New developments in non-post translationally modified microcins

Pons, Anne-Marie; Lanneluc, Isabelle; Cottenceau, Gilles; Sablé, Sophie

doi:10.1016/s0300-9084(02)01416-5

Cited by 55 publications

(52 citation statements)

References 32 publications

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“…14, final total protein concentration, 2.5 mg/ml) was carried out at 30°C in the presence of 100 mM Hepes KOH, pH 8.0, 2% PEG8000, 0.1 mM EDTA, 2 mM DTT, 0.5 g/liter of bulk E. coli tRNA, 0.1 g/liter of folinic acid, 0.1 mM luciferin, 20 mM acetyl phosphate, 20 mM phosphoenolpyruvate, 1 mM GTP, 1 mM UTP, 1 mM CTP, 1.4 mM ATP, 11.5 mM Mg(OAc) 2 , 237 mM KOAc, 0.5 mM of each amino acid, 15 M pT7luc plasmid (15), 0.04 g/liter of pyruvate kinase, and 2 units/l of T7 RNA polymerase. The reaction mixture was placed into the cell of a Hidex Triathler luminometer and the appearance of luciferase activity in the course of translation was monitored.…”

Section: Preparation Of Cell Extract For MCC Processing-mcc-sensitivementioning

confidence: 99%

“…16, final total protein concentration 3 mg/ml) was carried out at 30°C in the presence of 26.1 mM Hepes KOH, pH 7.6, 2% glycerol, 1.6 mM DTT, 0.05 g/liter of bulk yeast tRNA, 0.1 mM luciferin, 16 mM creatine phosphate, 0.4 mM GTP, UTP, CTP, 1 mM ATP, 3 mM Mg(OAc) 2 , 70 mM KOAc, 0.2 mM each amino acid, 0.45 mM spermidine, 74 nM luciferase mRNA, and 0.1 g/liter of creatine kinase. The reaction progression was monitored as above.…”

Section: Preparation Of Cell Extract For MCC Processing-mcc-sensitivementioning

confidence: 99%

“…In Vitro Translation of Poly(U) RNA-Poly(U) RNA translation in 3 mg/ml E. coli S30 extract (14) was carried out at 30°C in the presence of 26.1 mM Hepes KOH, pH 7.6, 4% PEG8000, 1.7 mM DTT, 0.175 g/liter of bulk E. coli tRNA, 0.03 g/liter of folinic acid, 0.1 mM luciferin, 80 mM creatine phosphate, 0.8 mM GTP, 1.2 mM ATP, 10 mM Mg(OAc) 2 , 125 mM KOAc, ϳ74 nM poly(U) RNA (molecular mass 800 -1000 kDa, Sigma), 0.25 g/liter of creatine kinase. The reaction mixture contained [ 14 C]Phe.…”

Section: Preparation Of Cell Extract For MCC Processing-mcc-sensitivementioning

confidence: 99%

“…Cells were collected by centrifugation, and washed with 40 mM Tris-HCl, pH 7.6, 10 mM Mg(OAc) 2 , 50 mM KOAc, 0.1 mM EDTA, 1 mM DTT. The cell pellet was resuspended in an equal volume of the same buffer and disrupted by a French press (pressure 1000 bar).…”

Section: Preparation Of Cell Extract For MCC Processing-mcc-sensitivementioning

confidence: 99%

“…Whereas some microcins are active as unmodified peptides (2), others are produced as polypeptide precursors that are heavily modified by dedicated maturation enzymes (3). Interest is attached to such post-translationally modified microcins due to their highly unusual structures and the fact that they target important cellular processes that are attractive targets for antibacterial drug development.…”

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confidence: 99%

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Aspartyl-tRNA Synthetase Is the Target of Peptide Nucleotide Antibiotic Microcin C

Metlitskaya

Kazakov

Kommer

et al. 2006

Journal of Biological Chemistry

142

157

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Microcin C is a ribosome-synthesized heptapeptide that contains a modified adenosine monophosphate covalently attached to the C-terminal aspartate. Microcin C is a potent inhibitor of bacterial cell growth. Based on the in vivo kinetics of inhibition of macromolecular synthesis, Microcin C targets translation, through a mechanism that remained undefined. Here, we show that Microcin C is a subject of specific degradation inside the sensitive cell. The product of degradation, a modified aspartyl-adenylate containing an N-acylphosphoramidate linkage, strongly inhibits translation by blocking the function of aspartyl-tRNA synthetase.Microcins are a class of small (Ͻ10 kDa) ribosomally synthesized peptide antibiotics produced by Enterobacteriaceae (1). Whereas some microcins are active as unmodified peptides (2), others are produced as polypeptide precursors that are heavily modified by dedicated maturation enzymes (3). Interest is attached to such post-translationally modified microcins due to their highly unusual structures and the fact that they target important cellular processes that are attractive targets for antibacterial drug development.Genes responsible for microcin production are usually plasmidborne. Plasmids encoding microcin structural and maturation genes also encode determinants of immunity specific to the microcin produced. Based on cross-immunity, post-translationally modified microcins can be subdivided into the B, C, and J types. Microcin B (MccB) 4 is a 43-residue peptide with 8 thiazole and oxazole rings that are synthesized by the McbBCD maturation enzyme complex from multiple serine and cysteine residues present in the MccB precursor (4). MccB is a potent inhibitor of DNA gyrase; it traps the enzyme at the stage of DNA strand passage (5). Microcin J, a 21-amino acid peptide, contains an unusual lactam bond between its N-terminal glycine and the ␦-carboxyl group of an internal glutamate; it assumes a highly unusual threaded-lasso structure (6 -8). MccJ inhibits bacterial RNA polymerase by occluding a narrow channel that is used to traffic transcription substrates, NTPs, to the catalytic center of the enzyme (9, 10).The structure of the subject of this study, Microcin C (McC) is shown in Fig. 1A. McC is a heptapeptide containing a modified adenosine monophosphate covalently attached to its C terminus through an N-acylphosphoramidate linkage (11, 12). The phosphoramidate group of the nucleotide part of McC is additionally modified by a propylamine group. Additionally, in mature McC, the peptide moiety, which is encoded by the mccA gene, is modified and the C-terminal asparagine residue specified by mccA is converted to an aspartate (18, 19), through an unknown mechanism. In vivo, McC appears to target translation (12). Guijarro et al. (12) also reported that large concentrations of McC, as well as of synthetic peptide of the same sequence but without the nucleotide modification, mildly inhibit translation in vitro. They therefore concluded that the peptide part of McC is responsible for transl...

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Section: Preparation Of Cell Extract For MCC Processing-mcc-sensitivementioning

confidence: 99%

Section: Preparation Of Cell Extract For MCC Processing-mcc-sensitivementioning

confidence: 99%