A biaryl-linked tripeptide from Planomonospora reveals a widespread class of minimal RiPP gene clusters

•N-Acetylated tripeptides with an unusual Tyr-His biaryl bridging•Ribosomally synthetized from a pentapeptide precursor and modified by cytochrome P450•Encoded the 18-bp bycA gene, smallest coding gene ever reported•Widespread distribution of related two-gene clusters in actinobacterial genomes Microbial natural products impress their bioactivity, structural diversity, ingenious biosynthesis. While screening less exploited genus Planomonospora, two cyclopeptides were discovered, featuring bridging across tripeptide scaffold, sequences N-acetyl-Tyr-Tyr-His N-acetyl-Tyr-Phe-His. Planomonospora pointed toward ribosomal synthesis cyclopeptide encoded bytA, to our knowledge reported. Closely linked bytA is bytO, encoding P450 monooxygenase likely responsible for installment. In Streptomyces, bytAO segment was sufficient direct production crosslinked N-acetylated Tyr-Tyr-His tripeptide. Bioinformatic analysis monooxygenases indicated that they constitute widespread family enzymes, corresponding genes are closely 5-amino acid approximately 200 (actino)bacterial genomes, all potential linkage between amino acids 1 3. We propose named biarylitides this RiPPs. not only potent but also chemical diversity originality. Structurally novel compounds have high chance interacting targets or mechanisms action different known (Yera et al., 2011Yera E.R. Cleves A.E. Jain A.N. Chemical novelty: on-targets off-targets.J. Med. Chem. 2011; 54: 6771-6785Crossref PubMed Scopus (50) Google Scholar; Gfeller 2013Gfeller D. Michielin O. Zoete V. Shaping interaction landscape bioactive molecules.Bioinformatics. 2013; 29: 3073-3079https://doi.org/10.1093/bioinformatics/btt540Crossref (201) Chen 2020Chen Y. Mathai N. Kirchmair J. Scope 3D shape-based approaches predicting macromolecular structurally complex small molecules including macrocyclic ligands.J. Inf. Model. 2020; 60: 2858-2875https://doi.org/10.1021/acs.jcim.0c00161Crossref (9) Scholar). recent years, ribosomally synthesized post-translationally peptides (RiPPs) drawn attention due intricate, three-dimensional (3D) structures wide bacteria. RiPP biosynthetic (BGCs) compact, encompassing peptide, separated N-terminal leader C-terminal core peptide. After translation, peptide dedicated consecutive cleavage release mature (Arnison 2013Arnison P.G. Bibb M.J. Bierbaum G. Bowers A.A. Bugni T.S. Bulaj Camarero J.A. Campopiano D.J. Challis G.L. Clardy al.Ribosomally products: overview recommendations universal nomenclature.Nat. Prod. Rep. 30: 108-160Crossref (1222) Funk van der Donk, 2017Funk M.A. Donk W.A. Ribosomal products, tailored fit.Acc. Res. 2017; 50: 1577-1586https://doi.org/10.1021/acs.accounts.7b00175Crossref (43) RiPPs grouped into families on basis features post-translational modifications (Funk Several bioinformatic tools been developed automatic recognition BGCs microbial which usually based recognizing elements, such as diagnostic RiPP-processing enzymes (Russell Truman, 2020Russell A.H. Truman A.W. Genome mining strategies synthesised peptides.Computat. Struct. Biotechnol. 18: 1838-1851Crossref (25) study, we identified crosslinked, Tyr1-His3 bridging. producing strains led identification shortest BGC so far reported, just monooxygenase. Phylogenomic latter sequence revealed represent minimal present especially genomes. search secondary metabolites, investigated 72 belonging poorly characterized actinomycete (Monciardini 2014Monciardini P. Iorio M. Maffioli S.I. Sosio Donadio S. Discovering new sources.Microb. 2014; 7: 209-220Crossref (108) Scholar) extensive liquid chromatography coupled high-resolution ion electrospray ionization-tandem mass spectrometry-based metabolome extracts (Zdouc 2020Zdouc M.M. Crüsemann Preprint bioRxiv.2020: 210815https://doi.org/10.1101/2020.07.19.210815Crossref Molecular networking group (see Figure S1) observed samples four phylogenetically strains, 16S rRNA having 99.5%–99.6% identity algeriensis PM3 One strain, sp. ID82291, produced compound (1) m/z 522.198 [M + H]+ (calculated molecular formula C26H28N5O7), while other three (2) 506.203 C26H28N5O6). Additional variants found strain ID46116 (Figure further investigated. 2 showed UV absorption maxima at 270 315 nm, indicating chromophore. ID82291 selected investigation. From 15-L culture, 5 mg pure isolated. Extensive 1D 2D NMR analyses acetylated short A first set experiments, performed DMSO-d6, allowed observation aromatic spin systems containing CHα CH2β consistent tyrosine one histidine residues. The correlations NOESY experiment suggested N-acetyl-Tyr-Tyr-His, additional unsaturation, could be assigned overlapping signals. second CD3OD complete assignment non-exchangeable protons 1. TOCSY HSQC experiments residue lacked its characteristic proton position 5″ had quaternary carbon 142 ppm. HMBC experiment, cross-correlation both CH-5 CH-8 C5″, carbon-carbon bond C6 C5″ 1A). assignments spectra reported Figures S2 S3 Table S1. existence crosslink confirmed after hydrolysis 1, 335 H]+, expected carbon-carbon-linked histidine, 480 loss acetyl intact retained maximum Compound 2, isolates, deduced N-acetyl-Tyr-Phe-His, similarity tandem fragmentation neutral phenylalanine (−147.07 Da) being difference S2). identify literature precedent tyrosine-histidine bridge: aciculitins, cyclic nonapeptides decorated glycolipids marine sponge Aciculites orientalis (Bewley 1996Bewley C.A. He H. Williams D.H. Faulkner Aciculitins A−C: cytotoxic antifungal lithistid orientalis.J. Am. Soc. 1996; 118: 4314-4321Crossref (70) They contain connectivity than Interestingly, lack residues, show 310 matching those few metabolites described consist (N-acetylated) tripeptide: K-13 (3) Micromonospora halophytica ssp. exilisia Tyr-Tyr-Tyr ether oxygen Tyr-3 Tyr-1 (Kase 1987Kase Kaneko Yamada K. 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Among 1-related 4 inhibit proteases angiotensin-converting (ACE) B, respectively, apparent strict specificity, since ACE inhibitor. unable observe inhibition (data shown), suggesting and/or ring size can strongly influence activity. weak activity When grown D2O-supplemented medium, extensively labeled deuterium S4), formation requires de novo unaware any studies 5. To get insights biosynthesis, analyzed 7.58-Mbp genome presence antiSMASH-predicted NRPS (Blin 2019Blin Shaw Steinke Villebro R. Ziemert Lee S.Y. Medema M.H. Weber antiSMASH 5.0: updates metabolite pipeline.Nucleic Acids 2019; 2: W81-W87https://doi.org/10.1093/nar/gkz310Crossref (1527) Scholar), specify However, proved unsuccessful, formed recognized tools. Searching six-frame translation single plausible candidate: open reading frame MRYYH, preceded binding site followed PLM4_2056, 1B). proposed designated bytO below), similar recently tryptorubin (6), hexapeptide carbon-nitrogen bonds residues (Wyche 2017Wyche T.P. Ruzzini A.C. Schwab L. Currie C.R. Tryptorubin A: polycyclic fungus-derived Streptomycete.J. 139: 12899-12902Crossref (19) Reisberg 2020Reisberg S.H. Gao Walker A.S. Helfrich E.J. Baran P.S. Total reveals atypical atropisomerism small-molecule product, A.Science. 367: 458-463Crossref (21) Also 6, encodes latter, however, 22-amino Similarly, cittilin (7), YIYY tetrapeptide Myxococcus xanthus, 27-amino adjacent enzyme, methyltransferase distant endopeptidase (Hug 2020Hug J.J. Dastbaz Adam Revermann Koehnke Krug Mueller Biosynthesis cittilins, xanthus.ACS Biol. 15: 2221-2231https://doi.org/10.1021/acschembio.0c00430Crossref (12) Cytochrome install crosslinks peptides, as, e.g., NRPS-generated glycopeptides (Greule 2018Greule Stok J.E. De Voss Cryle Unrivalled diversity: many roles reactions bacterial cytochromes metabolism.Nat. 2018; 35: 757-791Crossref These precedents made depicted 1B candidate Apart possible regulator, five upstream downstream do partake organization ID107089, producers (Figures S1). regions almost identical: there nucleotide (A-T) genes, Y-F peptides; equally spaced, sites; share 97% identity, intergenic (120 bp) 95% identical. prove indeed governed bytAO, cloned 1,303-bp pSET152 conjugated plasmid expression host Streptomyces coelicolor M1152. Cultivation pSET HP20 extraction high-performance chromatography-tandem spectrometry analysis, resulted detection peak 522.199, same spectrum, retention time, spectrum 1C S4). This unambiguously confirms heterologous cluster making it knowledge, peptide-encoding 24-bp mccA, microcin C7 Escherichia coli, devoid (González-Pastor 1994González-Pastor San Millán J.L. Moreno F. gene.Nature. 1994; 369: 281https://doi.org/10.1038/369281a0Crossref (55) Guijarro 1995Guijarro J.I. González-Pastor Baleux Castilla Rico Delepierre structure antibiotic C7.J. 1995; 270: 23520-23532Abstract Full Text PDF (115) With 18 bp (including stop codon), codons smaller mccA thus peptide-coding Tree Life. Since infer biosynthesis recruits more generic involved trimming acid(s) N terminus N-acetylation. cluster-encoded Actinobacteria seems exception (Chen 2019Chen Xu B. E. Wang Lu Ge Zn-dependent bifunctional processing class III lanthipeptides.Proc. Natl. Acad. Sci. U S 116: 2533-2538https://doi.org/10.1073/pnas.1815594116Crossref (29) As observation, Actinobacteria, free fungi myxobacteria. reasoned PLM4_2056 would selective installing belong distinct phylogenetic branch family. If cognate pentapeptide-encoding then easily 1B. conducted analogous CDSs (coding DNA sequences) flanking PLM4_2056. Assuming conservation bond, restricted pentapeptides tyrosine/histidine 3,300 PLM_2056-related sequences, specified majority Actinobacteria. increase confidence hits, filtered detected CDS Shine-Dalgarno motif 6–8 sequence, trees 4. where occurred vicinity half (indicated filled circles 4). Furthermore, clade seen example replicate motifs (denoted “2x” insight distribution, aligned using program MUSCLE. Peptides preceding variation, strong preference basic comparison global alignment Intriguingly, genomic loci PLM_2056 homologs together bytA-like motifs, suggests these encode much wider modifications, co-localization methyltransferases, sulfotransferases, ATP-grasp tailoring Together, represents treasure trove discovery efforts unexplored family.Figure 4Summary phylogeny P450s, associated biarylitide biosynthesisShow full captionIdentical hits colored printed right tree. Filled unfilled indicate without RBS, respectively. Selected byt-like illustrating variety contexts shared potentially (highlighted blue). Expanded clades detailed annotations accessible at: https://itol.embl.de/tree/2131271061176991591098016 (Letunic Bork, 2019Letunic I. Bork Interactive Life (iTOL) v4: developments.Nucleic W256-W259https://doi.org/10.1093/nar/gkz239Crossref (2715) Scholar).View Large Image ViewerDownload Hi-res image Download (PPT) Identical side BytO ranges 40% 50%. contrast, shows 20%–25% glycopeptide crosslinks. name tripeptides, carrying chains Accordingly, become YYH, YFH, YYY, YPW, comprehensive years ago several uncovered, mostly through searching peptide-related (Kloosterman 2020Kloosterman A.M. Shelton K.E. Wezel G.P. Mitchell D.A. RRE-finder: genome-mining tool class-independent discovery.bioRxiv. 14: 992123https://doi.org/10.1101/2020.03.14.992123Crossref (0) Montalbán-López 2020Montalbán-López Scott T.A. Ramesh Rahman I.R. Heel A.J. Viel J.H. Bandarian Dittmann Genilloud Goto al.New developments discovery, enzymology engineering.Nat. https://doi.org/10.1039/d0np00027bCrossref (155) can, current because extremely gene. highlights importance metabolomics unveil chemistry unexpected BGCs. here Russell Scholar, hindsight data, via homology occurring Pyxidicoccus (myxobacteria) strain. searches MxYxY/H-encoding within ±500 architecture variations short, exists expand families. byt small, poses nonetheless some biochemical challenges specificity crosslink. acid, often −1, might act signal processing. Further will needed understand timing requirements terminus. imparts proteolytic stability, understanding important applications stabilizing applications. Our biological activities limited. protease inhibitory suggest bioactivity. Thus, properties role organisms await discovery. established consisting genes: pentapeptide, representing Tabled 1REAGENT RESOURCESOURCEIDENTIFIERBacterial virus strainsEscherichia coli DH5α cellsBiolineBio-85026Escherichia ET12567/pUZ8002Macneil 1992NAStreptomyces M1152Flinspach 2010NAPlanomonospora ID82291Naicons collectionNAPlanomonospora ID107089Naicons ID114239Naicons ID46116Naicons collectionNAChemicals, recombinant proteinsApramycin sulfateSigma-AldrichCat# A2024-5GNalidixic acidSigma-AldrichCat# 1451000Diaion HP-20Sigma-AldrichCat# 13,605α-chymotrypsin bovine pancreasSigma-AdrichCat# C4129Chymostatin, microbialSigma-AldrichCat# C7268Deuterium oxideSigma-AldrichCat# 151882Critical commercial assaysPhusion polymeraseNEBCat# M0530STaq ligaseNEBCat# M0208ST5 exonucleaseNEBCat# M0663SAngiotensin Converting Enzyme Activity Assay KitSigma-AldrichCat# CS0002Deposited dataPlanomonospora genomeZdouc ScholarGenBank: JABTEX000000000biarylitide-YYH clusterthis paperGenBank: MW201788biarylitide-YFH MW201789iTOL-webserverthis paperhttps://itol.embl.de/tree/21312710611256691590408100iTOL- cladesthis paperhttps://itol.embl.de/tree/2131271061176991591098016OligonucleotidespSET_fwd: CCTCTCTAGAGTCGACCTGCAGCthis paperNApSET_rev: CCTTCCGTACCTCCGTTGCTthis paperNAbytAO_fwd: AGCAACGGAGGTACGGAAGGAGGAGGTGTGCGATGCGCthis paperNAbytAO_rev: GCAGGTCGACTCTAGAGAGGCTAGCGGGGGAGAAGGACthis paperNApSET-bytAO_fwd: GCGTCGATTTTTGTGATGCTCGthis paperNApSET-bytAO_rev: CAGCGAATTCGGAAAACGGCthis paperNARecombinant DNApSET152_ermE∗Bierman 1992Bierman M.I. Stein K.L. Snyder J.V. Plasmid cloning vectors conjugal transfer spp.Gene. 1992; 43-49Crossref (1194) ScholarNApSET-bytAOthis paperNASoftware algorithmsantiSMASH 5.0Blin al