Maturation of the Translation Inhibitor Microcin C

Metlitskaya, Anastasia; Kazakov, Teymur; Vondenhoff, Gaston; Новикова, М. А.; Shashkov, Alexander S.; Zatsepin, Timofei S.; Semenova, Ekaterina; Зайцева, Наталья Владимировна; Ramensky, Vasily; Severinov, Konstantin

doi:10.1128/jb.00999-08

Cited by 39 publications

(57 citation statements)

References 16 publications

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“…Both formylated (compound 26) and nonformylated (compound 3) MRTGNAD-SA variants were approximately 10 times less effective than McC. This difference is likely due to the lack of the aminopropyl group, which was shown to increase the efficiency of target inhibition by processed McC (9). Interestingly, uninduced Ara-Yej cells were partially inhibited by compound 18 at concentrations above 2.5 M. This could be explained by low expression levels of the araBAD::yej fusion caused by traces of arabinose in the LB medium or by the presence of a yet-unknown additional, low-affinity transporter.…”

Section: Resultsmentioning

confidence: 97%

Characterization of Peptide Chain Length and Constituency Requirements for YejABEF-Mediated Uptake of Microcin C Analogues

et al. 2011

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Microcin C (McC), a natural antibacterial compound consisting of a heptapeptide attached to a modified adenosine, is actively taken up by the YejABEF transporter, after which it is processed by cellular aminopeptidases, releasing the nonhydrolyzable aminoacyl adenylate, an inhibitor of aspartyl-tRNA synthetase. McC analogues with variable length of the peptide moiety were synthesized and evaluated in order to characterize the substrate preferences of the YejABEF transporter. It was shown that a minimal peptide chain length of 6 amino acids and the presence of an N-terminal formyl-methionyl-arginyl sequence are required for transport.In the current ongoing quest for new antibiotics, aminoacyltRNA synthetases (aaRSs) have been regarded as promising targets (5,11,14). The natural antibiotic microcin C (McC) (Fig. 1, compound 1a) targets an aaRS and has been envisaged as a lead compound for further development as an antibacterial agent (17). McC consists of a heptapeptide that is covalently linked through a phosphoramidate bond to adenosine, with, in addition, an aminopropyl moiety esterified to the phosphoramidate linker (4). Once inside a sensitive cell, McC is processed by peptide deformylase and several peptidases that remove the N-terminal formyl group and the peptide part, respectively (7). As a result of intracellular processing, the active compound (compound 2), a modified nonhydrolysable aspartyl-adenylate, is released. Processed McC is a potent inhibitor of aspartyl-tRNA synthetase (AspRS) (8).McC penetrates the outer membrane of the Escherichia coli cell mostly through the OmpF porin, but also through other, yet-unidentified transport systems (M. Novikova, A. Metlitskaya, and K. Severinov, unpublished data), and is subsequently transported through the inner membrane by the YejABEF transporter (10). YejABEF is the only complex responsible for McC transport, since yej mutants are highly resistant to McC and its maturation intermediates and chemical analogues. While intact McC inhibits the growth of sensitive E. coli cells at low micromolar concentrations, processed McC does not affect cell growth, even at millimolar concentrations. Thus, the peptide chain enables McC to function through a Trojan-horse mechanism by promoting active uptake via the YejABEF transporter. The recently improved synthetic approach for the production of McC analogues has led us to investigate the uptake properties of the Yej transporter in more detail. The results obtained could be important for further drug development, where peptides function as carrier moieties for drugs that otherwise would not be able to penetrate the bacterial membranes. Here, we used a number of McC analogues truncated either from their C-or N-terminal sides, or otherwise modified, to determine the minimal peptide chain length sufficient for facilitated transport by Yej. MATERIALS AND METHODSChemistry. Reagents and solvents were purchased from commercial suppliers (Acros, Sigma-Aldrich, Bachem, and Novabiochem) and used as provided unless otherwise indicated....

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Section: Resultsmentioning

confidence: 97%

Characterization of Peptide Chain Length and Constituency Requirements for YejABEF-Mediated Uptake of Microcin C Analogues

et al. 2011

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“…Microcin C and its maturation derivative McC(1120) were purified as described elsewhere (11). Albomycin was a gift of G. Katrukha (Gause Institute of Antibiotics, Moscow, Russia).…”

Section: Methodsmentioning

confidence: 99%

The RimL Transacetylase Provides Resistance to Translation Inhibitor Microcin C

et al. 2014

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MccE is homologous to chromosomally encoded acetyltransferases RimI, RimJ, and RimL, which acetylate, correspondingly, the N termini of ribosomal proteins S18, S5, and L12. Here, we show that E. coli RimL, but not other Rim acetyltransferases, provides a basal level of resistance to McC and various toxic nonhydrolyzable aminoacyl adenylates. RimL acts by acetylating processed McC, which along with ribosomal protein L12 should be considered a natural RimL substrate. When overproduced, RimL also makes cells resistant to albomycin, an antibiotic that upon intracellular processing gives rise to a seryl-thioribosyl pyrimidine that targets seryl-tRNA synthetase. We further show that E. coli YhhY, a protein related to Rim acetyltransferases but without a known function, is also able to detoxify several nonhydrolyzable aminoacyl adenylates but not processed McC. We propose that RimL and YhhY protect bacteria from various toxic aminoacyl nucleotides, either exogenous or those generated inside the cell during normal metabolism.

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“…The antibacterial activities of MRTGNAX-SAs 7 to 9 and corresponding XSAs 4 to 6 were determined by monitoring the appearance of growth inhibition zones (anabiosys halos) on lawns of McC-sensitive E. coli K-12 BW28357 cells (10). As controls, McC and its derivative without the N-terminal formyl and the aminopropyl groups were used; as we have described elsewhere (16), such a derivative is produced by E. coli cells lacking aminopeptidases A, B, and N and harboring an McC-producing plasmid with a disrupted mccD and/or mccE gene). The results, presented in Fig.…”

Section: Design and Synthesis Of MCC Analogsmentioning

confidence: 99%