POSTTRANSLATIONAL HETEROCYCLIC BACKBONE MODIFICATIONS IN THE 43-PEPTIDE ANTIBIOTIC MICROCIN B17 - STRUCTURE ELUCIDATION AND NMR-STUDY OF A C-13,N-15-LABELED GYRASE INHIBITOR

Microcin B17 (McB17), the first known gyrase inhibitor of peptidic nat ure, is produced by ribosomal synthesis and post-translational modific ation of the 69-residue precursor protein by an Escherichia coli strai n. To elucidate the chemical structure of the mature 43-residue peptid e antibiotic, fermentation and purification protocols were established and optimized which allowed the isolation and purification of substan tial amounts of highly pure McB17 (non-labelled, N-15-labelled and C-1 3/N-15-labelled peptide. By ultraviolet-absorption spectroscopy, HPLC- electrospray mass spectrometry and GC-mass spectrometry, amino acid an alysis, protein sequencing, and, in particular, multidimensional NMR, we could demonstrate and unequivocally prove that the enzymic modifica tion of the precursor backbone at Gly-Cys and Gly-Ser segments leads t o the formation of 2-aminomethylthiazole-4-carboxylic acid and 2-amino methyl-oxazolyl-4-carboxylic acid, respectively. In addition, two bicy clic modifications 2-(2-aminomethyl-oxazolyl)thiazole-4-carboxylic aci d and 2-(2-aminomethylthiazolyl)oxazole-4-carboxylic acid were found t hat consist of directly linked thiazole and oxazole rings derived from one Gly-Ser-Cys and one Gly-Cys-Ser segment. Analogous to the thiazol e and oxazole rings found in antitumor peptides of microbial and marin e origin, these heteroaromatic ring systems of McB17 presumably play a n important role in its gyrase-inhibiting activity. e.g, interacting w ith the DNA to trap the covalent protein-DNA intermediate of the break age-reunion reaction of the gyrase.