Mechanism of Action of Ribosomally Synthesized and Post-Translationally Modified Peptides

Ongpipattanakul, Chayanid; Desormeaux, Emily; DiCaprio, Adam J; Donk, Wilfred A. van der; Mitchell, Douglas A.; Nair, Satish K.

doi:10.1021/acs.chemrev.2c00210

Cited by 89 publications

(74 citation statements)

References 931 publications

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“…Ribosomally synthesized and post-translationally modified peptides (RiPPs) comprise a natural product class exhibiting a wide array of unique molecular structures and biological activities. − A typical prokaryotic RiPP is derived from a precursor peptide that is composed of an N-terminal leader region and a C-terminal core region. While the post-translational modifications are installed in the core region, the leader region contains a recognition site that is bound by the biosynthetic proteins.…”

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confidence: 99%

Peptidase Activation by a Leader Peptide-Bound RiPP Recognition Element

et al. 2023

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The RiPP precursor recognition element (RRE) is a conserved domain found in many prokaryotic ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic gene clusters (BGCs). RREs bind with high specificity and affinity to a recognition sequence within the N-terminal leader region of RiPP precursor peptides. Lasso peptide biosynthesis involves an RRE-dependent leader peptidase, which is discretely encoded or fused to the RRE as a di-domain protein. Here we leveraged thousands of predicted BGCs to define the RRE:leader peptidase interaction through evolutionary covariance analysis. Each interacting domain contributes a three-stranded β-sheet to form a hydrophobic β-sandwich-like interface. The bioinformaticsguided predictions were experimentally confirmed using proteins from discrete and fused lasso peptide BGC architectures. Support for the domain−domain interface derived from chemical shift perturbation, paramagnetic relaxation enhancement experiments, and rapid variant activity screening using cell-free biosynthesis. Further validation of selected variants was performed with purified proteins. We developed a p-nitroanilide-based leader peptidase assay to illuminate the role of RRE domains. Our data show that RRE domains play a dual function. RRE domains deliver the precursor peptide to the leader peptidase, and the rate is saturable as expected for a substrate. RRE domains also partially compose the elusive S2 proteolytic pocket that binds the penultimate threonine of lasso leader peptides. Because the RRE domain is required to form the active site, leader peptidase activity is greatly diminished when the RRE domain is supplied at substoichiometric levels. Full proteolytic activation requires RRE engagement with the recognition sequence-containing portion of the leader peptide. Together, our observations define a new mechanism for protease activity regulation.

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Peptidase Activation by a Leader Peptide-Bound RiPP Recognition Element

et al. 2023

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“…Most importantly, these systems allow for the production of mature bacteriocins without the requirement of any other proteins, encoded in the bacteriocin gene clusters and involved in their processing, transport, regulation and immunity. However, although IV-CFPS-derived methods seems to be appropriate for production of the class II non-modified bacteriocins, these methods are not so efficient for production of the class I circular bacteriocins and other class I-derived bacteriocins from the group of RiPPs ( Montalbán-López et al, 2021 ; Ongpipattanakul et al, 2022 ), requiring post-translational proccessing and modifications for their antimicrobial activity.…”

Section: Discussionmentioning

confidence: 99%

“…The mature bacteriocin peptides were initially classified into two main classes: class I or lantibiotics, with lanthionine-containing post-translationally modified amino acid residues, and class II bacteriocins with unmodified amino acid residues ( Cotter et al, 2005 ). However, currently the class I group of modified bacteriocins includes all ribosomally synthesized and post-translationally modified peptides (RiPPS) that undergo enzymatic modification during biosynthesis, providing molecules with uncommon amino acids or structures with an impact on their properties (e.g., lanthipeptides, thiopeptides, lasso peptides, cyclized peptides, sactipeptides and others), whereas the class II group of bacteriocins, groups bacteriocins that do not require posttranslational modification enzymes for their maturation ( Alvarez-Sieiro et al, 2016 ; Chikindas et al, 2018 ; Montalbán-López et al, 2021 ; Ongpipattanakul et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

In vitro and in vivo production and split-intein mediated ligation (SIML) of circular bacteriocins

Peña

Bland²,

Sevillano

et al. 2022

Front. Microbiol.

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Circular bacteriocins are antimicrobial peptides produced by bacteria that after synthesis undergo a head-to-tail circularization. Compared to their linear counterparts, circular bacteriocins are, in general, very stable to temperature and pH changes and more resistant to proteolytic enzymes, being considered as one of the most promising groups of antimicrobial peptides for their potential biotechnological applications. Up to now, only a reduced number of circular bacteriocins have been identified and fully characterized, although many operons potentially coding for new circular bacteriocins have been recently found in the genomes of different bacterial species. The production of these peptides is very complex and depends on the expression of different genes involved in their synthesis, circularization, and secretion. This complexity has greatly limited the identification and characterization of these bacteriocins, as well as their production in heterologous microbial hosts. In this work, we have evaluated a synthetic biology approach for the in vitro and in vivo production combined with a split-intein mediated ligation (SIML) of the circular bacteriocin garvicin ML (GarML). The expression of one single gene is enough to produce a protein that after intein splicing, circularizes in an active peptide with the exact molecular mass and amino acid sequence as native GarML. In vitro production coupled with SIML has been validated with other, well described and not yet characterized, circular bacteriocins. The results obtained suggest that this synthetic biology tool holds great potential for production, engineering, improving and testing the antimicrobial activity of circular bacteriocins.

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“…In spite of valuable collective reviews focusing on RiPP biosynthetic mechanisms, intriguing/unique post-translationally modifying enzymes, as well as the novel structural scaffolds thereof, ,,,− relatively fewer summaries in regard to their diverse bioactivities, physiological/ecological functionalities, and/or mode of mechanism, are available in the literature. − Thus, this Review systematically covers the recent advances in bioactive RiPPs in terms of bioactivity and/or mode of mechanisms in the past decade. RiPPs exhibiting anti-Gram-positive bacteria, anti-Gram-negative bacteria, antitumor, antivirus, etc.…”

Section: Introductionmentioning

confidence: 99%

Recent Advances in Discovery, Bioengineering, and Bioactivity-Evaluation of Ribosomally Synthesized and Post-translationally Modified Peptides

Zhong

Wang

Yan

et al. 2022

ACS Bio Med Chem Au

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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are of increasing interest in natural products as well as drug discovery. This empowers not only the unique chemical structures and topologies in natural products but also the excellent bioactivities such as antibacteria, antifungi, antiviruses, and so on. Advances in genomics, bioinformatics, and chemical analytics have promoted the exponential increase of RiPPs as well as the evaluation of biological activities thereof. Furthermore, benefiting from their relatively simple and conserved biosynthetic logic, RiPPs are prone to be engineered to obtain diverse analogues that exhibit distinct physiological activities and are difficult to synthesize. This Review aims to systematically address the variety of biological activities and/or the mode of mechanisms of novel RiPPs discovered in the past decade, albeit the characteristics of selective structures and biosynthetic mechanisms are briefly covered as well. Almost one-half of the cases are involved in anti-Gram-positive bacteria. Meanwhile, an increasing number of RiPPs related to anti-Gram-negative bacteria, antitumor, antivirus, etc., are also discussed in detail. Last but not least, we sum up some disciplines of the RiPPs' biological activities to guide genome mining as well as drug discovery and optimization in the future.

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Mechanism of Action of Ribosomally Synthesized and Post-Translationally Modified Peptides

Cited by 89 publications

References 931 publications

Peptidase Activation by a Leader Peptide-Bound RiPP Recognition Element

Peptidase Activation by a Leader Peptide-Bound RiPP Recognition Element

In vitro and in vivo production and split-intein mediated ligation (SIML) of circular bacteriocins

Recent Advances in Discovery, Bioengineering, and Bioactivity-Evaluation of Ribosomally Synthesized and Post-translationally Modified Peptides

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