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

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.

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.

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 amino-terminal 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.