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

Abstract

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...

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

“…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).…”

Collision induced dissociation-based characterization of nucleotide peptides: Fragmentation patterns of microcin C7–C51, an antimicrobial peptide produced by Escherichia coli

“…This entity, which is the actual intracellularly active form of the microcin, is an inhibitor of translation [18]. It can be generated in vitro by specific enzymatic cleavage [18]. MccC7-C51, which is composed of two distinct parts, a peptidic moiety (the heptapeptide that we term PepC7-C51) (Scheme 1c) and a modified nucleotide moiety connected through a phosphoramidate bond, thus offering a unique opportunity to study the fragmentation of nucleotide peptides.…”

“…Indeed, a specific cleavage occurs inside the target bacteria at the microcin Ala6 -Asp7 peptide bond that yields a modified aspartyl-adenylate named MccC7-C51* (Scheme 1b). This entity, which is the actual intracellularly active form of the microcin, is an inhibitor of translation [18]. It can be generated in vitro by specific enzymatic cleavage [18].…”

“…It is a ribosomally-synthesized N-formylated heptapeptide including a C-terminal aspartic acid, which is covalently linked to a phosphoramidate group, itself substituted with both an adenosine moiety and a propylamine chain [17]. MccC7-C51 activity involves an original mechanism of "Trojan horse" type [18,19], where the microcin is allowed to enter, freely target bacteria, and is transformed when inside into a toxic entity. Indeed, a specific cleavage occurs inside the target bacteria at the microcin Ala6 -Asp7 peptide bond that yields a modified aspartyl-adenylate named MccC7-C51* (Scheme 1b).…”

Γονιδιωματική Ανάλυση Και Λειτουργικός Χαρακτηρισμός Ασύνηθων Συστημάτων Αμινοακυλίωσης Του Μεταφορικού RNA Σε Παθογόνους Μικροοργανισμούς

“…Αποτελεί ένα επταπεπτίδιο που συντίθεται μέσω του ριβοσώματος και περιέχει μια τροποποιημένη μονοφωσφορική αδενοσίνη ομοιοπολικά δεσμευμένη με το κατάλοιπο ασπαραγινικού οξέος του καρβοξυτελικού άκρου (Metlitskaya et al, 2006). Το πεπτίδιο αυτό αποικοδομείται στο εσωτερικό των κυττάρων και παράγεται έτσι ένα τροποποιημένο ασπαρτυλ-αδενυλικό, το οποίο αναστέλλει τη δράση της αμινοάκυλο-tRNA συνθετάσης τους ασπαραγινικού οξέος (AspRS).…”