PCP, 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... 

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

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.

Figure 3.112 Epothilone biosynthetic gene cluster from S. cellulosunu Modular organization of the epottiilone polyketide synthase (PKS) and model for epothilone formation. Abbreviations KS, p-ketoacyl ACP syntfiase KSy, p-ketoacyl ACP synthase containing a tyrosine substitiiion of the active-site cysteine AT, acylti-ansferase DH, dehydratase ER, enoylreductase KR, ketoreductase MT methyltcansferase ACP, acyl carrier protein TE, Ihioesterase C, condensation A, adenylation PCP, peptidyl carrier protein.

Figure 5.24 Schematic representation of the genetic organization of the 40-kb nostopeptohde A biosynthetic gene cluster from Nostoc sp. GSV224. A, adenylation (the predicted activated amino acids are reported as a subscript) C, condensation ACP, acyl carrier protein AT, acyl-transferase KS, p-ketoacyl-ACP synthase PCP, peptidyl carrier protein and TE, thioesterase.

PDB Protein Data Bank, PCP peptidyl carrier protein... 

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... 

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... 

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.

Then, the amino acid precursor 105 is selected by the adenylation domain A and attached to the peptidyl carrier protein PCP of the SalB didomain. PCP-bound 105 is subsequently oxidized by the cytochrome P450 hydroxylase SalD. Fusion of the PKS-and the NRPS-derived precursors by the C-terminal condensation domain of SalA, leads to a PCP-bound linear intermediate, which after bicyclization and concurrent release from the synthetase yields the fully assembled salinosporamide molecules by an unknown process. The observed structural diversity of the salinosporamide family is essentially due to the relaxed substrate specificity of the AT domains of SalA. Although selection of an acetate starter unit by ATL leads to the typical methyl substituent at C-3 of the salinosporamides, an alternative priming with propionate would produce the C-3-ethyl derivative salinosporamide 194. The promiscuity of ATj in turn facilitates the formation of the observed variability in the substitution pattern at C-2. For the assembly of 86, ATj incorporates the halogenated PKS extender unit chloroethylmalonyl-CoA, which is unique to the salinosporamide family. 

It is noteworthy that nonribosomal peptide synthetase is similarly posttrans-lationally modified by covalent attachment of the 4 -phosphopantetheine group to the peptidyl carrier protein (PCP) [193-198]. While the ACPS can modify various apo-ACPs [167,173,187-189,191,192],it failed to modify PCPs from a variety of peptide synthetases [189]. This led to the discovery of the second family of PPTases [189], such as EntD from E. coli [189, 199, 200], Sfp from Bacillus subtilis [189,200-204], PptT from M. tuberculosis [264], and Gsp from B. brevis [ 189,205,206], required for the biosynthesis of enterobactin, surfactin, mycobactin, and gramicidin S, respectively. In contrast to ACPS, proteins in the latter family, such as Sfp, showed broader substrate specificity, modifying apo-PCPs, apo-ACPs, as well as apo-aryl carrier proteins and utilizing both CoA, acyl CoAs, and CoA analogs [204]. 

A minimal NRPS module consists of an adenylation domain (A), condensation domain (C) and a peptidyl-carrier protein (PCP). In the first instance, the substrate-specific adenylation domain activates the carboxyl region of the amino acid with ATP, forming the mixed acyl-phosphoric acid anhydride with AMP, followed by loading onto the phosphopanthetheine moiety of the PCP. The condensation domain subsequently catalyses the nucleophific attack of the amino group of the previously activated amino acid, to the carbonyl of the tethered acyl group from the previous module [38, 39]. This results in the formation of a new peptide bond between the two units (Fig. 1.15). 

NRPS Metabolites and Peptides NRPS enzymes assemble amino acids, including nonproteinogenic ones, into oligopeptides. The enzymes contain several modnles, and especially an adenylation domain (A), which specifically selects and activates the amino acid to be transferred as a thioester on the nearby peptidyl carrier protein (PCP) [2]. A condensation module (C) then catalyzes the formation of the peptide bonds between the newly introduced amino acyl-PCP (bearing a free amine) and the elongated peptidyl-PCP thioester. At the end of the elongation, a cyclization can occur into cyclopeptides, but the peptide can also be... 

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