| The continuing need for new antibiotics demands a better understanding of the molecular mechanisms by which peptide antibiotics are biosynthesized. High resolution mass spectrometry now plays a greatly expanded role in the elucidation of mechanistic steps in both the nonribosomal and ribosomal biosynthetic pathways toward peptide antibiotics.;For determination of multiple covalent intermediates bound to the large enzymes responsible for biosynthesis via nonribosomal peptide synthesis, mass spectrometry is a promising method to provide new mechanistic insight. The application of Quadrupole-Fourier-Transform mass spectrometry (Q-FTMS) for direct analysis of aminoacyl intermediates is demonstrated for the first two modules (127 and 120 kDa) involved in the nonribosomal synthesis of gramicidin S. Cyanogen bromide digestion of recombinant proteins afforded detection of two active site peptides (both ∼13 kDa) that provided direct evidence for adenylation domains co-purifying with their preferred amino acid substrates. Given the ability to detect multiple covalent intermediates in tandem, a competition experiment among several non-natural substrates in parallel was performed using the first module. This defined mixture of acyl-enzyme intermediates was used to probe the selectivity of the condensation step producing a diversity of non-cognate dipeptides on the second module.;Q-FTMS also played a role in the elucidation of the specificity of the modifying enzyme, LctM, in the biosynthesis of the lantibiotic lacticin 481. MS/MS allowed for the localization of post-translationally introduced dehydrations (-18 Da each) as well as the ability to determine if the lanthionine rings were present. After establishing that the modifications had taken place on the wild-type substrate, the ability to modify mutant peptides was explored, thereby gaining insight into the specificity of the enzyme. Also the conversion of starting material to product was investigated by the alkylation of intermediates. Initial studies indicated that all dehydration events may occur before cyclization.;The following study describes the application of Q-FTMS to obtain mechanistic insight into the biosynthesis of peptide antibiotics produced either nonribosomally or by extensive post-translational modification of a ribosomally-encoded precursor. |
