Polypeptide biosynthesis from thioesters of amino acids

Abstract

Each of the two enzyme fractions for gramicidin S (GS) biosynthesis and the three for tyrocidine (Ty) biosynthesis form complexes with the corresponding substrate amino acids. These complexes, isolated by Sephadex G-50 gel filtration, contain equivalent amounts of aminoacyl adenylate and amino acid bound as thioester. As shown previously by denaturation through trichloroacetic acid (TCA) precipitation, (NH 4) 2SO 4 precipitation of these enzyme-amino acid complexes also discharges the noncovalently bound aminoacyl adenylate, retaining only the amino acids covalently bound as thioester, but without loss of activity. Combination of the thus prepared GS light enzyme charged with phenylalanine thioester and GS heavy enzyme charged with proline, valine, ornithine, and leucine thioester results in GS biosynthesis in the absence of aminoacyl adenylate. Moreover, the d-phenylalanine thioester of N-acetylcysteine or of thiophenol replaces the natural donor of activated amino acid, aminoacyl-AMP, produced by the reaction of ATP and amino acid, further substantiating the key role of thioester intermediates in antibiotic polypeptide biosynthesis.