title: Enzymatic Synthesis and Functional Characterization of Bioactive Microcin C-Like Compounds with Altered Peptide Sequence and Length.
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 extended-peptide-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.
  
  
  OBJECTIVE
  Enzymatic adenylation of chemically synthesized peptides allowed us to generate biologically active derivatives of the peptide-nucleotide antibiotic microcin C with improved bioactivity and altered entry routes into target cells, opening the way for development of various McC-based antibacterial compounds not found in nature.
authors.affiliation: Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia Peter the Great Saint Petersburg Polytechnical University, Saint Petersburg, Russia.
authors.forename: Olga
authors.initials: O
authors.lastname: Bantysh
authors.affiliation: Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia Moscow State University, Moscow, Russia.
authors.forename: Marina
authors.initials: M
authors.lastname: Serebryakova
authors.affiliation: Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia Peter the Great Saint Petersburg Polytechnical University, Saint Petersburg, Russia Waksman Institute, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey, USA.
authors.forename: Inna
authors.initials: I
authors.lastname: Zukher
authors.affiliation: Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia.
authors.forename: Alexey
authors.initials: A
authors.lastname: Kulikovsky
authors.affiliation: Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia.
authors.forename: Darya
authors.initials: D
authors.lastname: Tsibulskaya
authors.affiliation: Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia Skolkovo Institute of Science and Technology, Skolkovo, Russia.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Dubiley
authors.affiliation: Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia Peter the Great Saint Petersburg Polytechnical University, Saint Petersburg, Russia Waksman Institute, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey, USA Skolkovo Institute of Science and Technology, Skolkovo, Russia severik@waksman.rutgers.edu.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
mesh_terms: D000595:Amino Acid Sequence; D001430:Bacteriocins / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry; D008025:Ligases / Q000737:chemistry; D015394:Molecular Structure; D009154:Mutation
chemical_list: D001430:Bacteriocins; D029968:Escherichia coli Proteins; C020411:microcin; D008025:Ligases; C542496:MccB protein, E coli