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

Abstract

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).

The Role of the Phosphorus Atom in Drug Design

“…Microcin C( 230)i saribosomally synthesized antibiotic heptapeptide, which is covalently bound via ap hosphoramidate bond to adenosine formed by some strains of E. coli [205] Microcin Ca nd other closely related antibiotics can be considered as Trojan-horse peptide-adenylates targeting aspartyl-tRNAs ynthase. [206] The chemical structure of microcin Cw as established many years ago and is illustratedi nF igure 35.…”

The Novel Property of Heptapeptide of Microcin C7 in Affecting the Cell Growth of Escherichia coli

“…One reason for this could be the high concentration used here, compared to 200 μM in the previous study [ 15 ]. Additionally, the difference in the lethal concentration between MR and f-MR indicates that the N terminal modification has effects on the antibacterial activity, as reported by Olga et al [ 26 , 27 ].…”

Reiterative Synthesis by the Ribosome and Recognition of the N-Terminal Formyl Group by Biosynthetic Machinery Contribute to Evolutionary Conservation of the Length of Antibiotic Microcin C Peptide Precursor

“…In previous work, it was shown that E. coli MccB is able to modify a wide range of substrates in vivo and in vitro , including MBP protein with attached A7 peptide (14). To test MccB activity on elongated substrates encoded by pA11 and pA15, we incubated purified MccB with ATP and chemically synthesized A11 and A15 peptides and measured adenylated peptide yields by high-pressure liquid chromatography (HPLC) (Fig.…”

“…We speculate that this effect may be due to better transport of formylated peptides of appropriate length by the YejABEF transporter, which was previously shown to recognize formylated peptides (15). In the absence of the N-terminal formyl group, increasing the peptide length up to a certain point also increases the bioactivity (14). Interestingly, in the case of the Yersinia pseudotuberculosis mcc operon, which encodes a 42-amino-acid-long MccA precursor, the final peptide-nucleotide product is processed to an 11-amino-acid peptide with attached C-terminal nucleotide, and this processing is required for bioactivity (17).…”