Miriam, and Dillon, collaborators from the Mitchell and Nair labs published an article in mSystems describing a structure-based approach to improve the discovery and characterization of RiPP Recognition Elements (RREs), the conserved peptide-binding domains that direct ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthesis. By combining Foldseek structural searches, Hidden Markov Model refinement, and AlphaFold 3 modeling, they identified thousands of previously unrecognized RREs, expanded the RRE-Finder tool, and mapped 13 distinct precursor peptide recognition motifs across diverse RiPP classes. Experimental binding studies validated AlphaFold predictions and demonstrated the utility of the approach for identifying RRE–peptide interactions. This work substantially expands the known RRE landscape, improves genome mining of RiPP biosynthetic pathways, and provides new tools for discovering and engineering natural products.
Andrew and Yanqing, along with past and present members of the lab published an article in ACS Biochemistry identifying the activity of E. coli’s YcaO enzyme: thioamidation of ribosomal protein uL16. Through a proteome-wide AlphaFold3 analysis, they determined that uL16 is the only protein to interact with YcaO in a catalytically competent and confident manner. Genetic deletion, overexpression, in vitro reconstitution, and binding studies all support the observed function, and bioinformatics analyses highlight the widespread frequency of this modification in Pseudomonadota. Functional assessment identified a complex relationship with an adjacent ribosomal modification on uL16. This work elucidates the function of a long-misannotated enzyme and paves the way for both mechanistic characterization of sulfur relay and further understanding the effect(s) of this modification.
Riley and collaborators from the Mitchell lab published an article in ACS Chemical Biology about the discovery and characterization of several examples of partner protein-dependent graspetide biosynthesis. The graspetide RODEO module was updated to more robustly identify diverse graspetides and used to survey the protein co-occurrence for the >20,000 newly predicted graspetides. From this, two new graspetides with unusual co-occurring proteins were chosen to be investigated. The structure and biosynthetic requirements for these graspetides were determined using both in vivo and in vitro systems. In each case, the graspetide synthetase was found to require the co-occurring partner protein to produce graspetide linkages. Additionally, one of the graspetides contained several instances of an unusual, previously unseen 5-hydroxyisopeptide moiety.
Mayuresh and Miriam along with several former lab members (Shravan, Alex, and Xiaopeng) published an article in ACS Chemical Biology exploring a widespread group of multinuclear nonheme iron-dependent oxidative (MNIO) enzymes that modify peptide substrates. Using multiple analytical methods, we found that the largest MNIO family installs 5-thiooxazole moieties at peptidic Cys residues. These products appear to function in copper detoxification, leading us to name them captophorins (Cu-associated peptidic thiooxazole metallophores). After reconstituting MNIO activity in vitro, cell-free assays enabled clearer definitions of patterns in substrate engagement and turnover, allowing us to propose key substrate–enzyme contact points that explain how captophorin diversity is accommodated by MNIO sequence variation.
Shravan and collaborators from the Pamer lab have published an article in Cell Host & Microbe outlining the relationships between lanthipeptide-producing gut microbes and their effects on microbiome structure. Metagenomic data was collected and partially assembled from more than 1,000 clinical fecal samples. Using RODEO, Shravan searched these metagenomes for lanthipeptide biosynthetic gene clusters, and collaborators used this information to draw correlations between lanthipeptide production and microbiome colonization after antibiotic treatment. In vivo mouse studies further showed that lanthipeptide-producing microbes, like Blautia pseudococcoides SCSK, could prevent recovery of microbiome composition and metabolites. Blautia-induced dysbiosis could last for weeks, resulting in prolonged susceptibility to C. difficile and Klebsiella infection.
Dinh and Mayuresh, along with collaborators from the van der Donk, Suga, and Goto labs, have published a paper in the Journal of the American Chemical Society defining how peptide aminoacyl-tRNA ligases (PEARLs) recognize their aminoacyl-tRNA substrates. Using flexizyme-generated tRNAs carrying natural and noncanonical amino acids, they mapped substrate preferences of the Bacillus halodurans enzyme BhaBCala. They found that both the acceptor stem and anticodon arm of the tRNA are critical for recognition, while the enzyme tolerates a range of small amino acids, hydroxy acids, and mercaptocarboxylic acids. AlphaFold 3 modeling showed how these RNA elements interface with the active site, enabling future engineering of ligases for forming diverse peptide linkages.