Non-ribosomal Peptide Synthesis
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Nonribosomal peptides (NRP) are a class of
peptide Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides that have a molecular mass of 10,000 Da or more are called proteins. Chains of fewer than twenty am ...
secondary metabolites, usually produced by
microorganism A microorganism, or microbe, is an organism of microscopic scale, microscopic size, which may exist in its unicellular organism, single-celled form or as a Colony (biology)#Microbial colonies, colony of cells. The possible existence of unseen ...
s like
bacteria Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of Prokaryote, prokaryotic microorganisms. Typically a few micr ...
and
fungi A fungus (: fungi , , , or ; or funguses) is any member of the group of eukaryotic organisms that includes microorganisms such as yeasts and mold (fungus), molds, as well as the more familiar mushrooms. These organisms are classified as one ...
. Nonribosomal peptides are also found in higher organisms, such as
nudibranch Nudibranchs () are a group of soft-bodied marine gastropod molluscs, belonging to the order Nudibranchia, that shed their shells after their larval stage. They are noted for their often extraordinary colours and striking forms, and they have b ...
s, but are thought to be made by bacteria inside these organisms. While there exist a wide range of peptides that are not synthesized by
ribosome Ribosomes () are molecular machine, macromolecular machines, found within all cell (biology), cells, that perform Translation (biology), biological protein synthesis (messenger RNA translation). Ribosomes link amino acids together in the order s ...
s, the term ''nonribosomal peptide'' typically refers to a very specific set of these as discussed in this article. Nonribosomal peptides are synthesized by nonribosomal peptide synthetases, which, unlike the
ribosome Ribosomes () are molecular machine, macromolecular machines, found within all cell (biology), cells, that perform Translation (biology), biological protein synthesis (messenger RNA translation). Ribosomes link amino acids together in the order s ...
s, are independent of
messenger RNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the ...
. Each nonribosomal peptide synthetase can synthesize only one type of peptide. Nonribosomal peptides often have cyclic and/or branched structures, can contain non- proteinogenic
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...
s including D-amino acids, carry modifications like '' N''-methyl and ''N''-formyl groups, or are glycosylated, acylated, halogenated, or hydroxylated. Cyclization of amino acids against the peptide "backbone" is often performed, resulting in
oxazoline Oxazoline is a five-membered heterocyclic organic compound with the formula . It is the parent of a family of compounds called oxazolines (emphasis on plural), which contain non-hydrogenic substituents on carbon and/or nitrogen. Oxazolines are the ...
s and
thiazoline Thiazolines (; or dihydrothiazoles) are a group of isomeric 5-membered heterocyclic compounds containing both sulfur and nitrogen in the ring. Although unsubstituted thiazolines are rarely encountered themselves, their derivative (chemistry), der ...
s; these can be further oxidized or reduced. On occasion, dehydration is performed on
serine Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α- amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − ...
s, resulting in dehydroalanine. This is just a sampling of the various manipulations and variations that nonribosomal peptides can perform. Nonribosomal peptides are often dimers or trimers of identical sequences chained together or cyclized, or even branched. Nonribosomal peptides are a very diverse family of natural products with an extremely broad range of biological activities and pharmacological properties. They are often toxins, siderophores, or
pigment A pigment is a powder used to add or alter color or change visual appearance. Pigments are completely or nearly solubility, insoluble and reactivity (chemistry), chemically unreactive in water or another medium; in contrast, dyes are colored sub ...
s. Nonribosomal peptide
antibiotic An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting pathogenic bacteria, bacterial infections, and antibiotic medications are widely used in the therapy ...
s, cytostatics, and immunosuppressants are in commercial use.


Examples

*
Antibiotic An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting pathogenic bacteria, bacterial infections, and antibiotic medications are widely used in the therapy ...
s ** Actinomycin ** Bacitracin ** Calcium dependent antibiotic ** Daptomycin **
Vancomycin Vancomycin is a glycopeptide antibiotic medication used to treat certain bacterial infections. It is administered intravenously ( injection into a vein) to treat complicated skin infections, bloodstream infections, endocarditis, bone an ...
** Teixobactin ** Tyrocidine ** Gramicidin ** Zwittermicin A *
Antibiotic An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting pathogenic bacteria, bacterial infections, and antibiotic medications are widely used in the therapy ...
precursors ** ACV-Tripeptide * Cytostatics ** Epothilone ** Fabclavine ** Bleomycin * Immunosuppressants **
Ciclosporin Ciclosporin, also spelled cyclosporine and cyclosporin, is a calcineurin inhibitor, used as an immunosuppressant medication. It is taken Oral administration, orally or intravenously for rheumatoid arthritis, psoriasis, Crohn's disease, nephr ...
(Cyclosporine A) * Siderophores ** Pyoverdine ** Enterobactin ** Myxochelin A *
Pigment A pigment is a powder used to add or alter color or change visual appearance. Pigments are completely or nearly solubility, insoluble and reactivity (chemistry), chemically unreactive in water or another medium; in contrast, dyes are colored sub ...
s ** Indigoidine *
Toxin A toxin is a naturally occurring poison produced by metabolic activities of living cells or organisms. They occur especially as proteins, often conjugated. The term was first used by organic chemist Ludwig Brieger (1849–1919), derived ...
s ** Microcystins and ** Nodularins, cyanotoxins from
cyanobacteria Cyanobacteria ( ) are a group of autotrophic gram-negative bacteria that can obtain biological energy via oxygenic photosynthesis. The name "cyanobacteria" () refers to their bluish green (cyan) color, which forms the basis of cyanobacteri ...
. * Nitrogen storage polymers ** Cyanophycin – produced by some
cyanobacteria Cyanobacteria ( ) are a group of autotrophic gram-negative bacteria that can obtain biological energy via oxygenic photosynthesis. The name "cyanobacteria" () refers to their bluish green (cyan) color, which forms the basis of cyanobacteri ...
* Phytotoxins ** HC-toxin – a virulence factor made by the plant pathogenic fungus '' Cochliobolus (Helminthosporium) carbonum'' ** AM-toxin – made by the plant pathogenic fungus ''
Alternaria alternata ''Alternaria alternata'' is a fungus causing leaf spots, rots, and blights on many plant parts, and other diseases. It is an opportunistic pathogen on over 380 host species of plant. It can also cause upper respiratory tract infections and a ...
'' pv. ''Mali'' ** victorin – a chlorinated cyclic pentapeptide made by the pathogenic fungus '' Cochliobolus victoriae''. Its nonribosomal synthesis has not been established.


Biosynthesis

Nonribosomal peptides are synthesized by one or more specialized nonribosomal peptide-synthetase (NRPS)
enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...
s. The NRPS genes for a certain peptide are usually organized in one
operon In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splic ...
in bacteria and in gene clusters in
eukaryote The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...
s. However the first fungal NRP to be found was
ciclosporin Ciclosporin, also spelled cyclosporine and cyclosporin, is a calcineurin inhibitor, used as an immunosuppressant medication. It is taken Oral administration, orally or intravenously for rheumatoid arthritis, psoriasis, Crohn's disease, nephr ...
. It is synthesized by a single 1.6MDa NRPS. The enzymes are organized in modules that are responsible for the introduction 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 Biosynthesis, i.e., chemical synthesis occurring in biological contexts, is a term most often referring to multi-step, enzyme-Catalysis, catalyzed processes where chemical substances absorbed as nutrients (or previously converted through biosynthe ...
of nonribosomal peptides shares characteristics with the polyketide and
fatty acid In chemistry, in particular in biochemistry, a fatty acid is a carboxylic acid with an aliphatic chain, which is either saturated and unsaturated compounds#Organic chemistry, saturated or unsaturated. Most naturally occurring fatty acids have an ...
biosynthesis. Due to these structural and mechanistic similarities, some nonribosomal peptide synthetases contain polyketide synthase modules for the insertion of
acetate An acetate is a salt formed by the combination of acetic acid with a base (e.g. alkaline, earthy, metallic, nonmetallic, or radical base). "Acetate" also describes the conjugate base or ion (specifically, the negatively charged ion called ...
or propionate-derived subunits into the peptide chain. Note that as many as 10% percent of bacterial NRPS are not laid out as large modular proteins, but as separate enzymes. Some NRPS modules deviate from the standard domain structure, and some extra domains have been described. There are also NRPS enzymes that serve as a scaffold for other modifications to the substrate to incorporate unusual amino acids.


Modules

The order of modules and domains of a complete nonribosomal peptide synthetase is as follows: * ''Initiation'' or ''Starting'' module: /NMTA-PCP- * ''Elongation'' or ''Extending'' modules: -(C/Cy)- MTA-PCP- * ''Termination'' or ''Releasing'' module: -(TE/R) (Order:
N-terminus The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amin ...
to
C-terminus The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein Proteins are large biomolecules and macromolecules that comp ...
; ''[]'': optionally; ''()'': alternatively)


Domains

* F: Formylation (optional) * A: Adenylation (required in a module) * PCP: Thiolation and peptide carrier protein with attached 4'-phospho-pantetheine (required in a module) * C: Condensation forming the amide bond (required in a module) * Cy: Cyclization into thiazoline or oxazolines (optional) * Ox: Oxidation of thiazolines or oxazolines to thiazoles or oxazoles (optional) * Red: Reduction of thiazolines or oxazolines to thiazolidines or oxazolidines (optional) * E: Epimerization into D-amino acids (optional) * NMT: ''N''-methylation (optional) * TE: Termination by a thio-esterase (only found once in a NRPS) * R: Reduction to terminal aldehyde or alcohol (optional) * X: Recruits cytochrome P450 enzymes (optional)


Starting stage

* Loading: The first amino acid is activated with ATP as a mixed acyl-
phosphoric acid Phosphoric acid (orthophosphoric acid, monophosphoric acid or phosphoric(V) acid) is a colorless, odorless phosphorus-containing solid, and inorganic compound with the chemical formula . It is commonly encountered as an 85% aqueous solution, ...
anhydride An acid anhydride is a type of chemical compound derived by the removal of water molecules from an acid (chemistry), acid. In organic chemistry, organic acid anhydrides contain the functional group . Organic acid anhydrides often form when one ...
with AMP by the A-domain and loaded onto the
serine Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α- amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − ...
-attached 4'-phospho- pantethine (4'PP) sidechain of the PCP-domain catalyzed by the PCP-domain (thiolation). * Some A domains require interaction with MbtH-like proteins for their activity. * Sometimes the amino group of the bound amino acid is formylated by an F-domain or methylated by an NMT-domain.


Elongation stages

* Loading: Analogous to the starting stage, each module loads its specific amino acid onto its PCP-domain. *
Condensation Condensation is the change of the state of matter from the gas phase into the liquid phase, and is the reverse of vaporization. The word most often refers to the water cycle. It can also be defined as the change in the state of water vapor ...
: The C-domain catalyzes the
amide In organic chemistry, an amide, also known as an organic amide or a carboxamide, is a chemical compound, compound with the general formula , where R, R', and R″ represent any group, typically organyl functional group, groups or hydrogen at ...
bond formation between the
thioester In organic chemistry, thioesters are organosulfur compounds with the molecular structure . They are analogous to carboxylate esters () with the sulfur in the thioester replacing oxygen in the carboxylate ester, as implied by the thio- prefix ...
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 Condensation is the change of the state of matter from the gas phase into the liquid phase, and is the reverse of vaporization. The word most often refers to the water cycle. It can also be defined as the change in the state of water vapor ...
- 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 Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α- amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − ...
,
threonine Threonine (symbol Thr or T) is an amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form when dissolved in water), a carboxyl group (which is in the deprotonated −COO− ...
, or
cysteine Cysteine (; symbol Cys or C) is a semiessential proteinogenic amino acid with the chemical formula, formula . The thiol side chain in cysteine enables the formation of Disulfide, disulfide bonds, and often participates in enzymatic reactions as ...
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 stage

* Termination: The TE-domain (thio-esterase domain) hydrolyzes the completed polypeptide chain from the PCP-domain of the previous module, thereby often forming cyclic amides (
lactam A lactam is a Cyclic compound, cyclic amide, formally derived from an amino alkanoic acid through cyclization reactions. The term is a portmanteau of the words ''lactone'' + ''amide''. Nomenclature Greek_alphabet#Letters, Greek prefixes in alpha ...
s) or cyclic
ester In chemistry, an ester is a compound derived from an acid (either organic or inorganic) in which the hydrogen atom (H) of at least one acidic hydroxyl group () of that acid is replaced by an organyl group (R). These compounds contain a distin ...
s ( lactones). * Also, the peptide can be released by an R-domain that reduces the thioester bond to terminal
aldehyde In organic chemistry, an aldehyde () (lat. ''al''cohol ''dehyd''rogenatum, dehydrogenated alcohol) is an organic compound containing a functional group with the structure . The functional group itself (without the "R" side chain) can be referred ...
or
alcohol Alcohol may refer to: Common uses * Alcohol (chemistry), a class of compounds * Ethanol, one of several alcohols, commonly known as alcohol in everyday life ** Alcohol (drug), intoxicant found in alcoholic beverages ** Alcoholic beverage, an alco ...
.


Processing

The final peptide is often modified, e.g., by
glycosylation Glycosylation is the reaction in which a carbohydrate (or ' glycan'), i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor) in order to form a glycoconjugate. In biology (but not ...
, acylation, halogenation, or hydroxylation. The responsible enzymes are usually associated to the synthetase complex and their genes are organized in the same
operon In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splic ...
s or gene clusters.


Priming and deblocking

To become functional, the 4'-phospho-pantetheine 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).


Substrate specificities

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 that control substrate specificity and can be considered the '
codons Genetic code is a set of rules used by living cells to translate information encoded within genetic material ( DNA or RNA sequences of nucleotide triplets or codons) into proteins. Translation is accomplished by the ribosome, which links pro ...
' of nonribosomal peptide synthesis have been identified, and rational protein design has yielded methodologies to computationally switch the specificities of A-domains. The condensation C-domain is also believed to have substrate specificity, especially if located behind an epimerase E-domain-containing module where it functions as a 'filter' for the epimerized
isomer In chemistry, isomers are molecules or polyatomic ions with identical molecular formula – that is, the same number of atoms of each element (chemistry), element – but distinct arrangements of atoms in space. ''Isomerism'' refers to the exi ...
. Computational methods, such as SANDPUMA and NRPSpredictor2, have been developed to predict substrate specificity from DNA or protein sequence data.


Mixed with polyketides

Due to the similarity with polyketide synthases (PKS), many secondary metabolites are, in fact, fusions of NRPs and polyketides. In essence, this occurs when PK modules follow NRP modules, and vice versa. Although there is high degree of similarity between the Carrier (PCP/ACP) domains of both types of synthetases, the mechanism of condensation is different from a chemical standpoint: * PKS, carbon-carbon bond formation through Claisen condensation reaction * NRPs, the C domain catalyzes the amide bond formation between the amino acid it adds to the chain (on the PCP of one module) and the nascent peptide(on the PCP of the next module).


See also

* Epothilone * Esterase *
Ribosomally synthesized and post-translationally modified peptides Ribosomally synthesized and post-translationally modified peptides (RiPPs), also known as ribosomal natural products, are a diverse class of natural products of ribosomal origin. Consisting of more than 20 sub-classes, RiPPs are produced by a vari ...


References


Further reading

* * * {{refend Molecular biology Enzymes Glycopeptide antibiotics Antibiotics Peptides