MUTATIONAL ANALYSIS OF POSTTRANSLATIONAL HETEROCYCLE BIOSYNTHESIS IN THE GYRASE INHIBITOR MICROCIN B17 - DISTANCE DEPENDENCE FROM PROPEPTIDE AND TOLERANCE FOR SUBSTITUTION IN A GSCG CYCLIZABLE SEQUENCE

Microcin B17 (MccB17) is a peptidyl antibiotic that is secreted in sta tionary phase by several strains of Escherichia coli. The antibiotic e fficacy of this polypeptide depends on the posttranslational modificat ion of eight cysteine and serine residues to thiazoles and oxazoles, r espectively, within the 69 aa McbA structural gene product. Mono-and b isheterocycle formation is mediated by MccB17 synthetase, an enzyme co mplex composed of three proteins: McbB, -C, and -D. After substrate pr ocessing, an N-terminal 26 aa propeptide sequence is cleaved to afford the mature antibiotic, A method for the overexpression and rapid puri fication of microcin synthetase has been developed using a calmodulin- binding peptide tag. The determinants of substrate recognition and syn thetase-mediated heterocycle formation were investigated by a systemat ic evaluation of 15 MCbA(1-46) analogues representing minimal substrat es containing the first bisheterocyclization site (Gly(39)-Ser(40)-Cys (41)-Gly(42)) and variants thereof. Each substrate analogue was overex pressed and affinity-purified as fusions to maltose-binding protein, i ncubated with purified synthetase, and evaluated for processing by Wes tern blots, UV spectroscopy, and mass spectrometry. Insights gained in to the process of enzymatic heterocycle formation from cysteine and se rine residues are discussed, including the distance dependence of the first cyclized residue from the propeptide and the local sequence cont ext at the cyclizable sites. A model for McbA substrate recognition an d processing by MccB17 synthetase is proposed.