The Biosynthetic Landscape of Triceptides Reveals Radical SAM Enzymes That Catalyze Cyclophane Formation on Tyr- and His-Containing Motifs

Abstract

Peptide-derived cyclophanes inhabit a unique niche in the chemical space of macrocyclic peptides with several examples of pharmaceutical importance. Although both synthetic and biocatalytic methods are available for constructing these macrocycles, versatile (bio)­catalysts able to incorporate a variety of amino acids that compose the macrocycle would be useful for the creation of diverse peptide cyclophanes. In this report, we synergized the use of bioinformatic tools to map the biosynthetic landscape of radical SAM enzymes (3-CyFEs) that catalyze three-residue cyclophane formation in the biosynthesis of a new family of RiPP natural products, the triceptides. This analysis revealed 3940 (3113 unique) putative precursor sequences predicted to be modified by 3-CyFEs. Several uncharacterized maturase systems were identified that encode unique precursor types. Functional studies were carried out in vivo in Escherichia coli to identify modified precursors containing His and Tyr residues. NMR analysis of the products revealed that Tyr and His can also be incorporated into cyclophane macrocycles by 3-CyFEs. Collectively, all aromatic amino acids can be incorporated by 3-CyFEs, and the cyclophane formation strictly occurs via a C­(sp2)–C­(sp3) cross-link between the (hetero)­aromatic ring to Cβ. In addition to 3-CyFEs, we functionally validated an Fe­(II)/α-ketoglutarate-dependent hydroxylase, resulting in β-hydroxylated residues within the cyclophane rings. This study reveals the potential breadth of triceptide precursors and a systematic approach for studying these enzymes to broaden the diversity of peptide macrocycles.

Bioinformatic Atlas of Radical SAM Enzyme-Modified RiPP Natural Products Reveals an Isoleucine–Tryptophan Crosslink

“…More recently, manual inspection of clusters from an EFI-EST-based network of RaS enzymes led to the discovery of an enzyme that catalyzes the repeated formation of cyclopropylglycine from valine, a new reaction for RaS enzymes . Additionally, an SSN approach has been used to map the biosynthetic landscape of cyclophane-forming RaS enzymes, revealing new cyclophane macrocycles . To make these sorts of analyses easier, RadicalSAM.org was created as a web-based tool for the analysis of SSNs for the entire RaS family …”

“…35 Additionally, an SSN approach has been used to map the biosynthetic landscape of cyclophane-forming RaS enzymes, revealing new cyclophane macrocycles. 36 To make these sorts of analyses easier, RadicalSAM.org was created as a web-based tool for the analysis of SSNs for the entire RaS family. 34 We set out to develop a bioinformatics platform that could benefit from the use of SSNs but would also include computational filtering steps to limit the amount of manual analysis required.…”

Expanded sequence space of radical S-adenosylmethionine-dependent enzyme involved in post-translational macrocyclization

“…Broad substrate selectivity on other aliphatic residues further increases the enzyme-catalyzed chemical space of cyclic peptides. While this manuscript was under preparation, Brandon I. Morinaka and coworkers reported the HaaA precursor family with the same His-Ala crosslink, which resembles our ScaA family, and the YhhA family with similar conserved residues to the VguA family [31] . Together with our bioinformatic data, these findings suggested that histidine-involved post-translational cyclization is prevalent in bacteria.…”

“…Based on the residue-defined modification logic, three families expected to encode new chemical space were prioritized and characterized in this study (Figure 3, Table S29- 31). BlaA family was selected for functional characterization due to two conserved cysteines and two conserved glycine residues in the C-terminal, which may be catalyzed to form two Cys-to-Gly thioether bonds(Figure 3A).…”

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

“…While this manuscript was under preparation, Morinaka and coworkers reported the HaaA and the YhhA families which respectively resembled our ScaA family and VguA family. [33] Phylogenetic analysis of these cyclophane-forming rSAM enzymes revealed that a divergence event may have occurred in the FxS maturase family (TIGR04269) to give rise to ScaB and VguB families (Figure S64).…”