NPRS-domains’ core-motifs .
Nonribosomal peptides (NRPs) are a type of secondary metabolite with a wide range of pharmacological and biological activities including cytostatics, immunosuppressants or anticancer agents, antibiotics, pigments, siderophores, toxins. NRPs, unlike other proteins, are synthesized on huge nonribosomal peptide synthetase (NRPS) enzyme complexes that are not dependent on ribosomal machinery. Bacteria and fungi are the most common NRPs producers. Furthermore, the presence of these peptides has been confirmed in marine microbes. Nowadays, many of these peptides are used in the treatments of inflammatory, cancer, neurodegenerative disorders, and infectious disease for the development of new therapeutic agents. The structure, function, and synthesis of NRPs, as well as producer microorganisms and their several application areas, are covered in this chapter.
- biological activity
- nonribosomal peptide
- producer microorganisms
- secondary metabolite
Bioprocesses, which are consisted of a series of enzymatically catalyzed biochemical reactions in all living things, are necessary for survival. They have a high potential in terms of material synthesis, which has recently been performed by chemical techniques . Furthermore, the advancement of heterologous production systems and genetic engineering techniques has resulted in pioneering initiatives to manufacture usable biomaterials . These advancements also enabled the successful generation of primary and secondary metabolic pathway products in physiologically and genetically well-defined hosts, such as
NRPs are secondary metabolites that are synthesized outside of the ribosomal machinery and have a variety of properties such as cytostatics, immunosuppressants or anticancer agents, antibiotics, pigments, siderophores, toxins [3, 5, 6]. NRPs are typically produced by marine microorganisms and invertebrates, as well as soil-inhabiting microorganisms [5, 7, 8]. The majority of natural products produced by sponges, bryozoans, mollusks, and tunicates are members of the NRP or mixed polyketide–NRP families. Several of NRPs are being used in the development of new medicines for inflammatory, cancer, neurological diseases, and infectious disease nowadays .
Non-ribosomal peptide synthetases (NRPSs) enzymes are poly-functional mega-synthases that biosynthesize NRPs [7, 9, 10]. NRPSs, multi-modular enzyme or enzyme complexes from common bacteria, less common eukarya, and rare archaea, are capable of producing a wide range of natural pharmaceutical products (Bacitracin, antibiotics for skin infections; Bleomycin antitumor; Cyclosporin, antifungal, and immunosuppressive drugs; Daptomycin, antibiotics) [5, 7, 11]. NRPSs use both proteinogenic and nonproteinogenic amino acids (not encoded by DNA) as building blocks for the growing peptide chain [1, 7, 11, 12]. They catalyze multiple biosynthetic processes, each of which is responsible for particular one amino acid elongation on the growing peptide chain . This chapter looks at the structure, function, and synthesis of NRPs, as well as producer microorganisms and their various applications.
2. Synthesis, structure, and function of nonribosomal peptide (NRP)
NRPSs are responsible for nonribosomal peptide (NRP) synthesis. These are large multi-enzyme complexes that are modularly organized and serve as biosynthetic machinery and templates [5, 11, 12, 13, 14]. For example, a single NRPS of 1.6 MDa synthesizes the Cyclosporine A (7). In fungal systems, such as in the case of cyclosporine A (7), a single NRPS synthesizes peptides, whereas bacteria frequently use numerous NRPSs with genes grouped in an operon. NRPSs have a modular structure [14, 15].
In a genome mining research of 2699 genomes, Wang et al. found that more than half of the NRPS enzymes were non-modular NRPS enzymes . Nonmodular NRPS enzymes can be found in siderophore biosynthesis pathways, such as EntE and VibH in enterobactin and VibE in vibriobactin, or as a standalone peptidyl carrier protein, such as BlmI from the bleomycin gene cluster. NRPS enzymes are found frequently in bacteria, less frequently in eukaryotes, and infrequently in archaea. Actinobacteria, Cyanobacteria, Firmicutes, and Proteobacteria were the phyla with the greatest number of these enzymes in the bacterial domain. There was a correlation between genome size and the number of NRPS clusters [5, 17].
A module is a part of the NRPS polypeptide chain that is in charge of integrating one amino acid into the final product. Modules can further be separated into domains (Figure 1), which represent enzyme units that catalyze distinct steps of NRP synthesis. On the protein level, domains are defined by distinctive, greatly conserved order of patterns known as “core motifs.” In certain instances, biochemical and structural data were used to confirm the involvement of greatly conserved residues in domain function (Table 1) .
There are three domains in a module. These are 1) the adenylation (A) domain, 2) the peptidyl carrier protein (PCP) or thiolation (T) domain, and 3) the condensation (C) domain, all of which are responsible for the synthesis of NRPs. A module may include additional tailoring or altering domains incorporating epimerization (E), methylation and oxidation domains or a heterocyclization (Cy) domain in place of a C-domain. Finally, most NRPS termination modules have a TE-domain, which is in charge of releasing linear, cyclic, or branching cyclic peptides [5, 9, 10, 11, 15, 18, 19, 20, 21].
The order of the modules is frequently aligned with the sequences of the resulting peptides. NRP synthesis begins at the N-terminus and ends at the C-terminus, yielding peptides that are typically 3–15 amino acids long. The released peptides contain amino acids, that is, imino acids or ornithine and their structures are linear, cyclic-macrocyclic, branched-cyclic, branched-macrocyclic, dimers or trimers of identical structural elements .
The A-domain is responsible for the first step in biosynthesis, which involves recognizing and activating the amino acid substrate via adenylation with Mg-ATP, resulting in an aminoacyl adenylated intermediate. Around 550 amino acids make up domain A. It has 10 amino acid residues that serve as NRPS enzyme “codons” and are essential for substrate specificity. The D and L forms of the 20 amino acids used in ribosomal protein synthesis, as well as non-proteinogenic amino acids like imino acids, ornithine, and hydroxy acids like β-butyric acids and α-aminoadipic, are substrates recognized by the A-domain. The PCP-domain, which consists of about 80 amino acids and covalently attaches the activated amino acid to their cofactor 4′-phosphopantetheine (PP) arm via a thioester bond, completes the second step. And also, the active substrate and elongation intermediates are transferred to the C-domain via this domain. In the last step, C-domain, which contains approximately 450 amino acids, catalyzes the formation of peptide bonds between the carboxyl group of the incipiently synthesized peptide and the amino acid transported by the side module [5, 22]. Furthermore, this domain allows the expanding chain to be translocated to the next module. Following this step, the linear intermediate peptide is liberated in bacteria via internal cyclization or hydrolysis with the help of the Thioesterase (TE) domain. On the other hand, it appears less commonly in fungi’s NRPSs. Fungi use a variety of ways to release chains. The first is a thioesterase NADP(H)-dependent reductase domain (R), which catalyzes NADPH reduction to create an aldehyde and the second is a terminal C domain, which catalyzes release by forming intermolecular or intramolecular amide bonds. By N-, C-, and O-methylation, halogenation, acylation, hydroxylation, glycosylation, or heterocyclic ring formation, the primary product of this synthesis can be changed post-synthetically to reach its mature form by additional tailoring enzymes that are not part of the NRPS. The structural diversity of NRPs is formed in part by these enzymes and their reactions .
Because of their extensive multidomain organization, NRPS genes are easier to identify using recent genome mining technologies, and they are also relatively easy to detect. Secondary metabolites production genes are frequently found in bacterial and fungal gene clusters. The clusters’ core is thought to be NRPS genes. Nevertheless, they are linked to genes involved in building blocks synthesis, product ornamentation, self-resistance, and peptide export. For the purpose of analyzing and in silico exploration of NRPS pathways, advanced genome sequencing techniques have made genome mining methodologies available, which are assisted by a variety of bioinformatics tools, such as AntiSMASH, PRISM, and SMURF .
Nowadays, known NRP structures are divided into various categories, each with its own structural characteristics. Lipocyclopeptides with varied linkage patterns, such as fengycin, iturin, surfactin, and head-to-tail-cyclized peptides of varying ring sizes, such as cyclosporine, gramicidin S, maybe the largest group. There are also a lot of linear peptide configurations. They include tripeptides (such as sevadicin and bialaphos) as well as 20-mer peptides (e.g., alamethicin, peptaibols). The current highest size limit for NRPs is syringopeptin 25A, which has 25 amino acids (syringopeptin 25A). Tailoring enzymes modify the structure of some NRPs. The most structurally complicated molecules are probably bleomycins, ergopeptides, glycopeptide antibiotics, and β-lactams .
Figure 2 shows some NRPs with diverse structures and a wide spectrum of activities. ACV-tripeptide (6), for example, is a precursor to antibiotics of the penicillin and cephalosporin families. Gramicidin S (4), tyrocidine A (1), and vancomycin (5) are three other antibiotic-active substances. Cyclosporin A (7), an immunosuppressive drug, is used in the post-transplantation care of patients. Cancer is treated with cytostatic agents, such as bleomycin A2 (8) and epothilone (9). Enterobactin (10), bacillibactin (11), mixochelin A (12), yersiniabactin (13), and vibriobactin (14) are examples of iron chelating agents. These compounds, known as siderophores, are created in iron-deficient environments to provide bacteria with an iron source. Figure 2 also depicts the structures of pigments like indigodin (15), toxins like thaxtomin A (17), and peptides with uncertain functions like anabaenopeptilide 90-A (18) .
NRPs have a number of structural characteristics that distinguish them from ribosomal peptides. For example, non-proteinogenic amino acids, such as ornithine in 1, 2, and 4, hydroxyphenyl or dihydroxyphenyl-glycine in 5 and (4R)-4-[(E)-2-butenyl]-4-methyl-L. -threonine (Bmt) in 7, are included. Furthermore, the structures are frequently macrocyclic (1), branched macrocyclic (2), or dimers of two (4) or trimers of three (10, 11) structural components. Smaller heterocyclic rings, such as thiazole in 9, thiazoline in 13, or oxazoline in 14, are common structural properties of nonribosomal peptides. In addition, fatty acids (3), glycosylations (5), N-methylations (7), and N-formylations (18) may also be present, as well as the addition of propionate units (8) or acetate .
3. Overview of producer microorganisms for NRP
NRPs are typically produced by marine microorganisms, soil-inhabiting microorganisms, including
4. Application areas of NRPs
Novel peptide products’ biological functions are strictly associated with their chemical structure, which is constrained by a peptide sequence that ensures specific interaction with a specific molecular target. Chemical alterations, such as the incorporation of fatty acid chains, D-amino acids, glycosylated amino acids, and heterocyclic rings, as well as cyclization or oxidative cross-linking of side chains, add a lot to these unique interactions. Bacitracin, fengycin, pristinamycin, surfactin, tyrocidine, and vancomycin are examples of novel peptides with antibacterial and antifungal properties .
When the ribosomal code was deciphered in the 1960s, Tatum and coworkers discovered that ribosomes had no effect on cell-based tyrocidine production [23, 26]. The first NRPs agent is tyrocidine, a cyclic decapeptide that is biosynthesized outside of the
|Compound||Biosynthetic class of agent||Source||Disease/Molecular target|
|Bacitracin||Cyclic peptide||Antibiotic/dephosphorylation of C55-isoprenyl pyrophosphate|
|Bleomycin||Hybrid peptide||Antibiotic/inhibition of DNA synthesis|
|Capreomycin||Cyclicpeptide||Antibiotic/protein synthesis inhibitor|
|Carbapenems||Synthetic thienamycin||Antibacterial (multidrug resistant)/bacterial cell-wall biosynthesis (peptidoglycan;β-lactamase inhibition)|
|Cephalosporin||Antibiotic/Alters bacterial outer membrane|
|Chlorampheniol||Synthetic;further derivatives: thiamphenicol [c], florfenicol||Antibacterial/inhibition of ribosomal protein synthesis|
|Colistin (Polymyxin E)||—||Antibacterial/binding to lipopolysaccharide (outer membrane), interaction with the cytoplasmic membrane|
|Dalbavancin||Semisynthetic teicoplanin derivative||—||Antibacterial (Gram-positive)/membrane anchoring; disruption of cell membrane and inhibition of bacterial cell wall biosynthesis|
|Daptomycin||Lipopeptide||Antibiotic (Gram-positive)/disrupts the cell membrane|
|Gramicidin||Linear pentadecapeptide||Antibiotic/ion-channel formation, increasing the permeability of the membrane|
|Lincomycin||—||Antibacterial (patients allergic to penicillin) inhibition of the ribosomal protein synthesis (50S-subunit, dissociation of peptidyl-tRNA from the ribosome)|
|Monobactams||—||Antibacterial (Gram-negative)/bacterial cell-wall biosynthesis|
|Oritavancin||—||Semi synthetic||Antibiotic/disrupts the cell membrane|
|Polymyxin B||Polypeptides||Antibacterial (Gram-negative)/binding to lipopolysaccharide (outer membrane), interaction with cytoplasmic membrane|
|Pristinamycin||Depsipeptide||Antibacterial (Gram-positive)/ribosomal biosynthesis (50S-subunit, peptidyl transfer, and elongation of protein synthesis)|
|Teicoplanin||Glycopeptide||Antibiotic/inhibit cell wall synthesis|
|Telavancin||—||Antibacterial (Gram-positive) disruption of cell membrane and inhibition of bacterial cell-wall biosynthesis|
|Tyrothricin||—||Antibacterial (Gram-positive)/disruption of cell membrane|
|Vancomycin||Glycopeptide||Antibiotic/inhibit cell wall synthesis|
|Virginiamycin||—||Antibacterial/ribosomal biosynthesis (50S-subunit, peptidyl transfer, and elongation of protein synthesis)|
As demonstrated in Table 2, systemic and topical antibacterials are the most often used NRPs-based drugs, accounting for billions of dollars in the chemical and pharmaceutical industry sales. Table 3 lists their other applications, which include anticancer agents, antifungals, animal feed additives, immunosuppressants (cyclosporine), obstetrics (ergometrine), and pain management (ergotamine).
|Agent||Origin||Properties and area of application|
|Actinomycin D (Dactinomycin)||Antitumor/DNA intercalator, inhibition of transcription|
|Bialaphos||Herbicide/tripeptide prodrug, inhibitor of glutamine synthetase|
|Bleomycin A2, B2||Antitumor/metal-dependent oxidative cleavage of DNA in presence of molecular oxygen|
|Capreomycin||Antituberculous/ inhibition of the ribosomal protein synthesis (16S and 23S-rRNA)|
|Carfilzomib||Synthetic derivative of epoxomycin (||Anticancer/proteasome inhibitor|
|Caspofungin||Antifungal (candidiasis, aspergillosis) fungal cell-wall integrity ((1-3)-β-D-glucan synthase)|
|Cyclosporine A||Immunosuppressant/cyclophilin binding, inhibition of IL-2 expression (inhibition of T-cell activation)|
|Emodepside||Anthelmintic/Slo-1 receptor (K+ channel)|
|Enduracidin (Enramycin)||Antibacterial, food additive/inhibition of MurG (essential for cell-wall biosynthesis in Gram positive bacteria), inhibition of the transglycosylation step of peptidoglycan biosynthesis|
|Enniatins (fusafungine)||Antibacterial (topical), antifungal, anti-inflammatory/ ionophore (NH4+) membrane depolarization|
|Ergometrine (ergonovine)||Obstetrics/interaction with a-adrenergic, dopaminergic and serotonin receptors|
|Ergotamine||Migraine vasoconstrictive (5-HT1B receptor, but also dopamine and noradrenaline receptors)|
|Romidepsin||Antitumor/histone deacetylase inhibitor (inducing apoptosis)|
|Trabectedin||Bacterial symbiont of ||Antitumor (antiproliferative, treatment of soft tissue sarcoma) DNA binder, blocks binding of transcription factors|
In the medical field, NRP-based marketed drugs, such as Cyclosporin A and Bleomycin A2, have high selling prices. The cost of these medicines is $107 for 25 mg of Cyclosporine A (98% purity) obtained from
The 70% discovery of NRPs with antibacterial, antiviral, cytostatic, immunosuppressive, antimalarial, antiparasitic, animal growth promoters, and natural insecticides activity is mostly attributed to marine organisms . NRPs obtained from marine organisms (sponges, tunicates, and their associated phyla, such as Acidobacteria, Actinobacteria, Bacteriodetes, Chloroflexi, Cyanobacteria, Nitrospira, Planctomycetes, Poribacteria, Proteobacteria, Verrucomicrobia, and Archaea) have excellent binding properties, low off-target toxicity, and high stability and these properties make them a promising molecule for the development of new therapeutics pharmacologically active in many clinical searches. Table 4 shows the chemical structure and source of various NRPs isolated from marine sponges and tunicates.
|NRPs agents||Chemical class||Origin||Target|
|Miraziridine A||Linear pentapeptide||Cancer/inhibit protease cathepsin B|
|Haligramides A-B||Cyclic hexapeptides||Cancer/A-549 (lung), HCT-15 (colon), SF-539 (CNS), SNB-19 (CNS)|
|Prepatellamide A||Cyclic peptide||Cancer/P388 murine leukemia cell lines|
|Tamandarins A-B||Depsipeptides||Cancer/pancreatic carcinoma BX-PC3, prostatic cancer DU-145, head and neck carcinoma UMSCC10b|
|Microsclerodermins F–I||Cyclic peptides||Cancer/HCT-116 cell line|
|Wainunuamide||Cyclic hexapeptide||Cancer/A2780 ovarian, K562 leukemia cancer cells|
|Leucamide A||Cyclic hexapeptide||Cancer/Tumor cell lines HM02, HepG2, Huh7|
|Axinellin C||Cyclic octapeptide||Cancer/A2780 ovarian, K562 leukemia cancer cells|
|Milnamide D||Linearpeptide||Cancer/HCT-116 cells|
|Kapakahines E–G||—||Cancer/P388 murine leukemia cells|
|Didmolamides A-B||Cyclic hexapeptides||Cancer Tumor cell lines (A549, HT29 and MEL28)|
|Bistratamides E–J||Cyclic hexapeptides||Cancer/Human colon tumor (HCT-116) cell line|
|Milnamide C||—||Cancer/MDA-MB-435cancer cells|
|Scleritodermin A||Cyclic peptide||Cancer|
|Microcionamids A-B||—||Cancer/Human breast tumor cell lines MCF-7 and SKBR-3|
|Kendarimide A||Linear peptide||Cancer/KB-C2 cells|
|Phakellistatin 14||Cyclo heptapeptide||Cancer/Murine lymphocytic leukemia P388 cell line|
|Polytheonamides A-B||Polypeptides||Cancer/P388 murine leukemia cells|
|Neopetrosiamides A-B||Tricyclic peptides||Cancer|
|Theopapuamide||Cyclic depsipeptide||Cancer/CEM-TART, HCT-116 cell lines|
|Azumamide A-E||Cyclo tetrapeptides||Cancer|
|Callyaerin G||Cyclic peptide||Cancer/Mouselymphoma cell line (L5178Y) and HeLa cells|
|Stylopeptide 2||Cyclo decapeptide||Cancer/BT-549 and HS578T breast cancer cell lines|
|Ciliatamides A-C||Lipopeptides||Cancer/HeLa cells|
|Diazonamides C–E||Macrocyclic peptides||Cancer/Human tumor cell lines (A549, HT29, MDA-MB231)|
|Rolloamide A-B||Cyclic heptapeptides||Cancer|
|Callyaerin A–F and H||Cyclic peptides||Cancer/L5178Y cell line|
|Papuamides E-F||Depsipeptides||Cancer/Brine shrimp|
|Stylissamide X||Octapeptide||Cancer/HeLa cells|
|Gombamide A||Hexapeptide||Cancer/K562 and A549 cell lines|
|Neamphamide A||Cyclic depsipeptide||HIV|
|Mirabamides A-D||Cyclic depsipeptide||HIV|
|Homophymine A||Cyclodepsipeptide||HIV/PBMC cell line|
|Celebeside A-C||Depsipeptides||HIV/Colon carcinoma (HCT-116) cells|
|Theopapuamides B–D, Mutremdamide A, Koshikamides C-H||Cyclic depsipeptide||HIV|
|Halipeptin A-B||Cyclic depsipeptide||Inflammation|
|Solomonamide A- B||Cyclic peptide||Inflammation|
|Stylissatin A||Cyclic peptide||Murine macrophage RAW264.7|
|Citronamides A- B||—|
In the NCBI database, there are currently about 1.164 distinct non-ribosomal peptides that form over 500 different monomers including both proteinogenic and non-proteinogenic L- and D-amino acids, as well as amines and carboxylic acids. These complex secondary metabolites’ linear, cyclic, branching, or other complicated primary structures are frequently altered to enhance clinical qualities and/or bypass resistance mechanisms. Indeed, the nucleotide sequence modification of a native NRPS gene or mixing modules from multiple NRPSs makes them more efficient with pharmacological properties. Several bioengineering and molecular techniques have been developed during the last few decades to produce modified NRPs with improved physicochemical characteristics and bioactivity .
In this chapter, we discussed the significance, synthesis, and application areas of NRPs-based agents, which have received a lot of interest as a new source of pharmaceutical agents. NRPs with unique chemical structures and diverse biological actions, such as antibacterials (penicillin, vancomycin), anticancer compounds (bleomycin), and immunosuppressants (cyclosporine), have been researched as novel compounds for new drug discovery and development throughout the last several decades.
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