Ribosomally synthesized and post-translationally modified peptides
Ribosomally synthesized and post-translationally modified peptides, 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 variety of organisms, including prokaryotes, eukaryotes, and archaea, and they possess a wide range of biological functions.
As a consequence of the falling cost of genome sequencing and the accompanying rise in available genomic data, scientific interest in RiPPs has increased in the last few decades. Because the chemical structures of RiPPs are more closely predictable from genomic data than are other natural products, their presence in sequenced organisms can, in theory, be identified rapidly. This makes RiPPs an attractive target of modern natural product discovery efforts.
Definition
RiPPs consist of any peptides that are ribosomally-produced and undergo some degree of enzymatic post-translational modification. This combination of peptide translation and modification is referred to as "post-ribosomal peptide synthesis" in analogy with nonribosomal peptide synthesis.Historically, the current sub-classes of RiPPs were studied individually, and common practices in nomenclature varied accordingly in the literature. More recently, with the advent of broad genome sequencing, it has been realized that these natural products share a common biosynthetic origin. In 2013, a set of uniform nomenclature guidelines were agreed upon and published by a large group of researchers in the field. Prior to this report, RiPPs were referred to by a variety of designations, including post-ribosomal peptides, ribosomal natural products, and ribosomal peptides.
The acronym "RiPP" stands for "ribosomally synthesized and post-translationally modified peptide".
Prevalence and applications
RiPPs constitute one of the major superfamilies of natural products, like alkaloids, terpenoids, and nonribosomal peptides, although they tend to be large, with molecular weights commonly in excess of 1000 Da. The advent of next-generation sequencing methods has made genome mining of RiPPs a common strategy. In part due to their increased discovery and hypothesized ease of engineering, the use of RiPPs as drugs is increasing. Although they are ribosomal peptides in origin, RiPPs are typically categorized as small molecules rather than biologics due to their chemical properties, such as moderate molecular weight and relatively high hydrophobicity.The uses and biological activities of RiPPs are diverse.
RiPPs in commercial use include nisin, a food preservative, thiostrepton, a veterinary topical antibiotic, and nosiheptide and duramycin, which are animal feed additives. Phalloidin functionalized with a fluorophore is used in microscopy as a stain due to its high affinity for actin. Anantin is a RiPP used in cell biology as an atrial natriuretic peptide receptor inhibitor.
In 2012-2013, a derivatized RiPP in clinical trials was LFF571. Phase II clinical trials of LFF571, a derivative of the thiopeptide GE2270-A, for the treatment of Clostridioides difficile infections, with comparable safety and efficacy to vancomycin, was terminated early as the results were unfavorable. Also recently in clinical trials was the NVB302 which is used for the treatment of Clostridioides difficile infection. Duramycin has completed phase II clinical trials for the treatment of cystic fibrosis.
Other bioactive RiPPs include the antibiotics cyclothiazomycin and bottromycin, the ultra-narrow spectrum antibiotic plantazolicin, and the cytotoxin patellamide A. Streptolysin S, the toxic virulence factor of Streptococcus pyogenes, is also a RiPP. Additionally, human thyroid hormone itself is a RiPP due to its biosynthetic origin as thyroglobulin.
Classifications
Amatoxins and phallotoxins
Amatoxins and phallotoxins are 8- and 7-membered natural products, respectively, characterized by N-to-C cyclization in addition to a tryptathionine motif derived from the crosslinking of Cys and Trp. The amatoxins and phallotoxins also differ from other RiPPs based on the presence of a C-terminal recognition sequence in addition to the N-terminal leader peptide. α-Amanitin, an amatoxin, has a number of posttranslational modifications in addition to macrocyclization and formation of the tryptathionine bridge: oxidation of the tryptathionine leads to the presence of a sulfoxide, and numerous hydroxylations decorate the natural product. As an amatoxin, α-amanitin is an inhibitor of RNA polymerase II.Bottromycins
Bottromycins contain a C-terminal decarboxylated thiazole in addition to a macrocyclic amidine.There are currently six known bottromycin compounds, which differ in the extent of side chain methylation, an additional characteristic of the bottromycin class. The total synthesis of bottromycin A2 was required to definitively determine the structure of the first bottromycin.
Thus far, gene clusters predicted to produce bottromycins have been identified in the genus Streptomyces. Bottromycins differ from other RiPPs in that there is no N-terminal leader peptide. Rather, the precursor peptide has a C-terminal extension of 35-37 amino acids, hypothesized to act as a recognition sequence for posttranslational machinery.
Cyanobactins
Cyanobactins are diverse metabolites from cyanobacteria with N-to-C macrocylization of a 6–20 amino acid chain. Cyanobactins are natural products isolated from cyanobacteria, and close to 30% of all cyanobacterial strains are thought to contain cyanobacterial gene clusters. However, while thus far all cyanobactins are credited to cyanobacteria, there exists the possibility that other organisms could produce similar natural products.The precursor peptide of the cyanobactin family is traditionally designated the "E" gene, whereas precursor peptides are designated gene "A" in most RiPP gene clusters. "A" is a serine protease involved in cleavage of the leader peptide and subsequent macrocyclization of the peptide natural product, in combination with an additional serine protease homologue, the encoded by gene "G". Members of the cyanobactin family may bear thiazolines/oxazolines, thiazoles/oxazoles, and methylations depending on additional modification enzymes. For example, perhaps the most famous cyanobactin is patellamide A, which contains two thiazoles, a methyloxazoline, and an oxazoline in its final state, a macrocycle derived from 8 amino acids.
Lanthipeptides
Lanthipeptides are one of the most well-studied families of RiPPs. The family is characterized by the presence of lanthionine and 3-methyllanthionine residues in the final natural product. There are four major classes of lanthipeptides, delineated by the enzymes responsible for installation of Lan and MeLan. The dehydratase and cyclase can be two separate proteins or one multifunctional enzyme. Previously, lanthipeptides were known as "lantipeptides" before a consensus was reached in the field.Lantibiotics are lanthipeptides that have known antimicrobial activity. The founding member of the lanthipeptide family, nisin, is a lantibiotic that has been used to prevent the growth of food-born pathogens for over 40 years.
Lasso peptides
Lasso peptides are short peptides containing an N-terminal macrolactam macrocycle "ring" through which a linear C-terminal "tail" is threaded. Because of this threaded-loop topology, these peptides resemble lassos, giving rise to their name. They are a member of a larger class of amino-acid-based lasso structures. Additionally, lasso peptides are formally rotaxanes.The N-terminal "ring" can be from 7 to 9 amino acids long and is formed by an isopeptide bond between the N-terminal amine of the first amino acid of the peptide and the carboxylate side chain of an aspartate or glutamate residue. The C-terminal "tail" ranges from 7 to 15 amino acids in length.
The first amino acid of lasso peptides is almost invariably glycine or cysteine, with mutations at this site not being tolerated by known enzymes. Thus, bioinformatics-based approaches to lasso peptide discovery have thus used this as a constraint. However, some lasso peptides were recently discovered that also contain serine or alanine as their first residue.
The threading of the lasso tail is trapped either by disulfide bonds between ring and tail cysteine residues, by steric effects due to bulky residues on the tail, or both. The compact structure makes lasso peptides frequently resistant to proteases or thermal unfolding.
Linear azol(in)e-containing peptides
Linear azolee-containing peptides contain thiazoles and oxazoles, or their reduced thiazoline and oxazoline forms. Thiazoles are the result of cyclization of Cys residues in the precursor peptide, while oxazoles are formed from Thr and Ser. Azole and azoline formation also modifies the residue in the -1 position, or directly C-terminal to the Cys, Ser, or Thr. A dehydrogenase in the LAP gene cluster is required for oxidation of azolines to azoles.Plantazolicin is a LAP with extensive cyclization. Two sets of five heterocycles endow the natural product with structural rigidity and unusually selective antibacterial activity. Streptolysin S is perhaps the most well-studied and most famous LAP, in part because the structure is still unknown since the discovery of SLS in 1901. Thus, while the biosynthetic gene cluster suggests SLS is a LAP, structural confirmation is lacking.
Microcins
are all RiPPs produced by Enterobacteriaceae with a molecular weight <10 kDa. Many members of other RiPP families, such as microcin E492, microcin B17 and microcin J25 are also considered microcins. Instead of being classified based on posttranslational modifications or modifying enzymes, microcins are instead identified by molecular weight, native producer, and antibacterial activity. Microcins are either plasmid- or chromosome-encoded, but specifically have activity against Enerobacteriaceae. Because these organisms are also often producers of microcins, the gene cluster contains not only a precursor peptide and modification enzymes, but also a self-immunity gene to protect the producing strain, and genes encoding export of the natural product.Microcins have bioactivity against Gram-negative bacteria but usually display narrow-spectrum activity due to hijacking of specific receptors involved in the transport of essential nutrients.