Austin and collaborators from the Broderick and Guo labs have published an article in ACS Bio & Med Chem Au investigating an archaeal radical SAM enzyme that modifies a cysteine-rich protein. A bioinformatic analysis of high cysteine archaea (mainly consisting of methanogens) led to the prioritization of the Crp BGC which contains a cystine-rich protein (CrpA) and a radical SAM enzyme (RS; CrpC). CrpC was reconstituted and characterized in vitro and shown to be an active RS-SPASM enzyme, while extensive mass spectrometry and chemical labeling determined that CrpC installs thioether crosslinks on CrpA. This work provides a new bioinformatic process for natural product discovery and expands on the limited characterization of archaeal BGCs and proteins with RiPP-like modifications.
Dinh and collaborators from the van der Donk lab published an article in Angewandte Chemie International Edition on ApyO, the cytochrome P450 that macrocyclizes the aminopyruvatide precursor peptide. AlphaFold 3 modeling and cell-free expression identified the C-terminal 10 residues of ApyA as a competent substrate, with a conserved Arg required for turnover. Replacing Tyr6 with Trp redirected catalysis to an N–C bond with the Trp indole, while Leu7 substitutions gave isomer pairs with either the native C–C or a nonnative C–O linkage. The apy pathway product desmethylaminopyruvatide A inhibited cathepsin L, cathepsin B, and papain with IC50 values of 0.6, 0.5, and 0.3 nM. This work shows that residues adjacent to a crosslink can redirect P450 chemistry, expanding the macrocyclic scaffolds accessible enzymatically.
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.
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.
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.
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.