Amino Acid Residues Required for Maturation, Cell Uptake, and Processing of Translation Inhibitor Microcin C

Kazakov, Teymur; Metlitskaya, Anastasia; Severinov, Konstantin

doi:10.1128/jb.01609-06

Cited by 22 publications

(29 citation statements)

References 11 publications

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“…One therefore could speculate that a positive charge is beneficial at this position. However, the latter result is in contrast with the McC variants obtained by mutagenesis, where Ala 2 , Ser 2 , and Trp 2 variants also proved active (6). In addition, and more curiously, compounds 23 and 7b displayed antibacterial activity, albeit at high concentrations, which may be explained by their small size and hence their ability to diffuse through the bacterial membrane.…”

Section: Discussioncontrasting

confidence: 51%

“…Site-specific mutagenesis of the mccA gene coding for the peptide moiety of McC and providing substitution of the internal amino acid positions generated multiple active McC variants. Only the C-terminal asparagine, which is converted to aspartic acid upon maturation of McC, proved indispensable (6). The N-terminal formyl methionine was not included in the analysis, since it is essential for (17) and probing of the importance of the N-terminal methionine residue.…”

Section: Discussionmentioning

confidence: 99%

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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: Discussioncontrasting

confidence: 51%

Section: Discussionmentioning

confidence: 99%

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

et al. 2011

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“…To characterize compounds produced by cells harboring pUHAB and its derivatives, culture medium of producing cells was subjected to the standard McC purification procedure, whose final stage is reverse-phase high-performance liquid chromatography (HPLC) (8,10). All purified compounds with strong adsorption at 260 nm were characterized by matrixassisted laser desorption ionization mass spectrometry.…”

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Maturation of the Translation Inhibitor Microcin C

et al. 2009

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“…Several peptides matched mccA mutants previously tested in vivo (15). These included derivatives with substitutions of the terminal residue (MRTGNAD and MRTGNAQ) and substitutions in the third (MRAGNAN, MRLGNAN, MRKGNAN, MRS GNAN, and MRCGNAN) and fourth (MRTWNAN) positions of the MccA peptide.…”

Section: Use Of In Vitro Adenylation By Mccb To Prepare Mutant MCC Vamentioning

confidence: 99%

“…Structure-activity analysis of the McC peptide was carried out by introduction of point substitutions in the mccA gene (15). Using this approach, codons 2 to 7 of mccA were each systematically substituted for codons coding for 19 remaining standard amino acids, and the effects of these substitutions on the ability of cells harboring mutated mcc operons to produce mutant microcins were determined.…”

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Enzymatic Synthesis and Functional Characterization of Bioactive Microcin C-Like Compounds with Altered Peptide Sequence and Length

et al. 2015

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T he antibiotic microcin C (McC) (Fig. 1) is produced by Escherichia coli strains harboring a plasmid-borne mcc operon. McC consists of a ribosomally synthesized heptapeptide that is covalently linked through a phosphoramidate bond to adenosine; the phosphoramidate linker is esterified with an aminopropyl moiety (1). Inside a sensitive cell, the N-terminal formyl group of the McC peptide is removed by peptide deformylase, after which the peptide part is processed by aminopeptidases (2). As a result, processed McC, a modified nonhydrolyzable aspartyl-adenylate, is released. Processed McC is a potent inhibitor of aspartyl-tRNA synthetase (AspRS), an essential enzyme (3).McC penetrates the inner membrane of E. coli cells through the YejABEF transporter (4). While intact McC inhibits the growth of sensitive E. coli cells at low micromolar concentrations (5, 6), processed McC does not affect cell growth, even at millimolar concentrations (3, 7). Thus, the peptide chain enables processed McC function through a Trojan horse mechanism by promoting its active uptake via YejABEF. YejABEF is uniquely responsible for McC transport, since yej mutants are fully resistant to McC (4). The biological function of YejABEF (other than McC transport) is presently unknown. In Salmonella, it confers resistance to some antimicrobial peptides and allows proliferation inside activated macrophages, thus contributing to virulence (8). The latter property may be related to the fact that YejABEF interferes with peptide presentation on major histocompatibility complex (MHC) class I molecules (9).

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Amino Acid Residues Required for Maturation, Cell Uptake, and Processing of Translation Inhibitor Microcin C

Cited by 22 publications

References 11 publications

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

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

Maturation of the Translation Inhibitor Microcin C

Enzymatic Synthesis and Functional Characterization of Bioactive Microcin C-Like Compounds with Altered Peptide Sequence and Length

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