BIOCHEMICAL-CHARACTERIZATION OF PEPTIDES CARRIER PROTEIN (PCP), THE THIOLATION DOMAIN OF MULTIFUNCTIONAL PEPTIDE SYNTHETASES

Background: A structurally diverse group of bioactive peptides is synt hesized by peptide synthetases which act as templates for a growing pe ptide chain, attached to the enzyme via a thioester bond. The protein templates are composed of distinctive substrate-activating modules, wh ose order dictates the primary structure of the corresponding peptide product. Each module contains defined domains that catalyze adenylatio n, thioester and peptide bond formation, as well as substrate modifica tions. To show that a putative thiolation domain (PCP) is involved in covalent binding and transfer of amino acyl residues during non-riboso mal 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. T he protein was expressed as a His(6) fusion protein, and purified in a single step by affinity chromatography. Incorporation of beta-[H-3]al anine, a precursor of coenzyme A, demonstrated the modification of PCP with the cofactor 4'-phosphopantetheine. When an adenylation domain i s present to supply the amino adenylate moiety, PCP can be acylated in vitro. Conclusions: PCP can bind covalently to the cofactor phosphopa ntetheine and can subsequently be acylated, strongly supporting the mu ltiple carrier model of non-ribosomal peptide synthesis. The adenylati on 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 co mplexes.