ATP GTP HYDROLYSIS IS REQUIRED FOR OXAZOLE AND THIAZOLE BIOSYNTHESIS IN THE PEPTIDE ANTIBIOTIC MICROCIN B17/

In the maturation of the Escherichia coil antibiotic Microcin B17, the product of the mcbA gene is modified posttranslationally by the multi meric Microcin synthetase complex (composed of McbB, C, and D) to cycl ize four Cys and four Ser residues to four thiazoles and four oxazoles , respectively. The purified synthetase shows an absolute requirement for ATP or GTP in peptide substrate heterocyclization, with GTP one-th ird as effective as ATP in initial rate studies. The ATPase/GTPase act ivity of the synthetase complex is conditional in that ADP or GDP form ation requires the presence of substrate; noncyclizable versions of Mc bA bind to synthetase, but do not induce the NTPase activity. The stoi chiometry of ATP hydrolysis and heterocycle formation is 5:1 for a sub strate that contains two potential sites of modification. However, at high substrate concentrations (>50K(m)) heterocycle formation is inhib ited, while ATPase activity occurs undiminished, consistent with uncou pling of NTP hydrolysis and heterocycle formation at high substrate co ncentrations. Sequence homology reveals that the McbD subunit has moti fs reminiscent of the Walker B box in ATP utilizing enzymes and of mot ifs found in small G protein GTPases. Mutagenesis of three aspartates to alanine in these motifs (D132, D147, and D199) reduced Microcin B17 production in vivo and heterocycle formation in vitro, suggesting tha t the 45 kDa McbD has a regulated ATPase/GTPase domain in its N-termin al region necessary for peptide heterocyclization.