Editing of non-cognate aminoacyl adenylates by peptide synthetases

Non-ribosomally formed peptides display both highly conserved and variable amino acid positions, the variations leading to a wide range of peptide fam ilies. Activation of the amino acid substrate proceeds in analogy to the ri bosomal biosynthetic mechanism generating aminoacyl adenylate and acyl inte rmediates. To approach the mechanism of fidelity of amino acid selection, t he stability of the aminoacyl adenylates was studied by employing a continu ous coupled spectrophotometric assay. The ape-form of tyrocidine synthetase 1 (apo-TY1) was used, generating an L-phenylalanyl-adenylate intermediate stabilized by the interaction of two structural subdomains of the adenylati on domain. Adenylates of substrate analogues have shown variable and reduce d degrees of stability, thus leading to an enhanced generation of pyrophosp hate due to hydrolysis and continuous adenylate formation. Discrimination o f the non- aromatic amino acids L-Leu and L-Met, or L-Phe analogues such as p-amino- and p-chloro-L-Phe derivatives, as well as the stereospecific :se lection of L-Phe, is supported by less-stable adenylate intermediates exhib iting elevated susceptibility to hydrolysis. Breakdown of the L-phenylalany l intermediate utilizing 2'-deoxy-ATP as the nucleotide substrate was signi ficantly enhanced Compared with the natural analogue. Apo-TY1 engineered at positions involved in adenylate formation showed variable protection again st hydrolysis, The results imply that stability of the aminoacyl intermedia tes may act as an essential factor in substrate selection and fidelity of n on-ribosomal-peptide-forming systems.