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

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 ß-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 Cß 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.

Semi-enzymatic synthesis of pseudouridine.

Modifications of RNA molecules have a significant effect on their structure and function. One of the most common modifications is the isomerization from uridine to pseudouridine. Despite its prevalence in natural RNA sequences, organic synthesis of pseudouridine has been challenging because of the stereochemistry requirement and the sensitivity of reaction steps to moisture. Herein, a semi-enzymatic synthetic route is developed for the synthesis of pseudouridine using adenosine 5'-monophosphate and uracil as the starting materials and a reverse reaction catalyzed by the pseudouridine monophosphate glycosidase. This synthetic route has only three steps and the overall yield of ß-pseudouridine production was 68.4%.

DNAzymes for amine and peptide lysine acylation.

DNAzymes were previously identified by in vitro selection for a variety of chemical reactions, including several biologically relevant peptide modifications. However, finding DNAzymes for peptide lysine acylation is a substantial challenge. By using suitably reactive aryl ester acyl donors as the electrophiles, here we used in vitro selection to identify DNAzymes that acylate amines, including lysine side chains of DNA-anchored peptides. Some of the DNAzymes can transfer a small glutaryl group to an amino group. These results expand the scope of DNAzyme catalysis and suggest the future broader applicability of DNAzymes for sequence-selective lysine acylation of peptide and protein substrates.

Benzylic Radical Stabilization Permits Ether Formation During Darobactin Biosynthesis.

The Gram-negative selective antibiotic darobactin A has attracted interest owing to its intriguing fused bicyclic structure and unique mode of action. Biosynthetic studies have revealed that darobactin is a ribosomally synthesized and post-translationally modified peptide (RiPP). During maturation, the darobactin precursor peptide (DarA) is modified by a radical S-adenosyl methionine (rSAM)-dependent enzyme (DarE) to contain ether and C-C crosslinks. In this work, we describe the enzymatic tolerance of DarE using a panel of DarA variants, revealing that DarE can install the ether and C-C crosslinks independently and in different locations on DarA. These efforts produced 57 darobactin variants, 50 of which were enzymatically modified. Several new variants with fused bicyclic structures were characterized, including darobactin W3Y, which replaces tryptophan with tyrosine at the twice-modified central position, and darobactin K5F, which displays a fused diether ring pattern. Three additional darobactin variants contained fused diether macrocycles, leading us to investigate the origin of ether versus C-C crosslink formation. Computational analyses found that more stable and long-lived Cß radicals found on aromatic amino acids correlated with ether formation. Further, molecular docking and calculated transition state structures provide support for the different indole connectivity observed for ether (Trp-C7) and C-C (Trp-C6) crosslink formation. We also provide experimental evidence for a ß-oxotryptophan modification, a proposed intermediate during ether crosslink formation. Finally, mutational analysis of the DarA leader region and protein structural predictions identified which residues were dispensable for processing and others that govern substrate engagement by DarE. Our work informs on darobactin scaffold engineering and sheds additional light on the underlying principles of rSAM catalysis.