T. Stachelhaus et al., BIOCHEMICAL-CHARACTERIZATION OF PEPTIDES CARRIER PROTEIN (PCP), THE THIOLATION DOMAIN OF MULTIFUNCTIONAL PEPTIDE SYNTHETASES, Chemistry & biology, 3(11), 1996, pp. 913-921
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.