Peptide synthetases are large multienzyme complexes that catalyze the non-ribosomal synthesis of a structurally diverse family of bioactive peptides. They possess a multidomain structure and employ the thiotemplate mechanism to activate, modify and link together by amide or ester bonds the constituent amino acids of the peptide product. The domains, which represent the functional building units of peptide synthetases, appear to act as independent enzymes whose specific linkage order forms the protein-template that defines the sequence of the incorporated amino acids.
Two types of domains have been characterized in custom peptide synthesis of bacterial and fungal origin: type I comprises about 600 amino acids and contains at least two modules involved in substrate recognition, adenylation and thioester formation, whereas type II domains carry in addition an insertion of about 430 amino acids that may function as a N-methyltransferase module. The role of other genes associated with bacterial operons encoding peptide synthetases is also discussed.
structurally diverse group of bioactive peptides is synthesized by peptide synthetases which act as templates for a growing peptide chain, attached to the enzyme via a thioester bond. The protein templates are composed of distinctive substrate-activating modules, whose order dictates the primary structure of the corresponding peptide product. Each module contains defined domains that catalyze adenylation, thioester and peptide bond formation, as well as substrate modifications. To show that a putative thiolation domain (PCP) is involved in covalent binding and transfer of amino acyl residues during non-ribosomal peptide synthesis, we have cloned and biochemically characterized that region of tyrocidine synthetase 1, TycA. Results: The 327-bp gene fragment encoding PCP was cloned using its homology to the genes for the acyl carrier proteins of fatty acid and polyketide biosynthesis. The protein was expressed as a His-6 fusion protein, and purified in a single step by affinity chromatography. Incorporation of beta-(3H)alanine, a precursor of coenzyme A, demonstrated the modification of PCP with the cofactor 4'-phosphopantetheine. When an adenylation domain is present to supply the amino adenylate moiety, PCP can be acylated in vitro. Conclusions: PCP can bind covalently to the cofactor phosphopantetheine and can subsequently be acylated, strongly supporting the multiple carrier model of non-ribosomal custom peptide synthesis. The adenylation and thiolation domains can each act as independent multifunctional enzymes, further confirming the modular structure of peptide synthases, and can also perform sequential steps in trans, as do multienzyme complexes.