Biochemical characterization of peptidyl carrier protein (PCP), the thiolation domain of multifunctional peptide synthetases.
BACKGROUND
A 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 His6 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 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.
Stachelhaus
T
T
Biochemie-Fachbereich Chemie, Philipps-University of Marburg, Hans-Meerwein-Str., D-35032 Marburg, Germany. marahiel@ps1515.chemie.uni-marburg.de
Hüser
A
A
Marahiel
M A
MA
D000215:Acylation; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D013438:Sulfhydryl Compounds; D010453:Peptide Synthases; C035180:tyrocidine synthetase