Nonribosomal peptide synthetases NRPSs

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... 

Three categories of synthetases are distinguished, based on their substrate specificity and mode of product synthesis. The two known types of polyketide synthetases (PKSs) (Type I and II) utilize acyl-coenzyme A (CoA) monomers while nonribosomal peptide synthetases (NRPSs) use amino acids and their analogs as substrates. Type I PKS and NRPS oligomerize these building blocks by a modular assembly-line arrangement while type II PKS iteratively assembles monomeric units. 

Nonribosomal peptide synthetases (NRPSs) function similarly to the ribosome in a second way They act processively to build a peptide chain. In both cases, the amine group of the incoming aminoacyl monomer attacks the (thio)ester bond that links the nascent peptide to its carrier, which translocates the chain while elongating it by one monomer (22). 

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. 

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.

One of the many unique features found in NRPs is that they are not limited to the 20 standard common amino acids. Rather, NRPs often contain unusual structural elements including D-amino acids, peptide-derived heterocycles, and A-methyl groups." Access to these moieties is the consequence of the way in which NRPs are biosynthesized by nonribosomal peptide synthetases (NRPSs). Perhaps not surprisingly, the molecular logic underpinning NRP synthesis is very similar to that in the PKS systems." ... 

Another approach comes from Professor Christopher T. Walsh s laboratory at Harvard Medical School. The Walsh laboratory studies the biosynthesis of natural products (natural products are small molecules created by nature). What does natural product biosynthesis have to do with cellular imaging Like the AGT method where a DNA repair protein is used, it turns out that some of the proteins involved in natural product assembly are useful for labeling the cell surface. Peptide carrier proteins (PCPs) are 80- to 120-amino acid domains of nonribosom peptide synthetases (NRPSs). NRPSs are protein megacomplexes used by many microbial species, like Pseudomonas and Streptomyces, to biosynthesize natural products from common amino acid precursors. An enzyme called a phosphop-antetheinyl transferase will covalently attach the 4 -phosphopantethei-nyl moiety of coenzyme A to a specific serine residue in the PCP domain. One phosphopantetheinyl transferase, Sfp from a microbe... 

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. 

Figure 3.4 Improvement of the activity of chimeric NRPSs using directed evolution. (1) A heterologous A domain is swapped into an NRPS, typically resulting in a significant loss of synthetase activity. (2) A library of chimeric synthetase mutants is constructed in which the heterologous A domain has been diversified (for example, by error-prone PCR). (3) The library is subjected to an in vivo screen for production of the unnatural nonribosomal peptide derivative. (4) Clones showing improved production are characterized and subjected to further rounds of diversification and screening...

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