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, inhibitor of viral entry, therapeutic for cystic fibrosis, and tumor and vasculature imaging agent. Duramycin contains a β-hydroxylated Asp (Hya) and four macrocycles, including an essential lysinoalanine (Lal) crosslink. The mechanism of Lal formation is not known. We here 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. 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. Duramycin and closely related compounds are ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by various actinomycetes 1-5 and marine symbionts 6 . Duramycin binds phosphatidylethanolamine (PE), a major structural phospholipid in mammalian and microbial cell membranes 7, 8 . Duramycin contains 19 amino acids, and five final posttranslational modifications (PTMs), including one lanthionine (Lan), two methyllanthionines (MeLan), an erythro-3-hydroxy-L-aspartic acid (Hya) resulting from the hydroxylation of Asp15, and one (2S,9S)-Lal (Figure 1). The carboxylate and hydroxyl groups of Hya15 make key interactions with the ethanolamine head group of PE, providing

Genomic analysis of siderophore β-hydroxylases reveals divergent stereocontrol and expands the condensation domain family

“…Several families of enzymes are responsible for the β-hydroxy amino acids found in nonribosomal peptides, namely Fe-heme monooxygenases (18), diiron monooxygenases (19), and nonheme Fe(II)/α-ketoglutarate-dependent dioxygenases (20). Only this last class of Fe NH /αKG dioxygenases have been found to hydroxylate aspartic acid (20)(21)(22). OrbG, encoded in the biosynthetic gene cluster of the siderophore ornibactin (SI Appendix, Fig.…”

Structure and chemistry of lysinoalanine crosslinking in the spirochaete flagella hook

“…20,21 For Lal formation within the lanthipeptide duramycin by DurN, an additional enzyme DurX generates an essential cofactor -erythro-3-hydroxy-L-aspartic acid (Hya) -to assist in Lal formation. 23 Hya acts as a general base to deprotonate the Lal precursor lysine ε-amine for nucleophilic attack on DHA, which is previously formed via serine phosphorylation and elimination. 23 In contrast to Lal formation in duramycin, a very different biochemical mechanism produces Lal in the spirochaete flagellar hook.…”

“…23 Hya acts as a general base to deprotonate the Lal precursor lysine ε-amine for nucleophilic attack on DHA, which is previously formed via serine phosphorylation and elimination. 23 In contrast to Lal formation in duramycin, a very different biochemical mechanism produces Lal in the spirochaete flagellar hook. Through our structural and biochemical experiments, we show that an active center composed from the FlgE subunit interface is sufficient to selfcatalyze formation of the unusual Lal crosslink, and does so without the need for additional substrates, enzymes, or cofactors.…”

“…21,22 Recently, isolation and characterization of the duramycin biosynthetic gene cluster from Streptomyces cinnamoneus revealed that as many as three different enzymes are required for Lal biosynthesis, with the terminal enzyme DurN catalyzing a substrate-assisted addition in order to form Lal. 21,23 In contrast to Lal biogenesis in lanthipeptides, Lal biosynthesis by spirocheate FlgE is autocatalytic and requires no other enzymes or cofactors. 7 In the oral pathogen Treponema denticola (Td), FlgE is a 50 kDa protein composed of four distinct domains: D0, Dc, D1, and D2 (Fig.…”