Nine Post-translational Modifications during the Biosynthesis of Cinnamycin

Ökesli, Ayşe; Cooper, Lisa E.; Fogle, Emily J.; Donk, Wilfred A. van der

doi:10.1021/ja205783f

Cited by 101 publications

(140 citation statements)

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“…In 2003, the cinnamycin biosynthetic gene cluster from Streptomyces cinnamoneus DSM 40005 was reported and used to produce cinnamycin (6). More recently, we characterized the biochemical processes involved in cinnamycin biosynthesis both in E. coli and in vitro (25). Although cinnamycin and duramycin differ by only a single amino acid (Fig.…”

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confidence: 99%

Insights into the Biosynthesis of Duramycin

Huo

Ökesli

Zhao

et al. 2017

Appl Environ Microbiol

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Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that are characterized by the thioether cross-linked bisamino acids lanthionine (Lan) and methyllanthionine (MeLan). Duramycin contains 19 amino acids, including one Lan and two MeLans, an unusual lysinoalanine (Lal) bridge formed from the -amino group of lysine 19 and a serine residue at position 6, and an erythro-3-hydroxy-L-aspartic acid at position 15. These modifications are important for the interactions of duramycin with its biological target, phosphatidylethanolamine (PE). Based on the binding affinity and specificity for PE, duramycin has been investigated as a potential therapeutic, as a molecular probe to investigate the role and localization of PE in biological systems, and to block viral entry into mammalian cells. In this study, we identified the duramycin biosynthetic gene cluster by genome sequencing of Streptomyces cinnamoneus ATCC 12686 and investigated the dur biosynthetic machinery by heterologous expression in Escherichia coli. In addition, the analog duramycin C, containing six amino acid changes compared to duramycin, was successfully generated in E. coli. The substrate recognition motif of DurX, an ␣-ketoglutarate/iron(II)-dependent hydroxylase that carries out the hydroxylation of aspartate 15 of the precursor peptide DurA, was also investigated using mutagenesis of the DurA peptide. Both in vivo and in vitro results demonstrated that Gly16 is important for DurX activity.IMPORTANCE Duramycin is a natural product produced by certain bacteria that binds to phosphatidylethanolamine (PE). Because PE is involved in many cellular processes, duramycin is an antibiotic that kills bacteria, but it has also been used as a molecular probe to detect PE and monitor its localization in mammalian cells and even whole organisms, and it was recently shown to display broad-spectrum inhibition of viral entry into host cells. In addition, the molecule has been evaluated as treatment for cystic fibrosis. We report here the genes that are involved in duramycin biosynthesis, and we produced duramycin by expressing those genes in Escherichia coli. We show that duramycin analogs can also be produced. The ability to access duramycin and analogs by production in E. coli opens opportunities to improve duramycin as an antibiotic, PE probe, antiviral, or cystic fibrosis therapeutic.KEYWORDS antibiotic, antiviral agents, biosynthesis, duramycin, phosphatidylethanolamine L antibiotics are ribosomally synthesized and posttranslationally modified peptides (RiPPs) exhibiting antimicrobial activity. They are characterized by the thioether cross-linked amino acids lanthionine (Lan) and/or methyllanthionine (MeLan) (1), which are formed by dehydration of Ser and Thr residues, followed by intramolecular Michaeltype addition of Cys thiols to the resulting dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively (2-4) (Fig. 1a). These posttranslational modifications take place in the core peptide region of the precursor...

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Insights into the Biosynthesis of Duramycin

Huo

Ökesli

Zhao

et al. 2017

Appl Environ Microbiol

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“…This phylum has been the source and/or inspiration for the majority of pharmaceutically useful natural products (24,25). Although Actinobacteria constitute only a small proportion of known lanthipeptide producers, they encode a diverse set of posttranslational modifications that have not been observed for lanthipeptides from other phyla (2) (e.g., lysinoalanine formation in cinnamycin [26], tryptophan chlorination and proline dihydroxylation in microbisporicin [27,28, also called NAI-107 [29], and glycosylation in NAI-112 [30]). The unique modifications in actinobacterial lanthipeptide biosynthesis suggest great potential for exploring unknown lanthipeptide chemical space in this phylum.…”

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confidence: 99%

Expanded Natural Product Diversity Revealed by Analysis of Lanthipeptide-Like Gene Clusters in Actinobacteria

Zhang

Doroghazi

Zhao

et al. 2015

Appl Environ Microbiol

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Lanthionine-containing peptides (lanthipeptides) are a rapidly growing family of polycyclic peptide natural products belonging to the large class of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Lanthipeptides are widely distributed in taxonomically distant species, and their currently known biosynthetic systems and biological activities are diverse. Building on the recent natural product gene cluster family (GCF) project, we report here large-scale analysis of lanthipeptidelike biosynthetic gene clusters from Actinobacteria. Our analysis suggests that lanthipeptide biosynthetic pathways, and by extrapolation the natural products themselves, are much more diverse than currently appreciated and contain many different posttranslational modifications. Furthermore, lanthionine synthetases are much more diverse in sequence and domain topology than currently characterized systems, and they are used by the biosynthetic machineries for natural products other than lanthipeptides. The gene cluster families described here significantly expand the chemical diversity and biosynthetic repertoire of lanthionine-related natural products. Biosynthesis of these novel natural products likely involves unusual and unprecedented biochemistries, as illustrated by several examples discussed in this study. In addition, class IV lanthipeptide gene clusters are shown not to be silent, setting the stage to investigate their biological activities. Lanthipeptides are a rapidly growing family of polycyclic peptides characterized by the presence of the thioether crosslinked amino acids lanthionine and methyllanthionine (1-5). These compounds are widely distributed in taxonomically distant species and display very diverse biological activities, ranging from antimicrobial to antiallodynic (5-7). Antibacterial lanthipeptides, such as the commercially used food preservative nisin, are termed lantibiotics (8). Lanthipeptides are generated from a ribosomally synthesized linear precursor peptide (generically termed LanA) and therefore belong to the large class of natural products that are ribosomally synthesized and posttranslationally modified peptides (RiPPs) (9). The precursor peptide LanA consists of a C-terminal core peptide where all posttranslational modifications take place and an N-terminal leader peptide that is important for posttranslational modifications and that is subsequently removed by proteolysis (10, 11). The installation of the (methyl)lanthionine thioether bridges is achieved by the initial dehydration of Ser and Thr residues in the precursor peptides, followed by stereoselective intramolecular Michael-type addition of Cys thiols to the newly formed dehydroamino acids (Fig. 1A).Four classes of biosynthetic enzymes are known to catalyze lanthionine formation (2, 12) (Fig. 1B). Class I lanthionine synthetases consist of a dehydratase and a cyclase that are generically termed LanB and LanC, respectively (8). Class II enzymes are generically named LanMs, which are single polypeptides containing an N-termi...

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“…The div cluster is comprised of genes putatively encoding five biosynthetic proteins, an export pump, and two genes of unknown function (Supplementary Table 1). In addition to DivA and DivM, these include an α-ketoglutarate-dependent Fe(II) monooxygenase/β-Asp hydroxylase (DivX; ARD09204), a homolog of DurN (WP_071962205.1) shown to be involved in lysinoalanine formation 15–17 (DivN; ARD09209), a SAM-dependent methyltransferase (DivMT; ARD09206), and an ABC-type transporter (DivT; ARD09207).…”

Section: Resultsmentioning

confidence: 99%

“…17 In that event, a simple chemical method was developed wherein the lysinoalanine residue could be robustly produced by treatment with base. We followed this procedure with compounds 5–8 , leading to efficient production of the lysinoalanine residue (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, analysis of this contig showed unique coding sequences, such as a putative methyltransferase. Based on the minimum genes required for in vitro 4 and duramycin biosynthesis, 15,17 a second contig with a gene sharing 44% identity to durN , a protein shown to be involved in lysinoalanine formation, was included in the cluster for 1 . Extensions of the contigs using matching reads were then optimized to produce a single contig 17 kbp in length containing the entire cluster (12 kbp from precursor gene to the durN homolog, divN ).…”

Section: Methodsmentioning

confidence: 99%

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Accessing chemical diversity from the uncultivated symbionts of small marine animals

et al. 2018

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Chemistry drives many biological interactions between the microbiota and host animals, yet it is often challenging to identify the chemicals involved. This poses a problem, as such small molecules are excellent sources of potential pharmaceuticals, pretested by nature for animal compatibility. We discovered anti-HIV compounds from small, marine tunicates from the Eastern Fields of Papua New Guinea. Tunicates are a reservoir for novel bioactive chemicals, yet their small size often impedes identification or even detection of the chemicals within. We solved this problem by combining chemistry, metagenomics, and synthetic biology to directly identify and synthesize the natural products. We show that these anti-HIV compounds, the divamides, are a novel family of lanthipeptides produced by symbiotic bacteria living in the tunicate. Neighboring animal colonies contain structurally related divamides that differ starkly in their biological properties, suggesting a role for biosynthetic plasticity in a native context where biological interactions take place.

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Nine Post-translational Modifications during the Biosynthesis of Cinnamycin

Cited by 101 publications

References 48 publications

Insights into the Biosynthesis of Duramycin

Insights into the Biosynthesis of Duramycin

Expanded Natural Product Diversity Revealed by Analysis of Lanthipeptide-Like Gene Clusters in Actinobacteria

Accessing chemical diversity from the uncultivated symbionts of small marine animals

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