Characterization of the macrocyclase involved in the biosynthesis of RiPP cyclic peptides in plants

Chekan, Jonathan R.; Estrada, Paola; Covello, Patrick S.; Nair, Satish K.

doi:10.1073/pnas.1620499114

Cited by 59 publications

(87 citation statements)

References 56 publications

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“…The modified cyclotide precursor peptide is then cleaved proteolytically Nterminal of the cyclotide core peptide, and, finally, the C terminus of the core peptide is cleaved and cyclized with the N terminus by an asparagine-specific endopeptidase in the plant vacuole (19,23,24). Similarly, orbitides are derived from precursor peptides by endoproteolytic cleavage N-terminal of the orbitide core peptide followed by subsequent C-terminal proteolysis and cyclization catalyzed by serine proteases (19,25,26). Beyond head-to-tail cyclic peptides, the phytochemical repertoire of cyclic peptides suggests that the plant kingdom contains a largely undiscovered diversity of branched cyclic peptide chemistry with tremendous pharmacological potential and unknown biosynthetic mechanisms (27).…”

Section: Significancementioning

confidence: 99%

Gene-guided discovery and engineering of branched cyclic peptides in plants

Kersten

Weng

2018

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

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The plant kingdom contains vastly untapped natural product chemistry, which has been traditionally explored through the activityguided approach. Here, we describe a gene-guided approach to discover and engineer a class of plant ribosomal peptides, the branched cyclic lyciumins. Initially isolated from the Chinese wolfberry Lycium barbarum, lyciumins are protease-inhibiting peptides featuring an N-terminal pyroglutamate and a macrocyclic bond between a tryptophan-indole nitrogen and a glycine α-carbon. We report the identification of a lyciumin precursor gene from L. barbarum, which encodes a BURP domain and repetitive lyciumin precursor peptide motifs. Genome mining enabled by this initial finding revealed rich lyciumin genotypes and chemotypes widespread in flowering plants. We establish a biosynthetic framework of lyciumins and demonstrate the feasibility of producing diverse natural and unnatural lyciumins in transgenic tobacco. With rapidly expanding plant genome resources, our approach will complement bioactivity-guided approaches to unlock and engineer hidden plant peptide chemistry for pharmaceutical and agrochemical applications. lyciumins | BURP domain | ribosomal peptides | natural products | plant metabolism P lants have been an important source of traditional medicines in many cultures for millennia. Underlying the historic use of medicinal plants are bioactive natural products produced in these organisms for signaling and defense (1). Most plant natural products with potential pharmacological applications were discovered based on their bioactivity in bioprospecting studies inspired by traditional herbal medicines (2). However, bioactivityguided discovery of new plant natural products faces a major bottleneck in rediscovery of known structures after purification via bioassay-guided fractionation (3). Subsequent drug development of a target plant natural product is also hindered by low isolation yields from the source plant and by structural complexity of the target compounds, where large-scale total synthesis of these compounds via organic chemistry is often infeasible (4).Over the last two decades, advances in genome sequencing, analytical chemistry, and synthetic biology allowed researchers to address these problems in microbial and fungal natural product chemistry (5). An increasing resource of microbial and fungal genomes enabled the development of powerful computational discovery pipelines for new natural product chemistry, using geneguided discovery approaches such as genome mining, in which a predicted genotype is connected to a chemotype by applying biosynthetic knowledge (5-7). Furthermore, mass spectrometry-based metabolomics accelerated discovery of new chemotypes from microbes and fungi in the context of growing genomic information (6, 8), while heterologous expression and metabolic engineering of biosynthetic gene clusters enabled source organism-independent production and diversification of natural products (9). More recently, analogous gene-guided approaches have been developed to chara...

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Section: Significancementioning

confidence: 99%

Gene-guided discovery and engineering of branched cyclic peptides in plants

Kersten

Weng

2018

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

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“…However, a large and promising source of ligases was discovered within a third family: plant ligases producing ribosomally synthesized and posttranslationally modified peptides (RiPPs) (8). Many RiPPs contain a head-to-tail cyclic structure that requires either a cyclase or a ligase for posttranslational modification from the linear precursors (9)(10)(11)(12)(13). Cyclotide-processing enzymes such as asparaginyl endopeptidases (AEPs) (14) and cyclotide ligases, that are also asparaginyl-specific, were successfully isolated from the cyclotide-producing plant Clitoria ternatea and named butelase 1 and 2 (15).…”

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confidence: 99%

Structural determinants for peptide-bond formation by asparaginyl ligases

Hemu

Sahili

et al. 2019

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

100

269

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Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptideproducing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1′ sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1-3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs. peptide ligase | data mining | ligase-activity determinant

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“…It was not included in the most comprehensive review of plant cyclic peptides to date (21). Instead -perhaps due to the economic importance of flaxseed, from which it comes -cyclolinopeptide A is often cited as the first cyclic peptide (or sometimes the first orbitide) discovered in a plant (1,4,(21)(22)(23)(24)(25). However, cyclolinopeptide A was not isolated until 1959 (26) and its chemical structure solved only in 1966 (27).…”

Section: Introductionmentioning

confidence: 99%

The genetic origin of evolidine, the first cyclopeptide discovered in plants, and related orbitides

Fisher

Payne

Chetty

et al. 2020

Preprint

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Cyclic peptides are reported to have antibacterial, antifungal and other bioactivities. Several genera of the Rutaceae family are known to produce orbitides, which are small head-to-tail cyclic peptides composed of proteinogenic amino acids and lacking disulfide bonds. Melicope xanthoxyloides is an Australian rain forest tree of the Rutaceae family in which evolidine -the first plant cyclic peptide -was discovered. Evolidine (cyclo-SFLPVNL) has subsequently been all but forgotten in the academic literature, but here we use tandem mass spectrometry to rediscover evolidine and using de novo transcriptomics we show its biosynthetic origin to be from a short precursor just 48 residues in length. In all, seven M. xanthoxyloides orbitides were found and they had atypically diverse C-termini consisting of residues not recognized by either of the known proteases plants use to macrocyclize peptides. Two of the novel orbitides were studied by nuclear magnetic resonance spectroscopy and although one had definable structure, the other did not. By mining RNA-seq and whole genome sequencing data from other species, it was apparent that a large and diverse family of peptides is encoded by sequences like these across the Rutaceae.

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Characterization of the macrocyclase involved in the biosynthesis of RiPP cyclic peptides in plants

Cited by 59 publications

References 56 publications

Gene-guided discovery and engineering of branched cyclic peptides in plants

Gene-guided discovery and engineering of branched cyclic peptides in plants

Structural determinants for peptide-bond formation by asparaginyl ligases

The genetic origin of evolidine, the first cyclopeptide discovered in plants, and related orbitides

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