Nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) generate a vast number of clinically relevant natural products. These modular systems are amenable to genetic engineering to generate novel metabolites. A cyclization (Cy) domain mediates amide bond formation and cyclodehydration of an amino acid containing a side chain with a beta-heteroatom to introduce a thiazoline or oxazoline ring in the structure. The rates of peptide bond formation and subsequent cyclodehydration will be determined for the natural substrates of four Cy domains from the NRPS subunits EpoB, PchE, PchF, and VibF. This will enable a comparison of cyclization reactions for L- and D- amino acid side chains, neighboring heterocycles, is well as a comparison of thiazoline and oxazoline ring formation. The ability of these Cy domains to accept alternate substrates will also be evaluated, identifying Cy domains as potential tools for combinatorial biosynthesis. Finally, conserved amino acids of Cy domains will be mutated, and the effects of those mutations analyzed for effects on the two reactions of amide bond formation and cyclodehydration.
