Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-translational Modification

Cao, Li; Beiser, Moshe; Koos, Joseph D.; Orlova, M.; Elashal, Hader E.; Schroeder, Heidi; Link, A. James

doi:10.1101/2021.05.19.444711

Cited by 9 publications

(20 citation statements)

References 65 publications

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“…32,37 The addition of an electrophilic moiety like the succinimide to such inhibitors could convert them into suicide inhibitors that covalently attach to a protease target. In addition to OEPs as described here, this novel class of Asp-modifying O-methyltransferases has also been found in lasso peptide BGCs (see companion paper by Cao et al) 47 as well as in lanthipeptide BGCs. 65 While all three examples of these RiPP modifying methyltransferases start off with Asp methylation and formation of aspartimide, the fate of the aspartimide residue differs.…”

Section: Discussionmentioning

confidence: 55%

“…46 In a closely related organism, Thermobifida cellulosilytica, there is a lasso peptide biosynthetic gene cluster (BGC) with an Omethyltransferase in addition to the minimal biosynthetic genes for lasso peptides. 47 We searched T. fusca for similar O-methyltransferases and found one associated with an ATP-grasp enzyme and a putative RiPP precursor. In a previous bioinformatic survey of ATP-grasp enzyme prevalence, the colocalization of an ATP-grasp enzyme, an O-methyltransferase, and a precursor protein was noted in actinobacteria.…”

Section: A Microviridin/oep-like Bgc In T Fusca Harbors An Uncharacte...mentioning

confidence: 99%

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Biosynthesis and characterization of fuscimiditide, an aspartimidylated graspetide

et al. 2022

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Microviridins and other ω−ester linked peptides (OEPs) are characterized by sidechain-sidechain linkages installed by ATP-grasp enzymes. Here we describe the discovery of a new family of OEPs, the gene clusters of which also encode an O-methyltransferase with homology to the protein repair catalyst protein L-isoaspartyl methyltransferase (PIMT). We produced the first example of this new ribosomally synthesized and post-translationally modified peptide (RiPP), fuscimiditide, via heterologous expression. NMR analysis of fuscimiditide revealed that the peptide contains two ester crosslinks forming a stem-loop macrocycle. Furthermore, an unusually stable aspartimide moiety is found within the loop macrocycle. We have also fully reconstituted fuscimiditide biosynthesis in vitro establishing that ester formation catalyzed by the ATP-grasp enzyme is an obligate, rate-limiting first biosynthetic step. Aspartimide formation from aspartate is catalyzed by the PIMT homolog in the second step. The aspartimide moiety embedded in fuscimiditide hydrolyzes regioselectively to isoaspartate (isoAsp). Surprisingly, this isoAspcontaining protein is also a substrate for the PIMT homolog, thus driving any hydrolysis products back to the aspartimide form. Whereas aspartimide is often considered a nuisance product in protein formulations, our data here suggest that some RiPPs have aspartimide residues intentionally installed via enzymatic activity.

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Section: Discussionmentioning

confidence: 55%

Section: A Microviridin/oep-like Bgc In T Fusca Harbors An Uncharacte...mentioning

confidence: 99%

Biosynthesis and characterization of fuscimiditide, an aspartimidylated graspetide

et al. 2022

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“…Members of this family are related to IPR000682, which converts Lisoaspartyl and D-aspartyl residues to L-Asp, and IPR027573, which frequently co-occurs with lanthipeptide biosynthetic enzymes. 34 Recently, members of the latter family, OlvS A , TceM, and LihM, were implicated in RiPP biosynthesis 35,36 OlvS A catalyzes a three-step reaction (methylation, cyclization, and hydrolysis) of a highly conserved L-Asp to the corresponding L-isoaspartyl residue in OlvA, the cognate lanthipeptide precursor peptide. TceM and LihM introduce stable aspartimide moieties in the lasso peptides, cellulonodin-2 and lihuanodin, respectively.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Bioinformatics-Guided Expansion and Discovery of Graspetides

et al. 2021

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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.

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“…22 RiPP-associated PIMTs also harbor an additional Cterminal domain of >100 aa relative to canonical PIMTs. In our previous work on the PIMT homologues that aspartimidylate lasso peptides, 20 we showed that this C-terminal extension to PIMT is important for RiPP substrate recognition.…”

Section: ■ Introductionmentioning

confidence: 82%

Mechanistic Analysis of the Biosynthesis of the Aspartimidylated Graspetide Amycolimiditide

et al. 2022

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Several classes of ribosomally synthesized and post-translationally modified peptides (RiPPs) are composed of multiple macrocycles. The enzymes that assemble these macrocycles must surmount the challenge of installing a single specific set of linkages out of dozens of distinct possibilities. One class of RiPPs that includes multiple macrocycles are the graspetides, named after the ATP-grasp enzymes that install ester or amide linkages between pairs of nucleophilic and electrophilic side chains. Here, using heterologous expression and NMR spectroscopy, we characterize the connectivity and structure of amycolimiditide, a 29 aa graspetide with a stem-loop structure. The stem includes four esters and extends over 20 Å. The loop of amycolimiditide is distinguished by the presence of an aspartimide moiety, installed by a dedicated O-methyltransferase enzyme. We further characterize the biosynthesis of amycolimiditide in vitro, showing that the amycolimiditide ATP-grasp enzyme AmdB operates in a strict vectorial manner, installing esters starting at the loop and proceeding down the stem. Surprisingly, the O-methyltransferase AmdM that aspartimidylates amycolimiditide prefers a substrate with all four esters installed, despite the fact that the most distal ester is ∼30 Å away from the site of aspartimidylation. This study provides insights into the structure and diversity of aspartimidylated graspetides and also provides fresh insights into how RiPP biosynthetic enzymes engage their peptide substrates.

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Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-translational Modification

Cited by 9 publications

References 65 publications

Biosynthesis and characterization of fuscimiditide, an aspartimidylated graspetide

Biosynthesis and characterization of fuscimiditide, an aspartimidylated graspetide

Bioinformatics-Guided Expansion and Discovery of Graspetides

Mechanistic Analysis of the Biosynthesis of the Aspartimidylated Graspetide Amycolimiditide

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