A multi-iron enzyme installs copper-binding oxazolone/thioamide pairs on a nontypeable
            <i>Haemophilus influenzae</i>
            virulence factor

Manley, Olivia M.; Shriver, Tucker J.; Xu, Tian; Melendrez, Isaac A.; Palacios, Philip; Robson, Scott A.; Guo, Yisong; Kelleher, Neil L.; Ziarek, Joshua J.; Rosenzweig, Amy C.

doi:10.1073/pnas.2408092121

Cited by 2 publications

References 43 publications

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A widespread family of ribosomal peptide metallophores involved in bacterial adaptation to metal stress

Leprevost,

Jünger,

Lippens

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a structurally diverse group of natural products that bacteria employ in their survival strategies. Herein, we characterized the structure, the biosynthetic pathway, and the mode of action of a RiPP family called bufferins. With thousands of homologous biosynthetic gene clusters throughout the bacterial phylogenetic tree, bufferins form by far the largest family of RiPPs modified by multinuclear nonheme iron-dependent oxidases (MNIO, DUF692 family). Using Caulobacter vibrioides bufferins as a model, we showed that the conserved Cys residues of their precursors are transformed into 5-thiooxazoles, further expanding the reaction range of MNIO enzymes. This rare modification is installed in conjunction with a partner protein of the DUF2063 family. Bufferin precursors are rare examples of bacterial RiPPs found to feature an N-terminal Sec signal peptide allowing them to be exported by the ubiquitous Sec pathway. We reveal that bufferins are involved in copper homeostasis, and their metal-binding propensity requires the thiooxazole heterocycles. Bufferins enhance bacterial growth under copper stress by complexing excess metal ions. Our study thus describes a large family of RiPP metallophores and unveils a widespread but overlooked metal homeostasis mechanism in bacteria.

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A widespread family of ribosomal peptide metallophores involved in bacterial adaptation to metal stress

Leprevost,

Jünger,

Lippens

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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Cysimiditides: RiPPs with a Zn-tetracysteine motif and aspartimidylation

Zhu,

Cao,

et al. 2024

Preprint

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Aspartimidylation is a post-translational modification found in multiple families of ribosomally synthesized and post-translationally modified peptides (RiPPs). We recently reported on the imiditides, a new RiPP family in which aspartimidylation is the class-defining modification. Imiditide biosynthetic gene clusters encode a precursor protein and a methyltransferase that methylates a specific Asp residue, converting it to aspartimide. A subset of imiditides harbor a tetracysteine motif, so we have named these molecules cysimiditides. Here, using genome mining we show that there are 56 putative cysimiditides predicted in publicly available genome sequences, all within actinomycetota. We successfully heterologously expressed two examples of cysimiditides and showed that the major products are aspartimidylated and that the tetracysteine motif is necessary for expression. Cysimiditides bind a Zn2+ ion, presumably at the tetracysteine motif. Using in vitro reconstitution of the aspartimidylation reaction, we show that Zn2+ is required for methylation and subsequent aspartimidylation of the precursor protein. An AlphaFold 3 model of the cysimiditide from Thermobifida cellulosilytica shows a hairpin structure anchored by the Zn2+-tetracysteine motif with the aspartimide site in the hairpin loop. An AlphaFold 3 model of this cysimiditide in complex with its cognate methyltransferase suggests that the methyltransferase recognizes the Zn2+-tetracysteine motif to correctly dock the precursor protein. Cysimiditides expand the set of experimentally-confirmed RiPPs harboring aspartimides, and represent the first RiPP class that has an obligate metal ion.

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A multi-iron enzyme installs copper-binding oxazolone/thioamide pairs on a nontypeable Haemophilus influenzae virulence factor

Cited by 2 publications

References 43 publications

A widespread family of ribosomal peptide metallophores involved in bacterial adaptation to metal stress

A widespread family of ribosomal peptide metallophores involved in bacterial adaptation to metal stress

Cysimiditides: RiPPs with a Zn-tetracysteine motif and aspartimidylation

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