Aminoacyl-CoAs as probes of condensation domain selectivity in nonribosomal peptide synthesis

In nonribosomal biosynthesis of peptide antibiotics by multimodular synthet ases, amino acid monomers are activated by the adenylation domains of the s ynthetase and loaded onto the adjacent carrier protein domains as thioester s, then the formation of peptide bonds and translocation of the growing cha in are effected by the synthetase's condensation domains. Whether the conde nsation domains have any editing function has been unknown. Synthesis of am inoacyl-coenzyme A (CoA) molecules and direct enzymatic transfer of aminoac yl-phosphopantetheine to the carrier domains allow the adenylation domain e diting function to be bypassed. This method was used to demonstrate that th e first condensation domain of tyrocidine synthetase shows low selectivity at the donor residue (D-phenylalanine) and higher selectivity at the accept or residue (L-proline) in the formation of the chain-initiating D-Phe-L-Pro dipeptidyl-enzyme intermediate.