Darobactin Substrate Engineering and Computation Show Radical Stability Governs Ether versus C–C Bond Formation

Abstract

The Gram-negative selective antibiotic darobactin A has attracted interest owing to its intriguing fused bicyclic structure and unique targeting of the outer membrane protein BamA. Darobactin, a ribosomally synthesized and post-translationally modified peptide (RiPP), is produced by a radical S-adenosyl methionine (rSAM)-dependent enzyme (DarE) and contains one ether and one C–C cross-link. Herein, we analyze the substrate tolerance of DarE and describe an underlying catalytic principle of the enzyme. These efforts produced 51 enzymatically modified darobactin variants, revealing that DarE can install the ether and C–C cross-links independently and in different locations on the substrate. Notable variants with fused bicyclic structures were characterized, including darobactin W3Y, with a non-Trp residue at the twice-modified central position, and darobactin K5F, which displays a fused diether ring pattern. While lacking antibiotic activity, quantum mechanical modeling of darobactins W3Y and K5F aided in the elucidation of the requisite features for high-affinity BamA engagement. We also provide experimental evidence for β-oxo modification, which adds support for a proposed DarE mechanism. Based on these results, ether and C–C cross-link formation was investigated computationally, and it was determined that more stable and longer-lived aromatic Cβ radicals correlated with ether formation. Further, molecular docking and transition state structures based on high-level quantum mechanical calculations support the different indole connectivity observed for ether (Trp-C7) and C–C (Trp-C6) cross-links. Finally, mutational analysis and protein structural predictions identified substrate residues that govern engagement to DarE. Our work informs on darobactin scaffold engineering and further unveils the underlying principles of rSAM catalysis.

Exploring the Darobactin Class of Antibiotics: A Comprehensive Review from Discovery to Recent Advancements

“…Further, if the third amino acid is serine (Ser) (Ω1-X2-S3), serine −OH is oxidized to aldehyde. This further supports the hypothesis proposed by Woodard et al that the radical stability of the intermediates drives the C–C cross-linking . Later, Ma et al identified several divergent darobactins, including photorhaptin A (Figure B) …”

“…The two proposed mechanisms differ in the position of installation of the initial hydroxy radical. Pathway A is further supported by Woodard et al…”

“…An interesting question arises here: Why does the ether bond formation precede the C–C cross-linking? Woodard et al through computational analysis supported by biosynthetic experiments, proposed that the stability of the radical is crucial in cross-linking . As the radicals in the Cβ-position of the aromatic amino acids in daropeptides are stable and are long-lived compared to the Cβ-alkyl radicals, a chemoselective radical formation takes place at the aryl Cβ-position.…”