E. coli microcin B17 (MccB17) is a posttranslationally modified peptide antibiotic that inhibits bacterial DNA gyrase. It contains four oxazole and four thiazole rings and is representative of a broad class of pharmaceutically important natural products with five membered heterocycles. Only for the MccB17 system have the oxazole and thiazole biosynthetic enzymes been identified, thus making this system amenable to characterization of the mechanism of heterocycle formation. Production and activity of MccB 17 require the presence of seven plasmid encoded genes, mcbABCDEFG, in addition to several chromosomally encoded genes. Biosynthesis of MccB17 requires the products of four genes, mcbABCD. mcbA codes for an inactive 69 amino acid precursor molecule, preMccB 17. Through the action of McbB, McbC, and McbD, preMccB17 is modified posttranslationally to produce proMccB17. This processing converts four cysteine and four serine residues to four thiazole and four oxazole heterocycles, respectively. Removal of a 26 amino acid leader peptide by a chromosomally encoded protease results in the conversion of proMccB17 to the active antibiotic, MccB17. The remaining three genes in the operon, mcbEFG are responsible for MccB 17 export and immunity. We have recently reported the purification of active E. coli microcin synthetase, using as a primary assay a polyclonal antibody that recognizes the oxazole and/or thiazole rings in modified microcin to establish that McbB, C, and D were constituents of the synthetase complex and to define conditions and cofactors for the cyclization, dehydration and desaturation that underlie each enzymatic posttranslational conversion of gly-ser and gly-cys dipeptide moieties into amino-methyloxazoles and thiazoles in the antibiotic. Each completed modification results in a loss of 20.03 Da, allowing differentiation of biosynthetic intermediates by mass spectrometry (MS). Current projects ongoing in the lab are aimed at elucidating the mechanism of oxazole and thiazole formation and establishing the role of each enzyme and the leader peptide in these unique reactions. These studies rely heavily on MS for the characterization of biosynthetic intermediates andfor the microcharacterization of the enzymes involved. Using the complementary instruments available at the BUSM facility for the analysis of selected samples, the questions about microcin B17processing outlined below will be addressed. The arrival of an FT-ICR MS at the BUSM Resource will markedly expand the capabilities of the lab, particularly for MS/MS of >5 kDa ions. 1. ATP Stoichiom,~ta. Among the requirements that we first noted for microcin synthetase activity was an absolute dependence ox ATP. Our immediate goal is to determine the stoichiometry of ATP hydrolysis to heterocycle formation. For these experiments, aliquots of a reaction following the modification of a 46 amino acid substrate will be taken, the substrate and products will be purified out of the reaction mix by HPLC, and the ratio of starting material (M) to the various products (M-20 and M-40) will be assessed by ESIMS. Comparison of this data to theamount of ATP hydrolyzed in a reaction run in parallel will provide a ratio of ATP hydrolysis to the number of heterocyles formed. 2. Overall Mechanism of Substrate Processing. For an enzyme performing multiple, similar modifications, a central question is how many modifications are completed for a single binding event? In a distributive mechanism, the answer is one, for a processive mechanism, the answer would be between two and eight for microcin. Initial ESI/FT/FTMS data acquired at Cornell University favors a distributive mechanism, although further experiments are required. . Directionali1y of Processing. Another important question that can be addressed by MS concerns the directionality, or regioselectivity, of processing. In other words, does microcin synthetase modify its substrate in a N-~C, C--+N, or in a random fashion. Tandem MS will play a key role in localization of cyclization sites in the biosynthetic intermediates.
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