Radical SAM-dependent ether crosslink in daropeptide biosynthesis

Abstract

Darobactin is a ribosomally synthesized and post-translationally modified peptide (RiPP), which possesses potent activity against various Gram-negative bacteria. Darobactin features a highly unique bicyclic scaffold, consisting of an ether crosslink between two Trp residues and a C–C crosslink between a Lys and a Trp. Here we report in vivo and in vitro activity of darobactin synthase DarE. We show DarE is a radical S-adenosylmethionine (rSAM) enzyme and is solely responsible for forming the bicyclic scaffold of darobactin. DarE mainly produced the ether-crosslinked product in vitro, and when the assay was performed in H218O, apparent 18O incorporation was observed into the ether-crosslinked product. These observations suggested an rSAM-dependent process in darobactin biosynthesis, involving a highly unusual oxygen insertion step from a water molecule and subsequent O–H and C–H activations. Genome mining analysis demonstrates the diversity of darobactin-like biosynthetic gene clusters, a subclade of which likely encode monocyclic products with only an ether linkage. We propose the name daropeptide for this growing family of ether-containing RiPPs produced by DarE enzymes.

Activity and cryo-EM structure guided biosynthetic pathway engineering yields non-natural Darobactin antibiotics with superior activity against Gram-negative pathogens

“…A new, fast, and less expensive core peptide modification strategy, based on the overlap-extension polymerase chain reaction (OE-PCR) method facilitated the generation of fifteen new darA gene variants, leading to promising derivatives with improved bioactivity and altered core sequences compared to Darobactin A, while conserving both tryptophans critical for bicyclization. [20] Most notably D22 shows significantly improved MIC values against critical pathogens, such as clinical isolates of carbapenem-resistant A. baumannii (up to 32-fold) and P. aeruginosa (up to 4-fold). Single-particle cryo-EM structure elucidation explained the binding behaviour of the most active new derivative compared to established Darobactin A and D9.…”

“…One encodes the precursor peptide DarA and the other encodes the radical S-adenosylmethionine (rSAM) enzyme DarE, catalyzing the bicyclic crosslinking. [20] Heterologous production rates of up to 25 mg/L in case of Darobactin A and 3 mg/L for the more active Darobactin 9 (D9), generated via biosynthetic pathway engineering, open the door for efficient large-scale production. [19] While the cryo-EM structure of Darobactin A in complex with BamA already exists, [15] no structure-activity relationship (SAR) rationale has been reported.…”

Expanded sequence space of radical S-adenosylmethionine-dependent enzyme involved in post-translational macrocyclization

“…The aromatic sidechain is another highly reactive group that can be modified to form structurally complex, highly strained macrocycles [3a, 7] . Tandem C(sp 2 )-C(sp 3 ) bonds [7] , biaryl [8] or C-O coupling can be installed by one single enzyme, exemplified by tryptorubin A [9] , cittilins [10] , darobactin [11] and Xenorceptide [7] . Side chain amino and carboxyl groups tend to undergo condensation to form iso-amide bonds or ester bonds, as observed in lasso peptide [12] and omega-ester containing peptides (OEPs) [13] .…”

“…Enzymes in this family generate a highly reactive 5'-deoxyadenosyl radical to abstract hydrogen of inert sp 3 carbon to activate substrate precursor peptide, and this activated carbon can fuse to cysteine thiol or aromatic ring to give a C-S or C-C bond [2, 6a, 7, 17] . One rSAM enzyme can install multiple C-S or C-C bonds or even oxygen insertion for peptide macrocyclization, as disclosed in the biosynthesis of sactipeptides and darobactin [11] .…”

Atroposelective Total Synthesis of Darobactin A

“…Presumably, these bonds are formed in Nature via post-translational macrocyclizations of a linear peptide. 1,14 These unusual features form the basis of a fused macrocylic bicycle (14-and 15-membered rings) containing atropisomeric indoles. [15][16] A fully synthetic route to darobactin A would not only increase the available supply of material for further study, but also provide a framework for medicinal chemistry explorations.…”