Structural, Biochemical, and Bioinformatic Basis for Identifying Radical SAM Cyclopropyl Synthases.

The importance of radical S-adenosyl-l-methionine (RS) enzymes in the maturation of ribosomally synthesized and post-translationally modified peptides (RiPPs) continues to expand, specifically for the RS-SPASM subfamily. We recently discovered an RS-SPASM enzyme that installs a carbon-carbon bond between the geminal methyls of valine residues, resulting in the formation of cyclopropylglycine (CPG). Here, we sought to define the family of cyclopropyl (CP) synthases because of the importance of cyclopropane scaffolds in pharmaceutical development. Using RadicalSAM.org, we bioinformatically expanded the family of CP synthases and assigned unique peptide sequences to each subclade. We identified a unique RiPP biosynthetic pathway that encodes a precursor peptide, TigB, with a repeating TIGSVS motif. Using LCMS and NMR techniques, we show that the RS enzyme associated with the pathway, TigE, catalyzes the formation of a methyl-CPG from the conserved isoleucine residing in the repeating motif of TigB. Furthermore, we obtained a crystal structure of TigE, which reveals an unusual tyrosyl ligation to the auxiliary I [4Fe-4S] cluster, provided by a glycine-tyrosine-tryptophan motif unique to all CP synthases. Further, we show that this unique tyrosyl ligation is absolutely required for TigE activity. Together, our results provide insight into how CP synthases perform this unique reaction.

Identification of a poly-cyclopropylglycine-containing peptide via bioinformatic mapping of radical S-adenosylmethionine enzymes.

Peptide-derived natural products are a large class of bioactive molecules that often contain chemically challenging modifications. In the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs), radical-SAM (rSAM) enzymes have been shown to catalyze the formation of ether, thioether, and carbon-carbon bonds on the precursor peptide. The installation of these bonds typically establishes the skeleton of the mature RiPP. To facilitate the search for unexplored rSAM-dependent RiPPs for the community, we employed a bioinformatic strategy to screen a subfamily of peptide-modifying rSAM enzymes which are known to bind up to three [4Fe-4S] clusters. A sequence similarity network was used to partition related families of rSAM enzymes into >250 clusters. Using representative sequences, genome neighborhood diagrams were generated using the Genome Neighborhood Tool. Manual inspection of bacterial genomes yielded numerous putative rSAM-dependent RiPP pathways with unique features. From this analysis, we identified and experimentally characterized the rSAM enzyme, TvgB, from the tvg gene cluster from Halomonas anticariensis. In the tvg gene cluster, the precursor peptide, TvgA, is comprised of a repeating TVGG motif. Structural characterization of the TvgB product revealed the repeated formation of cyclopropylglycine, where a new bond is formed between the γ-carbons on the precursor valine. This novel RiPP modification broadens the functional potential of rSAM enzymes and validates the proposed bioinformatic approach as a practical broad search tool for the discovery of new RiPP topologies.

How a Subfamily of Radical S-Adenosylmethionine Enzymes Became a Mainstay of Ribosomally Synthesized and Post-translationally Modified Peptide Discovery.

Radical S-adenosylmethionine (rSAM) enzymes are a large and diverse superfamily of enzymes, some of which are known to participate in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). Specifically, a subfamily of rSAM proteins with an elongated C-terminus known as a SPASM domain have become a fixation in the discovery of new RiPP natural products. Arguably, a structural study, a bioinformatic study, and a functional study built the foundation of the research for rSAM-SPASM-protein-modified RiPPs. In this Review, we focus on these three studies and how they initiated what has become an increasingly productive field. In addition, we discuss the current state of RiPPs that depends on rSAM-SPASM proteins and provide guidelines to consider in future research. Lastly, we discuss how genome mining tools have become a powerful means to identify and predict new RiPP natural products. Despite the state of our current knowledge, we do not completely understand the relationship of rSAM-SPASM chemistry, substrate recognition, and the structure-function relationship as it pertains to RiPP biosynthesis, and as such, there remain many interesting findings waiting to be discovered in the future.

Biosynthesis of the redox cofactor mycofactocin is controlled by the transcriptional regulator MftR and induced by long-chain acyl-CoA species.

Mycofactocin (MFT) is a ribosomally synthesized and post-translationally-modified redox cofactor found in pathogenic mycobacteria. While MFT biosynthetic proteins have been extensively characterized, the physiological conditions under which MFT biosynthesis is required are not well understood. To gain insights into the mechanisms of regulation of MFT expression in Mycobacterium smegmatis mc2155, we investigated the DNA-binding and ligand-binding activities of the putative TetR-like transcription regulator, MftR. In this study, we demonstrated that MftR binds to the mft promoter region. We used DNase I footprinting to identify the 27 bp palindromic operator located 5' to mftA and found it to be highly conserved in Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, and Mycobacterium marinum. To determine under which conditions the mft biosynthetic gene cluster (BGC) is induced, we screened for effectors of MftR. As a result, we found that MftR binds to long-chain acyl-CoAs with low micromolar affinities. To demonstrate that oleoyl-CoA induces the mft BGC in vivo, we re-engineered a fluorescent protein reporter system to express an MftA-mCherry fusion protein. Using this mCherry fluorescent readout, we show that the mft BGC is upregulated in M. smegmatis mc2155 when oleic acid is supplemented to the media. These results suggest that MftR controls expression of the mft BGC and that MFT production is induced by long-chain acyl-CoAs. Since MFT-dependent dehydrogenases are known to colocalize with acyl carrier protein/CoA-modifying enzymes, these results suggest that MFT might be critical for fatty acid metabolism or cell wall reorganization.