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Substrate-assisted enzymatic formation of lysinoalanine in duramycin.

ABSTRACT: Duramycin is a heavily post-translationally modified peptide that binds phosphatidylethanolamine. It has been investigated as an antibiotic, an inhibitor of viral entry, a therapeutic for cystic fibrosis, and a tumor and vasculature imaging agent. Duramycin contains a ?-hydroxylated Asp (Hya) and four macrocycles, including an essential lysinoalanine (Lal) cross-link. The mechanism of Lal formation is not known. Here we show that Lal is installed stereospecifically by DurN via addition of Lys19 to a dehydroalanine. The structure of DurN reveals an unusual dimer with a new fold. Surprisingly, in the structure of duramycin bound to DurN, no residues of the enzyme are near the Lal cross-link. Instead, Hya15 of the substrate makes interactions with Lal, suggesting it acts as a base to deprotonate Lys19 during catalysis. Biochemical data suggest that DurN preorganizes the reactive conformation of the substrate, such that the Hya15 of the substrate can serve as the catalytic base for Lal formation.

SUBMITTER: An L

PROVIDER: S-EPMC6372306 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Substrate-assisted enzymatic formation of lysinoalanine in duramycin.

An Linna L Cogan Dillon P DP Navo Claudio D CD Jiménez-Osés Gonzalo G Nair Satish K SK van der Donk Wilfred A WA

Nature chemical biology 20180903 10

Duramycin is a heavily post-translationally modified peptide that binds phosphatidylethanolamine. It has been investigated as an antibiotic, an inhibitor of viral entry, a therapeutic for cystic fibrosis, and a tumor and vasculature imaging agent. Duramycin contains a β-hydroxylated Asp (Hya) and four macrocycles, including an essential lysinoalanine (Lal) cross-link. The mechanism of Lal formation is not known. Here we show that Lal is installed stereospecifically by DurN via addition of Lys19 ...[more]

PMID: 30177849

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Project description: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.ImportanceDuramycin 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.

Dataset Information

Substrate-assisted enzymatic formation of lysinoalanine in duramycin.

Publications

Substrate-assisted enzymatic formation of lysinoalanine in duramycin.

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