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The aminocoumarin antibiotics clorobiocin and coumermycin A(sub 1) target the B subunit of DNA gyrase by presentation of the 5-methyl-pyrrolyl-2-carboxy ester moiety in the ATP-binding site of the enzyme. The transformation mass spectrometry analysis of the acyl-S-protein substrate/product mixture to establish that a two-electron oxidized pyrrolinyl-S-enzyme transiently accumulates on the way to the four-electron oxidized, heteroaromatic pyrrolyl-2-carboxy-S-PCP acyl enzyme product.

Results demonstrate that functional brochocin A and brochocin B are produced by Carnobacterium piscicola clones, whereas in Escherichia coli the brochocins are fragmented and weakly active or inactive. While brochocin A is expressed as a mature 59-amino acid peptide, brochocin B is produced as a fragment. Both brochocins show a high beta-sheet content in the secondary structure.

The aminocoumarin antibiotics clorobiocin and coumermycin A1 target the B subunit of DNA gyrase by presentation of the 5-methyl-pyrrolyl-2-Carboxy ester moiety in the ATP-binding site of the enzyme. The pyrrolyl pharmacophore is derived by a four electron oxidation of a prolyl unit while tethered in phosphopantetheinyl thioester linkage to a peptidyl carrier protein (PCP) subunit. L-Proline is selected and activated as L-prolyl-AMP by adenylation domain enzymes (CloN4 and CouN4) and then installed as the thioester on the holo form of the PCP proteins CIoN5 and CouN5. Enzymatic oxidation of the prolyl- S-PCP by the flavoprotein dehydrogenase CloN3 can be followed by rapid quench and subsequent electrospray ionization-Fourier transform mass spectrometry analysis of the acyl-S-protein substrate/ product mixture to establish that a two-electron oxidized pyrrolinyl-S-enzyme transiently accumulates on the way to the four-electron oxidized, heteroaromatic pyrrolyl-2-carboxy-S-PCP acyl enzyme product. [ABSTRACT FROM AUTHOR]