaces within cis- and trans-AT PKSs and their hybrids. It also reveals unexpected sequence/structure relationships in PKS DDs, identifying major current limitations to both accurately predicting and categorising these useful protein–protein interaction motifs.

Crystal structure of EF-Tu bound to Phe-tRNAphe (tan) and a GTP analog (green). Two antibiotics, kirromycin and enacyloxin IIa, are shown superimposed and therefore disrupting EF-Tu function and cellular translation. For details, see the MicroReview by Pr

Molecular MicrobiologyVolume 106, Issue 1 p. i-i Front CoverFree Access Crystal structure of EF-Tu bound to Phe-tRNAphe (tan) and a GTP analog (green). Two antibiotics, kirromycin and enacyloxin IIa, are shown superimposed and therefore disrupting EF-Tu function and cellular translation. For details, see the MicroReview by Prezioso et al. on pp. 22–34 of this issue. First published: 20 September 2017 https://doi.org/10.1111/mmi.13518AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume106, Issue1October 2017Pages i-i RelatedInformation

Enacyloxins Are Products of an Unusual Hybrid Modular Polyketide Synthase Encoded by a Cryptic Burkholderia ambifaria Genomic Island

Gram-negative Burkholderia cepacia complex (Bcc) isolates were screened for antimicrobial activity against cystic fibrosis microbial pathogens, and the ability of B.ambifaria to inhibit B.multivorans was identified. The activity was mapped to a cluster of cryptic, quorum-sensing-regulated modular polyketide synthase (PKS) genes. Enacyloxin IIa and its stereoisomer designated iso-enacyloxin IIa were identified as metabolic products of the gene cluster, which encoded an unusual hybrid modular PKS consisting of multiple proteins with sequence similarity to cis-acyltransferase (cis-AT) PKSs and a single protein with sequence similarity to trans-AT PKSs. The discovery of the potent activity of enacyloxins against drug-resistant bacteria and the gene cluster that directs their production provides an opportunity for engineered biosynthesis of innovative enacyloxin derivatives and highlights the potential of Bcc bacteria as an underexploited resource for antibiotic discovery.

GTPase activation of elongation factor EF-Tu by the ribosome during decoding

We have used single-particle reconstruction in cryo-electron microscopy to determine a structure of the Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF-Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin. This represents the state in the decoding process just after codon recognition by tRNA and the resulting GTP hydrolysis by EF-Tu, but before the release of EF-Tu from the ribosome. Progress in sample purification and image processing made it possible to reach a resolution of 6.4 A. Secondary structure elements in tRNA, EF-Tu and the ribosome, and even GDP and kirromycin, could all be visualized directly. The structure reveals a complex conformational rearrangement of the tRNA in the A/T state and the interactions with the functionally important switch regions of EF-Tu crucial to GTP hydrolysis. Thus, the structure provides insights into the molecular mechanism of signalling codon recognition from the decoding centre of the 30S subunit to the GTPase centre of EF-Tu.

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

Elongation factor (EF-) Tu.GTP is the carrier of aminoacyl-tRNA to the programmed ribosome. Enacyloxin IIa inhibits bacterial protein synthesis by hindering the release of EF-Tu.GDP from the ribosome. The crystal structure of the Escherichia coli EF-Tu.guanylyl iminodiphosphate (GDPNP).enacyloxin IIa complex at 2.3 A 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-Tu.GDP 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-Tu.GDPNP.enacyloxin IIa shows a disordered effector region that in the Phe-tRNAPhe.EF-Tu (Thermus aquaticus).GDPNP.enacyloxin IIa complex, solved at 3.1 A 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.

Intramolecular migration of long chain acyl group of Enacyloxin IIa methyl ester

Enacyloxin IIa methyl ester in non-protic solvent gave rearranged product in acidic conditions such as surface of TLC plate of silica gel or by treatment with a catalytic amount of p-toluenesulfonic acid. On the other hand, such a rearrangement did not occur in polar protic solvent. The structure of the rearranged product was elucidated as 4-acyl analog of the cyclohexane ring.

Mutant EF-tu species reveal novel features of the enacyloxin IIa inhibition mechanism on the ribosome

For clarification of the action of a new antibiotic, the analysis of resistant mutants is often indispensable. For enacyloxin IIa we discovered four resistant elongation factor Tu (EF-Tu) species in Escherichia coli with the mutations Q124K, G316D, Q329H, and A375T, respectively. They revealed that enacyloxin IIa sensitivity is dominant in a mixed population of resistant and wild-type EF-Tus. This points to an inhibition mechanism in which EF-Tu is the dominant target of enacyloxin IIa and in which a ribosome with a sensitive EF-Tu blocks mRNA translation for upstream ribosomes with resistant EF-Tus, a mechanism similar to that of the unrelated antibiotic kirromycin. Remarkably, the same mutations are also linked to kirromycin resistance, though the order of their levels of resistance is different from that for enacyloxin IIa. Among the mutant EF-Tus, three different resistance mechanisms can be distinguished: (i) by obstructing enacyloxin IIa binding to EF-Tu·GTP; (ii) by enabling the release of enacyloxin IIa after GTP hydrolysis; and (iii) by reducing the affinity of EF-Tu·GDP·enacyloxin IIa for aminoacyl-tRNA at the ribosomal A-site, which then allows the release of EF-Tu·GDP·enacyloxin IIa. Ala375 seems to contribute directly to enacyloxin IIa binding at the domain 1-3 interface of EF-Tu·GTP, a location that would easily explain the pleiotropic effects of enacyloxin IIa on the functioning of EF-Tu.

Antibiotic Inhibitors of Organellar Protein Synthesis in Plasmodium falciparum

Elongation factor Tu (EF-Tu) is encoded by the tuf gene of the plastid organelle of the malaria parasite Plasmodium falciparum. A range of structurally unrelated inhibitors of this GTP-dependent translation factor was shown to have antimalarial activity in blood cultures. The most active was the cyclic thiazolyl peptide amythiamicin A with an IC50 = 0.01 microM. Demonstrable complexes were formed in vitro between a recombinant version of P. falciparum EF-Tu(pl) and inhibitors that bind to different sites on EF-Tu; these included the antibiotics kirromycin, GE2270A and enacyloxin IIa.

Functional role of the noncatalytic domains of elongation factor Tu in the interactions with ligands.

Elongation factor (EF) Tu from Escherichia coli contains three domains, of which domain 1 (N-terminal domain) harbors the site for nucleotide binding and GTP hydrolysis. To analyze the function of domains 2 [middle (M) domain] and 3 [C-terminal (C) domain], EF-Tu(DeltaM) and EF-Tu(DeltaC) were engineered as GST-fused products and purified. Circular dichroism and thermostability showed that both constructs have conserved organized structures. Though inactive in poly(Phe) synthesis the two constructs could bind GDP and GTP with comparable micromolar affinities. Therefore, like the isolated N-terminal domain, they had lost a typical feature of EF-Tu, the >100 times stronger affinity for GDP than for GTP. EF-Tu(DeltaM) and EF-Tu(DeltaC) had an intrinsic GTPase activity comparable to that of wild-type EF-Tu. Ribosomes did not stimulate the GTPase activity of either factor, while kirromycin increased the GTPase activity of both constructs, particularly of EF-Tu(DeltaC), to a level, however, much lower than that of the intact molecule. The interaction with aa-tRNA of both mutants was >90% reduced. As a major result, their GDP-bound form could efficiently respond to EF-Ts. All four EF-Tu-specific antibiotics [kirromycin, pulvomycin, GE2270 A (=MDL 62 879), and enacyloxin IIa] retarded significantly the dissociation of EF-Tu(DeltaC).GTP, showing the same kind of effect as on EF-Tu.GTP, but they were little active on EF-Tu(DeltaM). GTP. Like EF-Tu(DeltaC).GTP, EF-Tu(DeltaM).GTP was, however, able to bind efficiently kirromycin and enacyloxin IIa, as determined via competition with EF-Ts. Together, these results enlight selective functions of domains 2 and 3, particularly toward the interaction with EF-Ts and antibiotics, and emphasize their functional cooperativity for an efficient interaction of EF-Tu with ribosomes and aa-tRNA and for maintaining the differential affinity for GTP and GDP.

Enacyloxin IIa, an inhibitor of protein biosynthesis that acts on elongation factor Tu and the ribosome.

This work analyzes the action of enacyloxin Ila, an inhibitor of bacterial protein biosynthesis. Enacyloxin IIa [IC50 on poly(Phe) synthesis approximately 70 nM] is shown to affect the interaction between elongation factor (EF) Tu and GTP or GDP; in particular, the dissociation of EF-Tu-GTP is strongly retarded, causing the Kd of EF- Tu-GTP to decrease from 500 to 0.7 nM. In its presence, the migration velocity of both GTP- and GDP-bound EF-Tu on native PAGE is increased. The stimulation of EF-Tu-GDP dissociation by EF-Ts is inhibited. EF- Tu-GTP can still form a stable complex with aminoacyl-tRNA (aa-tRNA), but it no longer protects aa-tRNA against spontaneous deacylation, showing that the EF-Tu-GTP orientation with respect to the 3' end of aa-tRNA is modified. However, the EF-Tu-dependent binding of aa-tRNA to the ribosomal A-site is impaired only slightly by the antibiotic and the activity of the peptidyl-transferase center, as determined by puromycin reactivity, is not affected. In contrast, the C-terminal incorporation of Phe into poly(Phe)-tRNA bound to the P-site is inhibited, an effect that is observed if Phe-tRNA is bound to the A-site nonenzymatically as well. Thus, enacyloxin IIa can affect both EF-Tu and the ribosomal A-site directly, inducing an anomalous positioning of aa-tRNA, that inhibits the incorporation of the amino acid into the polypeptide chain. Therefore, it is the first antibiotic found to have a dual specificity targeted to EF-Tu and the ribosome.

An extracellular quinoprotein oxidase that catalyzes conversion of enacyloxin IVa to enacyloxin IIa.

A new extracellular quinoprotein oxidase named enacyloxin oxidase (ENX oxidase), which is involved in biosynthesis of ENX IIa, a congener of ENX, was found in the culture supernatant of Frateuria sp. W-315 and purified as a homogeneous protein on SDS-PAGE. ENX oxidase was shown to have a molecular mass of 73 kDa by SDS-PAGE and 79 kDa by gel filtration. The enzyme was inhibited by various carbonyl reagents and the activity was stimulated by addition of PQQ. This is the first report on a quinoprotein oxidase that is secreted into the culture medium in the logarithmic growth phase, and acts for biosynthesis of the antibiotic.

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

The chemical structure of a unique polyenic antibiotic enacyloxin IIa (former name: fr. 2) produced by Frateuria (formerly Gluconobacter) sp. W-315 has been determined by extensive spectroscopic studies, in particular by NMR spectral analysis. It has a novel non-lactonic structure involving 3,4-dihydroxycyclohexanecarboxylic acid with a chlorine-containing polyenic and polyhydroxy acyl side chain attached as an ester to the 3-hydroxyl substituent of the acid.

New polyenic antibiotics active against gram-positive and gram-negative bacteria. VI. Non-lactonic polyene antibiotic, enacyloxin IIa, inhibits binding of aminoacyl-tRNA to A site of ribosomes.

New polyenic antibiotics active against gram-positive and gram-negative bacteria. VI. Non-lactonic polyene antibiotic, enacyloxin IIa, inhibits binding of aminoacyl-tRNA to A site of ribosomes.

New polyenic antibiotics active against gram-positive and gram-negative bacteria. VII. Isolation and structure of enacyloxin IVa, a possible biosynthetic intermediate of enacyloxin IIa.

New polyenic antibiotics active against gram-positive and gram-negative bacteria. VII. Isolation and structure of enacyloxin IVa, a possible biosynthetic intermediate of enacyloxin IIa.

New polyenic antibiotics active against Gram-positive and Gram-negative bacteria. V. Mode of action of enacyloxin IIa.

New polyenic antibiotics active against Gram-positive and Gram-negative bacteria. V. Mode of action of enacyloxin IIa.