Peptidyl carrier protein

Figure 11.5 Amino acid building blocks are incorporated into daptomycin backbone successively by NRPS subunits DptA, DptBC and DptD (a). Structural diversity of daptomycin peptide core can be obtained by genetic modifications of dpt gene cluster (b). C, condensation domain A, adenylation domain PCP, peptidyl carrier protein E, epimerase TE, thioesterase domain... 

Phosphopantetheine tethering is a posttranslational modification that takes place on the active site serine of carrier proteins - acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs), also termed thiolation (T) domains - during the biosynthesis of fatty acids (FAs) (use ACPs) (Scheme 23), polyketides (PKs) (use ACPs) (Scheme 24), and nonribosomal peptides (NRPs) (use T domain) (Scheme 25). It is only after the covalent attachment of the 20-A Ppant arm, required for facile transfer of the various building block constituents of the molecules to be formed, that the carrier proteins can interact with the other components of the different multi-modular assembly lines (fatty acid synthases (FASs), polyketide synthases (PKSs), and nonribosomal peptide synthetases (NRPSs)) on which the compounds of interest are assembled. The structural organizations of FASs, PKSs, and NRPSs are analogous and can be divided into three broad classes the types I, II, and III systems. Even though the role of the carrier proteins is the same in all systems, their mode of action differs from one system to another. In the type I systems the carrier proteins usually only interact in cis with domains to which they are physically attached, with the exception of the PPTases and external type II thioesterase (TEII) domains that act in trans. In the type II systems the carrier proteins selectively interact... 

Structure and Function of Peptidyl Carrier Protein Domains Structure and Function of Adenylation Domains Structure and Function of Condensation Domains Structure and Function of Thioesterase Domains Multidomain NRPS Structural Information PCP-C didomain structure PCP-TE didomain structure Structure of a C-A-PCP-TE termination module Pathways to Nonproteinogenic Amino Acids Incorporated into NRP Natural Nonproteinogenic Amino Acids Present as Cellular Metabolites Modification of Proteinogenic Amino Acids Nonproteinogenic Amino Acids Derived from Multistep Pathways Tailoring Enzymology in NRP Natural Products Chemical Approaches Toward Mechanistic Probes and Inhibitors of NRPS... 

Structure and Function of Peptidyl Carrier Protein Domains... 

A typical module consists of an adenylation (A) domain, a peptidyl carrier protein (PCP) domain, and a condensation (C) or elongation domain. The A domain activates a specific amino acid as an... 

A adenylation domain PCP peptidyl carrier protein domain C condensation domain E epimerization domain (Val module only) TE thioesterase domain... 

Each synthetase module contains three active site domains The A domain catalyzes activation of the amino acid (or hydroxyacid) by formation of an aminoacyl- or hydroxyacyl-adenylate, just as occurs with aminoacyl-tRNA synthetases. However, in three-dimensional structure the A domains do not resemble either of the classes of aminoacyl-tRNA synthetases but are similar to luciferyl adenylate (Eq. 23-46) and acyl-CoA synthetases.11 The T-domain or peptidyl carrier protein domain resembles the acyl carrier domains of fatty acid and polyketide synthetases in containing bound phos-phopantetheine (Fig. 14-1). Its -SH group, like the CCA-terminal ribosyl -OH group of a tRNA, displaces AMP, transferring the activated amino acid or hydroxy acid to the thiol sulfur of phosphopan-tetheine. The C-domain catalyzes condensation (peptidyl transfer). The first or initiation module lacks a C-domain, and the final termination module contains an extra termination domain. The process parallels that outlined in Fig. 21-11.1... 

LE Quadri, PH Weinreb, M Lei, MM Nakano, P Zuber, CT Walsh. Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases. Biochemistry 37 1585-1595, 1998. 

Figure 10.2 The PKS/NRPS biosynthetic paradigm, showing the most common domains and their relative positions within a modular PKS/NRPS enzyme. A = adenylation AT = acyl transferase C = condensation DH = dehydratase Ep = epimerase ER = enoyl reductase KR = ketoreductase KS = ketosynthase MT = methyltransferase PCP = peptidyl carrier protein TE = thioesterase.

Sieber SA, Walsh CT, Marahiel MA. Loading peptidyl-coenzyme A onto peptidyl carrier proteins a novel approach in characterizing macrocychzation by thioesterase domains. J. Am. Chem. Soc. 2003 125 10862-10866. 

Many natural peptides are synthesized by a sequence of enzyme-controlled processes carried out by a multifunctional enzyme of modular arrangement, similar to some polyketide synthases. These nonribosomal peptide synthetases (NRPSs) typically consist of an adenylation domain, a peptidyl carrier protein domain, and a condensation or elongation domain in order to carry out amide bond formation and some derivations of amino acid residues. 

Minimally, each module requires three domains - an adenylation domain (A), which activates a specific amino acid for incorporation a thiolation domain (T), also known as a peptidyl carrier protein (PCP), which transfers the growing peptide from one module to another, and the condensation (C) domain, which carries out the condensation of two residues. Analysis and comparison of the primary sequences of numerous adenylation domains has led to the establishment of a code that can be used to predict the amino acid activated by a particular adenylation domain. Genetic analysis of these sequences has shown that they clade together based on the amino acid encoded, rather than by species of origin. ... 

As with type I PKSs, NRPSs are built of repetitive catalytic units (modules), which are each responsible for the incorporation of one amino acid into the growing peptide chain. Although different chemistries are employed for activation and condensation of the substrates, the basic steps of NRPS chain elongation show striking similarities to the type I PKS mechanisms (1) recognition of the amino acid substrate and its activation as an aminoacyl adenylate (2) covalent binding of the residue as a thioester to a carrier protein and (3) condensation with the peptidyl residue attached to the upstream module. Consequently, a typical NRPS elongation module minimally comprises an adenylation (A) domain responsible for amino acid activation, a thiolation (T) domain (also known as a peptidyl-carrier protein (PCP)) to which the activated amino acid is covalently attached, and a condensation (C) domain, which catalyzes peptide bond formation. As in PKSs, a variety of optional domains, for example, MTs or epimerization (E) domains, further increase the structural... 

Figure 2 Enzymatic logic of nonribosomal peptide synthetases (NRPSs) and catalytic role of adenylation (A) domains. The A domain selects substrates from the cellular pool and tethers them as thioesters to peptidyl carrier protein (PCP) domains. In a typical NRPS, the A and PCP domains are always present. The condensation (C) domain is present in all chain extension modules. The epimerization (E) and the methyltransferase (MT) domains are optional. A final thioesterase (TE) domain generally catalyzes the release of the peptide from the NRPS.

Biochemical analyses of the assembly of the ergopeptines in C. purpurea have shown that ergopeptines are the products of an enzyme complex consisting of two nonribosomal peptide synthetase (NRPS) subunits (55). NRPSs generally exhibit modular structures, with each module responsible for the addition of an amino acid or other substituent. A typical module includes an adenylation (A-) domain, a thiolation (T-) domain (also known as a peptidyl carrier protein domain), and a condensation (C-) domain. The A-domain specifies the amino acid or other carboxylic acid substituent, and activates by it by an ATP-dependent adenylation reaction. The activated substituent then forms a thioester with the 4 -phosphopan-tetheine prosthetic group in the adjacent T-domain. Finally, the C-domain links the substituent to the next substituent in the chain. In a multimodular NRPS protein, the order in which substituents are added corresponds to the arrangement of modules from its N- to C terminus. 

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