Enzymatic synthesis and functional characterization of bioactive microcin C-Like compounds with altered peptide sequence and length

Olga Bantysh, Marina Serebryakova, Inna Zukher, Alexey Kulikovsky, Darya Tsibulskaya, Svetlana Dubiley, Konstantin Severinov

    Research output: Contribution to journalArticlepeer-review

    7 Citations (Scopus)

    Abstract

    Escherichia coli microcin C (McC) consists of a ribosomally synthesized heptapeptide attached to a modified adenosine. McC is actively taken up by sensitive Escherichia coli strains through the YejABEF transporter. Inside the cell, McC is processed by aminopeptidases, which release nonhydrolyzable aminoacyl adenylate, an inhibitor of aspartyl-tRNA synthetase. McC is synthesized by the MccB enzyme, which terminally adenylates the MccA heptapeptide precursor MRTGNAN. Earlier, McC analogs with shortened peptide lengths were prepared by total chemical synthesis and were shown to have strongly reduced biological activity due to decreased uptake. Variants with longer peptides were difficult to synthesize, however. Here, we used recombinant MccB to prepare and characterize McC-like molecules with altered peptide moieties, including extended peptide lengths. We find that N-terminal extensions of E. coli MccA heptapeptide do not affect MccB-catalyzed adenylation and that some extendedpeptide- length McC analogs show improved biological activity. When the peptide length reaches 20 amino acids, both YejABEF and SbmA can perform facilitated transport of toxic peptide adenylates inside the cell. A C-terminal fusion of the carrier maltose- binding protein (MBP) with the MccA peptide is also recognized by MccB in vivo and in vitro, allowing highly specific adenylation and/or radioactive labeling of cellular proteins.

    Original languageEnglish
    Pages (from-to)3133-3141
    Number of pages9
    JournalJournal of Bacteriology
    Volume197
    Issue number19
    DOIs
    Publication statusPublished - 2015

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