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

Kostenko, Anastasiia; Lien, Yi; Mendauletova, Aigera; Ngendahimana, Thacien; Novitskiy, Ivan M.; Latham, John

doi:10.1016/j.jbc.2022.101881

Cited by 12 publications

(15 citation statements)

References 47 publications

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“…Assessment of the unmodified and modified TigB3R by 1 H NMR revealed new features in the lower frequency range (∼3.65 ppm δ H and <1 ppm δ H , Figure A,B). The chemical shifts at δ H ∼0.58 and 0.15 ppm are consistent with those we previously reported for Val Cγ protons within the cyclopropane ring of the TVG RiPP . Therefore, we assigned these low frequency chemical shifts to the Ile-Cγ protons belonging to a cyclopropane ring.…”

Section: Resultssupporting

confidence: 90%

“…We expected that either TigD and/or TigE would catalyze the formation of a cyclopropane on the conserved Ile and/or Val residues, similar to TvgB. 25 Characterization of TigE and Its Activity. TigE was cloned into a pET28a vector, which allowed the expression of a fusion protein with an N-terminal, 6XHis-tag, allowing for facile anaerobic purification.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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

Lien,

Lachowicz,

Mendauletova

et al. 2024

ACS Chem. Biol.

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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.

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Section: Resultssupporting

confidence: 90%

Section: ■ Results and Discussionmentioning

confidence: 99%

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

Lien,

Lachowicz,

Mendauletova

et al. 2024

ACS Chem. Biol.

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“…Initial work by Haft and Basu showed that the family of RaS-RiPPs is diverse and widely encoded. , In recent years, sequence similarity networks (SSNs) and genomic enzymology pioneered by Gerlt and co-workers have become popular tools for identifying new RiPP BGCs. − As mentioned above, we previously used a streptococcal SSN of RaS-RiPPs to uncover novel RaS enzyme reactions and natural products. 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 .…”

Section: Resultsmentioning

confidence: 99%

“…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. 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.…”

Section: ■ Introductionmentioning

confidence: 99%

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

Clark

Seyedsayamdost

2022

J. Am. Chem. Soc.

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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of natural products with diverse activities and structures. RiPP classes are defined by the tailoring enzyme, which can introduce a narrow range of modifications or a diverse set of alterations. In the latter category, RiPPs synthesized by radical S-adenosylmethionine (SAM) enzymes, known as RaS-RiPPs, have emerged as especially divergent. A map of all RaS-RiPP gene clusters does not yet exist. Moreover, precursor peptides remain difficult to predict using computational methods. Herein, we have addressed these challenges and report a bioinformatic atlas of RaS-RiPP gene clusters in available microbial genome sequences. Using co-occurrence of RaS enzymes and transporters from varied families as a bioinformatic hook in conjunction with an in-house code to identify precursor peptides, we generated a map of ∼15,500 RaS-RiPP gene clusters, which reveal a remarkable diversity of syntenies pointing to a tremendous range of enzymatic and natural product chemistries that remain to be explored. To assess its utility, we examined one family of gene clusters encoding a YcaO enzyme and a RaS enzyme. We find the former is noncanonical, contains an iron–sulfur cluster, and installs a novel modification, a backbone amidine into the precursor peptide. The RaS enzyme was also found to install a new modification, a C–C crosslink between the unactivated terminal δ-methyl group of Ile and a Trp side chain. The co-occurrence search can be applied to other families of RiPPs, as we demonstrate with the emerging DUF692 di-iron enzyme superfamily.

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“…In RiPP biosynthetic pathways, rSAM enzymes typically catalyze side-chain cross-linking reactions (i.e., C–S/C–O/C–N/C–C bond formation) to install (hetero)cycloalkane or cyclophane units on the polypeptide substrate (Figure S1). – The ability of DarE to de novo construct an ether cross-link from an exogenous source of oxygen appears to be unprecedented in biochemistry. Furthermore, the mechanism by which a single DarE enzyme forms both the chemically distinct C–C cross-link and ether cross-link remains largely unknown.…”

Section: Introductionmentioning

confidence: 99%

Substrate-Controlled Catalysis in the Ether Cross-Link-Forming Radical SAM Enzymes

Ma,

Xi,

Wang

et al. 2023

J. Am. Chem. Soc.

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Darobactin is a heptapeptide antibiotic featuring an ether cross-link and a C−C cross-link, and both cross-links are installed by a radical S-adenosylmethionine (rSAM) enzyme DarE. How a single DarE enzyme affords the two chemically distinct cross-links remains largely obscure. Herein, by mapping the biosynthetic landscape for darobactin-like RiPP (daropeptide), we identified and characterized two novel daropeptides that lack the C−C cross-link present in darobactin and instead are solely composed of ether cross-links. Phylogenetic and mutagenesis analyses reveal that the daropeptide maturases possess intrinsic multifunctionality, catalyzing not only the formation of ether cross-link but also C−C cross-linking and Ser oxidation. Intriguingly, the different chemical outcomes are controlled by the exact substrate motifs. Our work not only provides a roadmap for the discovery of new daropeptide natural products but also offers insights into the regulatory mechanisms that govern these remarkably versatile ether cross-link-forming rSAM enzymes.

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

Cited by 12 publications

References 47 publications

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

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

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

Substrate-Controlled Catalysis in the Ether Cross-Link-Forming Radical SAM Enzymes

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