Nonribosomal peptides (NRP) are secondary metabolites, usually produced by microorganisms like bacteria and fungi. They are synthesized by nonribosomal peptide synthetases (NRPS) from amino acids. They often have a cyclic and/or branched structure, contain non-proteinogenic amino acids including D-amino acids, carry modifications like N-methyl and N-formyl groups, or are glycosylated, acylated, halogenated, or hydroxylated. Sometimes they are dimers or trimers of identical subunits.
Nonribosomal peptides are structurally a very diverse family of natural products with an extremely broad range of biological activities and pharmacological properties. They are mushroom and cyanobacterial toxins, siderophores, or pigments. Nonribosomal peptide antibiotics, cytostatics, and immunosuppressants are in commercial use.
- Nitrogen storage polymers
Nonribosomal peptides are synthesized by one or more specialized nonribosomal peptide-synthetase (NRPS) enzymes. The NRPS genes for a certain peptide are usually organized in one operon in bacteria and in gene clusters in eukaryotes. The enzymes are organized in modules that are responsible for the indroduction of one additional amino acid. Each module consists of several domains with defined functions, separated by short spacer regions of about 15 amino acids.
The biosynthesis of nonribosomal peptides shares similarities with the polyketide biosynthesis. Due to these structural and mechanistic similarities some nonribosomal peptide synthetases contain Polyketide synthase modules for the insertion of acetate or propionate derived subunits into the peptide chain.
The order of modules and domains of a complete nonribosomal peptide synthetase is as follows:
- Initiation or Starting module: [F/NMe]-A-PCP-
- Elongation or Extending modules: -(C/Cy)-[NMe]-A-PCP-[E]-
- Termination or Releasing module: -(TE/R)
(Order: N-terminus to C-terminus; : optionally; (): alternatively)
- F: Formylation (rare)
- A: Adenylation
- PCP: Thiolation and Peptide Carrier Protein with attached 4'-phospho-pantethein
- C: Condensation forming the amide bond
- Cy: Cylization into thiazolidine or oxazolidines (rare)
- E: Epimerization into D-amino acids (rare)
- TE: Termination by a thio-esterase
- R: Reduction to terminal aldehyde or alcohol (rare)
- Loading: The first amino acid is activated with ATP as a mixed acyl-phosphoric acid anhydride with AMP by the A-domain and loaded onto the serine-attached 4'-phospho-pantethein (4'PP) sidechain of the PCP-domain catalyzed by the PCP-domain (thiolation) .
- Sometimes the amino group of the bound amino acid is formylated by an F-domain or methylated by an NMe-domain.
- Loading: Analogous to the starting stage, each module loads its specific amino acid onto its PCP-domain.
- Condensation: The C-domain catalyzes the amide [Chemical bond|bond] formation between the thioester group of the growing peptide chain from the previous module with the amino group of the current module. The extended peptide is now attached to the current PCP-domain.
- Condensation-Cyclization : Sometimes the C-domain is replaced by a Cy-domain which, in addition to the amide bond formation, catalyzes the reaction of the serine, threonine, or cysteine sidechain with the amide-N, thereby forming oxazolidines and thiazolidine , respectively.
- Epimerization : Sometimes an E-domain epimerizes the innermost amino acid of the peptide chain into the D-configuration.
- This cycle is repeated for each elongation module.
- Termination: The TE-domain (thio-esterase domain) hydrolyzes the completed polyketide chain from the ACP-domain of the previous module, thereby often forming cyclic amides (lactams) or cyclic esters (lactones).
- Alternatively, the peptide can be released by an R-domain that reduces the thioester bond to terminal aldehyde or alcohol.
The final peptide is often modified, e.g. by glycosylation, acylation, halogenation, or hydroxylation. The responsible enzymes are usually associated to the synthetase complex and their genes are organized in the same operons or gene clusters .
Priming and Deblocking
To become functional, the 4'-phospho-pantethein sidechain of acyl-CoA molecules has to be attached to the PCP-domain by 4'PP transferases (Priming) and the S-attached acyl group has to be removed by specialized associated thioesterases (TE-II) (Deblocking).
Most domains have a very broad substrate specificity and usually only the A-domain determines which amino acid is incorporated in a module. Ten amino acids have been identified that control substrate specificity and can be considered the 'codons' of nonribosomal peptide synthesis. Sometimes the cyclization C-domain has a more narrow substrate specificity, especially if located behind an epimerase E-domain containing module where it functions as a 'filter' for the epimerized isomer.
- "Nonribosomal peptides: from genes to products" by Dirk Schwarzer, Robert Finking, and Mohamed A. Marahiel in Nat. Prod. Rep. 20(3):275-287 (2003) DOI: 10.1039/b111145k
- "Modular Peptide Synthetases Involved in Nonribosomal Peptide Synthesis" by Mohamed A. Marahiel, Torsten Stachelhaus, and Henning D. Mootz in Chem. Rev. 97(7):2651-2673 (1997) DOI: 10.1021/cr960029e