Peptide synthetases and acyl-CoA-synthetases form acyl adenylates which are transferred to CoA or enzyme-bound pantetheine. To verify the existence of an adenylate domain in peptide synthetases, a 60.8 kDa fragment of tyrocidine 1-synthetase was constructed by a 1629 bp deletion, expressed in Escherichia coli, and characterized. The truncated multienzyme activated phenylalanine and substrate analogues with comparable kinetics as the over-expressed synthetase, as judged by ATP-[32p]pp~ exchange reaction. Thus the N-terminal domain resembling an acyl-CoA-synthetase is an autonomous structural element. This N-terminal domain is followed by a cofactor binding domain, resembling acyl carrier proteins involved in polyketide formation.Key words." Tyrocidine synthetase; Multienzyme; Peptide synthetase; Acyl-CoA-synthetase; Pantetheine; CoA structed of modules, each contributing one amino-or hydroxy acid to the peptide structure. Each module contains an adenylate-forming domain, a cofactor binding site, and a condensation/modification domain. The adenylate forming domains display significant similarity to acyl-CoA-synthetases and related proteins. We have predicted, by alignment of sequences of these three classes and those of acyl carrier proteins also binding 4'-phosphopantetheine, the boundaries of adenylate and cofactor domains. The linker region is not rich in Ala, Pro and Glu residues, as in fatty acid synthases and 2-oxo-acid dehydrogenases containing similar cofactor domains. Still, amino acids frequently found in linker regions are dominating. Upon deletion of the cofactor and following functional domains of tyrocidine synthetase 1, a stable N-terminal fragment was obtained which catalysed adenylate formation with similar kinetic properties as the apo-form of the peptide synthetase [8].