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

The radical S-adenosylmethionine (rSAM) superfamily has become a wellspring for discovering new enzyme chemistry, especially regarding ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we report a compendium of nearly 15,000 rSAM proteins with high-confidence involvement in RiPP biosynthesis. While recent bioinformatics advances have unveiled the broad sequence space covered by rSAM proteins, the significant challenge of functional annotation remains unsolved. Through a combination of sequence analysis and protein structural predictions, we identified a set of catalytic site proximity residues with functional predictive power, especially among the diverse rSAM proteins that form sulfur-to-α carbon thioether (sactionine) linkages. As a case study, we report that an rSAM protein from Streptomyces sparsogenes (StsB) shares higher full-length similarity with MftC (mycofactocin biosynthesis) than any other characterized enzyme. However, a comparative analysis of StsB to known rSAM proteins using "catalytic site proximity" predicted that StsB would be distinct from MftC and instead form sactionine bonds. The prediction was confirmed by mass spectrometry, targeted mutagenesis, and chemical degradation. We further used "catalytic site proximity" analysis to identify six new sactipeptide groups undetectable by traditional genome-mining strategies. Additional catalytic site proximity profiling of cyclophane-forming rSAM proteins suggests that this approach will be more broadly applicable and enhance, if not outright correct, protein functional predictions based on traditional genomic enzymology principles.

Peptidase Activation by a Leader Peptide-Bound RiPP Recognition Element.

The RiPP precursor recognition element (RRE) is a conserved domain found in many prokaryotic ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic gene clusters (BGCs). RREs bind with high specificity and affinity to a recognition sequence within the N-terminal leader region of RiPP precursor peptides. Lasso peptide biosynthesis involves an RRE-dependent leader peptidase, which is discretely encoded or fused to the RRE as a di-domain protein. Here we leveraged thousands of predicted BGCs to define the RRE:leader peptidase interaction through evolutionary covariance analysis. Each interacting domain contributes a three-stranded ß-sheet to form a hydrophobic ß-sandwich-like interface. The bioinformatics-guided predictions were experimentally confirmed using proteins from discrete and fused lasso peptide BGC architectures. Support for the domain-domain interface derived from chemical shift perturbation, paramagnetic relaxation enhancement experiments, and rapid variant activity screening using cell-free biosynthesis. Further validation of selected variants was performed with purified proteins. We developed a p-nitroanilide-based leader peptidase assay to illuminate the role of RRE domains. Our data show that RRE domains play a dual function. RRE domains deliver the precursor peptide to the leader peptidase, and the rate is saturable as expected for a substrate. RRE domains also partially compose the elusive S2 proteolytic pocket that binds the penultimate threonine of lasso leader peptides. Because the RRE domain is required to form the active site, leader peptidase activity is greatly diminished when the RRE domain is supplied at substoichiometric levels. Full proteolytic activation requires RRE engagement with the recognition sequence-containing portion of the leader peptide. Together, our observations define a new mechanism for protease activity regulation.

Bioinformatics-Guided Expansion and Discovery of Graspetides.

Graspetides are a class of ribosomally synthesized and post-translationally modified peptide natural products featuring ATP-grasp ligase-dependent formation of macrolactones/macrolactams. These modifications arise from serine, threonine, or lysine donor residues linked to aspartate or glutamate acceptor residues. Characterized graspetides include serine protease inhibitors such as the microviridins and plesiocin. Here, we report an update to Rapid ORF Description and Evaluation Online (RODEO) for the automated detection of graspetides, which identified 3,923 high-confidence graspetide biosynthetic gene clusters. Sequence and co-occurrence analyses doubled the number of graspetide groups from 12 to 24, defined based on core consensus sequence and putative secondary modification. Bioinformatic analyses of the ATP-grasp ligase superfamily suggest that extant graspetide synthetases diverged once from an ancestral ATP-grasp ligase and later evolved to introduce a variety of ring connectivities. Furthermore, we characterized thatisin and iso-thatisin, two graspetides related by conformational stereoisomerism from Lysobacter antibioticus. Derived from a newly identified graspetide group, thatisin and iso-thatisin feature two interlocking macrolactones with identical ring connectivity, as determined by a combination of tandem mass spectrometry (MS/MS), methanolytic, and mutational analyses. NMR spectroscopy of thatisin revealed a cis conformation for a key proline residue, while molecular dynamics simulations, solvent-accessible surface area calculations, and partial methanolytic analysis coupled with MS/MS support a trans conformation for iso-thatisin at the same position. Overall, this work provides a comprehensive overview of the graspetide landscape, and the improved RODEO algorithm will accelerate future graspetide discoveries by enabling open-access analysis of existing and emerging genomes.