Nonribosomal peptide synthetase enzymes

It is likely that the biosynthesis of 113 is directed by a hybrid polyketide syn-thase/nonribosomal peptide synthetase enzyme system, as indicated in Figure 11.19. 

It is likely that the madurastatins are biosynthesized on a nonribosomal peptide synthetase, from salicylic acid as the starter acid. L-Serine is probably the precursor to the aziridine moiety, with epimerization occurring on the enzyme-bound amino acid as found for other nonribosomal peptides, with aziridine formation occurring at a late stage. Compounds 120 and 123 could therefore be biosynthetic precursors to 119 and 122, respectively. 

Several of these genes vere deduced to program for polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymes as well as novel tailoring enzymes, including those that create the various unusual functional groups in jamaicamide A. 

Both FASs and PKSs are structurally and mechanistically related to another class of multifunctional enzymes called nonribosomal peptide synthetases. These enzymes activate amino acids as aminoacyl thioesters, which subsequently undergo condensation via formation of amide bonds, leading to biosynthesis of peptide natural products. Enzyme-bound phosphopantetheinyl groups also play a central role in the peptide assembly process. For comparison, the genetics and biochemistry of peptide synthetases are also briefly reviewed here. 

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. 

Provisional understanding of the catalytic mechanism can be deduced by comparison with other ATP-binding enzymes from both prokaryotes and eukaryotes, such as peptide synthetases, luciferases, and acetyl-CoA and acyl-CoA synthetases. These enzymes either form an adenylate intermediate, which is further esterified with CoA as for 4CLs (e.g., acetyl-CoA synthetases (Acs), and acyl-CoA synthetases), or are oxidized by molecular oxygen (luciferases), or are enzyme-bound CoA derivatives, such as 4 -phosphopantheteine (nonribosomal peptide synthetases). 

PN is synthesized by three enzymes encoded by a cluster of three genes (Fig. 10.IB). The arrangement of PN genes in a cluster was first demonstrated in A. nidulans The first enzyme is a nonribosomal peptide synthetase, ACV synthetase (ACVS), catalyzing the condensation of L-a-aminoadipic acid (L-a-AAA), L-cysteine, and L-valine into a tripeptide, 8-(L-a-aminoadipyl)-L-cysteinyl-D-valine (ACV) (Fig. 10.4). In A. nidulans, ACV synthetase is encoded by the intronless. 

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. 

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... 

Additionally, a vast array of natural lipopeptides with remarkable structural diversity is produced by microorganisms living in different habitats, from aquatic to terrestrial environments. These products are not gene encoded, but are synthesized nonribosomally by large multifunctional enzymes, the peptide synthetases. 6"8 ... 

The catalytic mechanisms and molecular recognition properties of peptide synthetases have been studied for several decades [169]. Nonribosomal peptides are assembled on a polyenzyme-protein template, first postulated by Lipmann [170]. The polyenzyme model was refined into the thiotemplate mechanism (Fig. 11) in which the amino acid substrates are covalently bound via thioester linkages to active site sulfhydryls of the enzyme and condensed via a processive mechanism involving a 4 -phosphopantetheine carrier [171-173].The presence of a covalently attached pantetheine cofactor was first established in a cell-free system that catalyzed enzymatic synthesis of the decapeptides gramicidin S and tyrocidine. As in the case of fatty acid synthesis, its role in binding and translocating the intermediate peptides was analyzed [174,175]. 

In the case of linear gramicidin, the N-terminus of the nonribosomal peptide carries a formyl group (10). Just like in the bacterial ribosomal synthesis, only a formylated first building block is processed additionally by the corresponding enzymatic machinery. Thus, one can find a distinct formylation (F) domain at the very N-terminus of the synthetase. Another formylated NRPS product is coelichelin whose N-terminal ornithine residue is believed to be Nj-formylated in trans by a formyltransferase genetically associated with the NRPS (17). Formyl-tetrahydrofolate is used as source of the formyl group by these enzymes. 

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