KINETICS AND REGIOSELECTIVITY OF PEPTIDE-TO-HETEROCYCLE CONVERSIONS BY MICROCIN B17 SYNTHETASE

Background: The Escherichia coli peptide antibiotic microcin B17 (MccB 17) contains four oxazole and four thiazole rings introduced post-tran slationally in the 69 amino acid McbA gene product, an MccB17 precurso r, by the microcin B,C,D enzyme complex. Both monocyclic and 4,2-bis-h eterocyclic moieties are generated. The enzymatic cyclization involves 14 of the last 43 amino acids of McbA and requires the presence of th e first 26 amino acids that function as a specificity-conferring prope ptide. Results: We have constructed maltose-binding protein (MBP)-McbA (1-46) fusion proteins and have mutagenized the Gly39-Ser40-Cys41 (GSC ) wildtype sequence to assess the regioselectivity and chemoselectivit y of MccB17-synthetase-mediated heterocycle formation at the first two loci, residues 40 and 41 of McbA, Four single-site and four double-si te substrates showed substantial differences in turnover as assessed b y western assays, UV-visible spectroscopy and mass spectrometry. Cyste ine-derived thiazoles form at a greater rate than serine-derived oxazo les. Formation of bis-heterocycles is sensitive both to composition an d sequence context. Conclusions: The E. coli McbB,C,D MccB17 synthetas e is the first peptide heterocyclization enzyme to be characterized. T his study reveals substantial regioselectivity and chemoselectivity (t hiazole > oxazole) at the most aminoterminal bis-heterocyclization sit e of McbA, The heterocyclization of GSS and GCC mutants of McbA(1-46) by MccB17 synthetase demonstrates that the complex can efficiently gen erate tandem bis-oxazoles and bis-thiazoles, moieties not found in Mcc B17 but present in natural products such as hennoxazole and bleomycin. The observations suggest a common enzymatic mechanism for the formati on of peptide-derived heterocyclic natural products.