Expanded Sequence Space of Radical S‐Adenosylmethionine‐Dependent Enzymes Involved in Post‐translational Macrocyclization**

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent one of the largest but primarily underexplored natural product families in bacteria. The genetically encoded nature of RiPPs simplifies the prediction and prioritization of their biosynthetic gene clusters (BGCs). We report a small peptide and enzyme co-occurrence analysis workflow (SPECO), which allowed us to identify 32 220 prospective rSAM-catalyzed RiPP BGCs from 161 954 bacterial genomes and prioritize 25 families with new biosynthetic architectures or precursor patterns. We characterized three new enzymes that respectively catalyze cysteineglycine (BlaB), histidine-aliphatic side chain (ScaB), and tyrosine/histidine-arginine (VguB) cross-links. The cyclophane-forming enzyme ScaB exhibits broad substrate selectivity, allowing it to catalyze diverse triceptide formation. These results demonstrate the strength of the SPECO workflow in discovering new enzymes for peptide macrocyclization.

Catalytic Site Proximity Profiling for Functional Unification of Sequence-Diverse Radical S-Adenosylmethionine Enzymes

“…While the current manuscript was in preparation, a publication describing a new sactipeptide became available . The precursor peptide BlaA contains two conserved Cys and Gly residues and is related to StsA (Figures and S6).…”

“…While the current manuscript was in preparation, a publication describing a new sactipeptide became available. 39 The precursor peptide BlaA contains two conserved Cys and Gly residues and is related to StsA (Figures 3 and S6). Similarly, the rSAM enzyme BlaB is related to StsB and MftC (expectation values of 1 × 10 −68 and 3 × 10 −33 , respectively).…”

Genomic and metabolic analyses reveal antagonistic lanthipeptides in archaea

“…We next sought to identify putative lanthipeptide precursors by fetching the small peptide open reading frame (orf) adjacent to lanM genes [ 27 ]. A total of 348 putative precursors associated with 103 LanM proteins (Supplementary Table 1 and Supplementary data file ) were selected based on the presence of both Ser/Thr and Cys residues in their C-terminal core peptides, which are indispensable for forming the characteristic thioether crosslinks of lanthipeptides, and subjected to sequence similarity network (SSN) analysis (Fig.…”

Biosynthesis- and Metabolomics-guided discovery of antimicrobial cyclopeptides against drug-resistant clinical isolates

“…Following this rule, we analyzed 21,911 genomes of actinobacteria and 30,666 genomes of firmicutes for the ACP BGC identification using the SPECO pipeline 21 (Figure 1c). This analysis generated 1,172 unique putative precursor-flavoprotein pairs (Supplementary dataset).…”

“…In recent studies, our research focused on genomics-guided discovery of RiPP antibiotics, resulting in the discovery of antagonistic lanthipeptide from archaea and narrow-spectrum class II bacteriocins from the human microbiome 18, 19 . Moreover, applying rule-based genome mining strategies, we successfully revealed previously hidden peptidases 20 and untapped post-translational modification (PTM) enzymes 21, 22 for RiPP antibiotic biosynthesis. However, the challenge remains in the prioritization of BGCs of interest and the robust linking of metabolites to their corresponding BGCs prior to time-consuming isolation and identification.…”