Biology subjects

Dommaraju, S. R.

Publications and source records attributed to Dommaraju, S. R..

6 recordsLinked to original sources

bioRxiv2025

Biosynthesis of peptidic thiooxazole metallophores installed by multinuclear nonheme iron enzymes.

Significant effort has been directed toward characterization of nonheme iron enzymes owing to their breadth of unique reactivity. Through genome mining, we identified a conserved biosynthetic gene cluster within Pseudomonadota encoding one such family, the multinuclear nonheme iron-dependent oxidative enzymes (MNIO, formerly DUF692). Using a representative gene cluster from Fontimonas thermophila, we heterologously produced the post-translationally modified peptide fontiphorin, and detailed spectral analysis revealed MNIO-catalyzed installation of seven 5-thiooxazole (5TO) moieties. During our work, additional MNIO products were reported with conflicting structural assignments, so we investigated the related biosynthetic gene clusters from Haemophilus influenzae and Neisseria gonorrhoeae. Using alkylation-assisted HMBC correlations, we demonstrated that these products also contain 5TO resulting in a revision of the structure of oxazolin. We further provide evidence supporting a role for 5TO-containing peptides in copper detoxification and recommended this emerging class of Cu-associated peptidic thiooxazole metallophores be referred to as captophorins. To further explore the captophorins, we reconstituted fontiphorin biosynthesis in vitro and investigated its enzymatic requirements. Using cell-free production of single-site, double-site, and naturally occurring variants, we examined enzyme-substrate interactions to determine key sites governing catalysis by 5TO-forming MNIOs. Through our detailed spectroscopic approach for 5TO assignment and investigation of enzyme-substrate interactions, our work unifies tens of thousands of MNIOs in the biosynthesis of captophorins.

biochemistry↗

bioRxiv2025

A versatile enzymatic pathway for modification of peptide C-termini

Advances in bioinformatics have enabled the discovery of unique enzymatic reactions, particularly for ribosomally synthesized and post-translationally modified peptides (RiPPs). The recently discovered daptides, peptides with their C-terminus replaced by an amine, represent one such case, but the diversity, requirements, and engineering potential of daptide biosynthesis remain to be established. Using the daptide biosynthetic gene clusters from Thermobifida fusca and Streptomyces azureus, we reconstituted daptide biosynthesis in vitro, revealing the enzymatic requirements for successive oxidative decarboxylation, transamination, and N,N-dimethylation. In vitro and in vivo studies showed a tailoring family of YcaO enzymes convert a secondary amine intermediate to a C-terminal imidazoline. We further demonstrated enzymatic activity toward shortened, leader peptide-free, and non-native core peptides, highlighting a broad substrate tolerance. Using these insights, we directed the daptide pathway to install new C-termini, including a bioconjugation-compatible aminoacetone, on various peptide and protein substrates.

biochemistry↗

bioRxiv2025

Lantibiotic-producing bacteria impact microbiome resilience and colonization resistance

A subset of commensal bacterial strains secrete bacteriocins, such as lantibiotics, to establish and protect their niche in the gut. Because the antimicrobial spectrum of lantibiotics includes opportunistic pathogens, such as vancomycin-resistant Enterococcus faecium (VRE), they may provide an approach to reduce antibiotic-resistant infections. The impact of lantibiotic-producing bacteria on the complex microbial populations constituting the microbiome, however, remains poorly defined. We find that genes encoding lanthipeptides, including lantibiotics, are commonly present in the microbiomes of healthy humans and in dysbiotic microbiomes of hospitalized patients. In fecal samples collected from hospitalized patients, bacterial species encoding lantibiotic genes are present in greater abundance than lantibiotic-deficient strains of the same species. We demonstrate that the lantibiotic-producing bacterium, Blautia pseudococcoides SCSK, prevents intestinal recolonization of mice by a wide range of commensal species following antibiotic-induced dysbiosis and markedly reduces fecal concentrations of microbiota-derived metabolites associated with mucosal immune defenses. Lantibiotic-mediated dysbiosis results in sustained loss of colonization resistance against Klebsiella pneumoniae and Clostrioides difficile infection. Our findings reveal the potential impact of lantibiotic-producing bacterial species on microbiome resilience and susceptibility to infection following antibiotic treatment.

microbiology↗

bioRxiv2024

Computationally guided exploration of borosin biosynthetic strategies

Borosins are ribosomally synthesized and post-translationally modified peptides containing backbone -N-methylations. Identification of borosin precursor peptides is difficult because (1) there are no conserved sequence elements among borosin precursor peptides and (2) the biosynthetic gene clusters contain numerous domain architectures and peptide fusions. To tackle this problem, we updated the genome mining tool RODEO to automatically evaluate putative borosin BGCs and identify precursor peptides. Enabled by the new borosin module, we analyzed all borosin BGCs found in available sequence data and assigned precursor peptides to previously orphan borosin methyltransferases. Additionally, we bioinformatically predict and experimentally characterize a new fused borosin domain architecture, in which the modified core is N-terminal to the methyltransferase domain. Finally, we demonstrate that a borosin precursor peptide is the native substrate of shewasin A, a previously characterized pepsin-like aspartic peptidase whose native biological function was unknown.

bioinformatics↗

bioRxiv2023

Non-modular Fatty Acid Synthases Yield Unique Acylation in Ribosomal Peptides

Recent efforts in genome mining of ribosomally synthesized and post-translationally modified peptides (RiPPs) have expanded the diversity of post-translational modification chemistries1, 2. However, RiPPs are rarely reported as hybrid molecules incorporating biosynthetic machineries from other natural product families3-8. Here, we report lipoavitides, a class of RiPP/fatty acid hybrid lipopeptides that display a unique, membrane-targeting 4-hydroxy-2,4-dimethylpentanoyl (HMP)-modified N-terminus. The HMP is formed via condensation of isobutyryl-CoA and methylmalonyl-CoA catalyzed by a 3-ketoacyl-ACP synthase III enzyme, followed by successive tailoring reactions in the fatty acid biosynthetic pathway. The HMP and RiPP substructures are then connected by an acyltransferase exhibiting promiscuous activity towards the fatty acyl and RiPP substrates. Overall, the discovery of lipoavitides contributes a prototype of RiPP/fatty acid hybrids and provides possible enzymatic tools for lipopeptide bioengineering.

biochemistry↗

bioRxiv2023

Genome mining unveils a class of ribosomal peptides with two amino termini

The era of inexpensive genome sequencing and improved bioinformatics tools has reenergized the study of natural products, including the ribosomally synthesized and post-translationally modified peptides (RiPPs). In recent years, RiPP discovery has challenged preconceptions about the scope of post-translational modification chemistry, but genome mining of new RiPP classes remains an unsolved challenge. Here, we report a RiPP class defined by an unusual (S)-N2,N2-dimethyl-1,2-propanediamine (Dmp)-modified C-terminus, which we term the daptides. Nearly 500 daptide biosynthetic gene clusters (BGCs) were identified by analyzing the RiPP Recognition Element (RRE), a common substrate-binding domain found in half of prokaryotic RiPP classes. A representative daptide BGC from Microbacterium paraoxydans DSM 15019 was selected for experimental characterization. Derived from a C-terminal threonine residue, the class-defining Dmp is installed over three steps by an oxidative decarboxylase, aminotransferase, and methyltransferase. Daptides uniquely harbor two positively charged termini, and thus we suspect this modification could aid in membrane targeting, as corroborated by hemolysis assays. Our studies further show that the oxidative decarboxylation step requires a functionally unannotated accessory protein. Fused to the C-terminus of the accessory protein is an RRE domain, which delivers the unmodified substrate peptide to the oxidative decarboxylase. This discovery of a class-defining post-translational modification in RiPPs may serve as a prototype for unveiling additional RiPP classes through genome mining.

biochemistry↗