Kinetics and regioselectivity of peptide-to-heterocycle conversions by microcin B17 synthetase

Peter J. Belshaw, Ranabir Sinha Roy, Neil L. Kelleher, Christopher T. Walsh*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

41 Scopus citations

Abstract

Background: The Escherichia coli peptide antibiotic microcin B17 (MccB17) contains four oxazole and four thiazole rings introduced post-translationally in the 69 amino acid McbA gene product, an MccB17 precursor, by the microcin B,C,D enzyme complex. Both monocyclic and 4,2-bis-heterocyclic moieties are generated. The enzymatic cyclization involves 14 of the last 43 amino acids of McbA and requires the presence of the first 26 amino acids that function as a specificity-conferring propeptide. Results: We have constructed maltose-binding protein (MBP)-McbA1-46 fusion proteins and have mutagenized the Gly39-Ser40-Cys41 (GSC) wild-type sequence to assess the regioselectivity and chemoselectivity of MccB17-synthetase-mediated heterocycle formation at the first two loci, residues 40 and 41 of McbA. Four single-site and four double-site substrates showed substantial differences in turnover as assessed by western assays, UV-visible spectroscopy and mass spectrometry. Cysteine-derived thiazoles form at a greater rate than serine-derived oxazoles. Formation of bis-heterocycles is sensitive both to composition and sequence context. Conclusions: The E. coli McbB,C,D MccB17 synthetase is the first peptide heterocyclization enzyme to be characterized. This study reveals substantial regioselectivity and chemoselectivity (thiazole > oxazole) at the most aminoterminal bis-heterocyclization site of McbA. The heterocyclization of GSS and GCC mutants of McbA1-46 by MccB17 synthetase demonstrates that the complex can efficiently generate tandem bis-oxazoles and bis-thiazoles, moieties not found in MccB17 but present in natural products such as hennoxazole and bleomycin. The observations suggest a common enzymatic mechanism for the formation of peptide-derived heterocyclic natural products.

Original languageEnglish (US)
Pages (from-to)373-384
Number of pages12
JournalChemistry and Biology
Volume5
Issue number7
DOIs
StatePublished - Jul 1998

Keywords

  • Antibiotics
  • Microcin B17
  • Oxazole
  • Post-translational modification
  • Thiazole

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

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