dataesr/scientific-paragraphs-categorization huggingface dataset:
1. 1) in the core peptide (Fig. 78 ). Gene-deletion experiments led to the functional identication of the rst LanJ member, LtnJ, which is a zinc-dependent dehydrogenase responsible for the conversion of Dha to D-Ala during lacticin 3147 biosynthesis. 332 A recent investigation of carnolysin identied CrnJ, which is distinct from LtnJ in that it is capable of catalyzing the asymmetric reduction of Dhb residues to rare D-aminobutyrate residues (in addition to converting Dha residues to D-Ala). 61 Since CrnJ belongs to a different enzyme superfamily, the avin oxidoreductases,  333  LanJ enzymes are designated as being either LanJ A (zinc-dependent; InterPro family: IPR013149) or LanJ B (avin oxidoreductase). 333 Furthermore, a luciferase-like enzyme (LanJ C ) has been recently implicated in the reduction of dehydro amino acids in the biosynthesis of both class II and class V lanthipeptides.
Although additional biochemical and structural studies are required to further elucidate the TbtI mechanism, the data so far represent a fundamentally different use of SAM for this class of enzymes, reecting their exceptional catalytic diversity. 5. 3. 4 Cysteine sulfur methylation by PtyS in polytheonamide biosynthesis. 'Entotheonella factor', the uncultivated sponge symbiont that is the source of the polytheonamides (see Section 3. 12), contains in its genome a second proteusin-type BGC (pty locus). 369 Coexpression studies using precursor peptide-maturase gene combinations and E. coli as host revealed that PtyS, a SAM-dependent methyltransferase, catalyzes S-methylation of up to four Cys residues as the modication type. 369 Prior to these studies, S-methylation was known in non-ribosomal peptide biosynthesis but not for RiPPs.
377  In contrast, a range of synthetic heterocyclized plantazolicin analogs with truncated C-termini were all accepted, including a minimalistic Arg-Cys-based dipeptide with one thiazole moiety. 378, 379 The crystal structures of BamL and BpumL, which also included versions with bound truncated substrates (PDB: 4KVZ, 5DLY, 5DM0, 4KWC, and 5DM1), provided a rationale for this selectivity. 377 They revealed a plantazolicin-binding cle with a hydrophobic tunnel harboring the azoles units. Constrictions in the narrow cle provide an architecture that only allows access of heterocyclized substrates with planar azole moieties. The active-site pocket is sufficiently long to accommodate about the rst ve residues of the substrate. In agreement, K M values showed a tendency to increase with decreasing peptide length.
Phosphorylation during lasso peptide biosynthesis During efforts to expand the lasso peptide family (Section 3. 8) beyond members isolated and characterized from either proteobacteria or actinobacteria, a recent study focused on a family of BGCs found among rmicutes. Heterologous expression of a BGC from Paenibacillus dendritiformis led to the isolation of paeninodin, a lasso peptide bearing a phosphorylated Cterminal Ser residue. 162 Indeed, a unique feature conserved among the BGCs of rmicutes studied is a gene encoding a putative HPr kinase (InterPro family: IPR011104), which are typically involved in sugar uptake processes. Deletion of the HPr-related kinase gene padeK from the expression plasmid abolished production of phosphorylated paeninodin, which was recovered by in trans expression from an additional vector carrying padeK.
367 Genetic deletion of ptbO, a cytochrome P450 monooxygenase, resulted in the production of a GE2270 derivative lacking the hydroxyl moiety on the bcarbon of Phe8 implicating this enzyme in the hydroxylation process. To generate thiostrepton derivatives (Fig. 90B ), Streptomyces laurentii DtsrM (also called DtsrT), which cannot form quinaldic acid,  399  was fed with 7 0 or 8 0 -uorinated quinaldic acid analogs. 400 Rather than identifying thiostrepton analogs, only a truncated product was found in which the macrocycle containing the quinaldic acid was lacking. These ndings suggested that epoxidation of the quinaldic acid moiety is required prior to LP removal and subsequent cyclization by attack of the liberated amino group of Ile1 onto the epoxide (Fig. 90B ). TsrI (also called TsrP) and TsrK (also called TsrR) are homologous to cytochrome P450 enzymes (InterPro family: IPR001128) and are predicted to perform the bishydroxylation of Ile10 and epoxidation of the quinaldic acid.
Deletion of nosC resulted in an intermediate that lacked the hydroxyl on the pyridine ring, but also contained a bis-Dha tail. Recombinant NosC hydroxylated this intermediate on the pyridine ring. 401  cytochrome P450 enzyme is also believed to be responsible for the hydroxylation of a Pro residue in the biosynthesis of the lanthipeptide NAI-107,  402, 403  but this activity has not yet been reconstituted in vitro. P450-catalyzed hydroxylation of Phe has also been proposed in the biosynthesis of the thioamitide thioalbamide  11  and gene deletions in the thiovarsolin BGC suggest that VarO, a heme oxygenase (InterPro family: IPR016084), is involved in dehydrogenation of an arginine side chain. 14  5. 7. 3 Flavoprotein-catalyzed oxidation and halogenation 5. 7. 3. 1 Formation of the quinaldic acid of thiostrepton.
91 ). 5. 7. 3. 2 Oxime formation in azolemycins. Azolemycins are a family of LAPs (Section 3. 4) produced by Streptomyces sp. 404  The azolemycins contain unique oxime moieties installed on the N-terminal amine of Val1 with either the E or Z stereochemistry (Fig. 92 ). Subsequent experiments using Marfey's reagent permitted the conguration of all stereocenters in azolemycin A to be elucidated. In addition to genes encoding the canonical apparatus necessary for the introduction of the characteristic azoles in the biosynthesis of other LAPs (Section 3. 4), the BGC encodes a SAM-dependent MT (AzmE), and a avin-dependent monooxygenase (AzmF, InterPro family: IPR036661). 404  Subsitution of the azmF gene with a selection marker in Streptomyces sp. lead to abolishment of azolemycin A-D production.
2 ), lipolanthines (Fig. 11 ), lanthidins (Fig. 18A ), and linaridins (Fig. 27 ). Recent studies have explored the biosynthetic LanD avoenzymes (InterPro family: IPR003382) involved in the oxidative decarboxylation of the Cterminal Cys for various RiPPs. NAI-107 is a class I lanthipeptide containing a C-terminal AviCys that is installed by MibD, a avin-dependent decarboxylase. Recombinant MibD displayed decarboxylation activity of the terminal Cys residue in both full length MibA as well as in the MibA core peptide. Thus, the enzyme does not require the LP nor do the thioether rings need to be installed rst. However, MibD did not display the large substrate tolerance previously described in early studies of the lanthipeptide decarboxylase EpiD,  405  and instead tolerates a more limited sequence motif.
The oxidative decarboxylation of the linaridin cypemycin is catalyzed by CypD,  406  whose crystal structure was solved (PDB: 6JDD). The enzyme is structurally similar to previously characterized lanthipeptide LanD proteins despite low sequence similarity. Investigation of AviCys formation for the lanthipeptide mutacin 1140 showed that a lanthionine was formed in the absence of the MutD decarboxylase,  407  suggesting that oxidative decarboxylation is not required for ring formation. Co-expression of TvaA, the precursor peptide to thioviridamide, with the decarboxylase TvaF in E. coli led to the production of a C-terminal thioenol, consistent with observations for AviCys biosynthesis in lanthipeptides. TvaF can modify both the full length TvaA and a mutated core peptide, TvaA S8A (2-  13) , albeit at a signicantly lower rate, indicating that the leader peptide is not strictly required but likely assists with substrate recognition by TvaF.
129  The biosynthesis of the lanthidin cacaoidin has not yet been investigated but its BGC contains a LanD-like decarboxylase (Fig. 18B ). 54  For lipolanthines, the LanD decarboxylase MicD was shown to work in concert with the MicKC labionin synthetase, with the activity of both greatly affected by the presence of its partner. 88  5. 7. 3. 4 Chlorination of NAI-107 and ammosamide A. All known Trp halogenases (InterPro family: IPR006905) prior to 2013 used L-Trp as the substrate. Recently, two enzymes have been identied that act on Trp imbedded in a peptide. The lanthipeptide NAI-107 contains a 5-chloro-Trp residue that is generated by a FADH 2 -dependent halogenase, MibH. MibH collaborates with MibS, the cognate NADH-dependent avin reductase. 408 MibH exhibits unusual substrate specicity, because the enzyme was incapable of chlorinating free Trp, Trp in a linear peptide, and Trp in mutacin 1140, which has a similar set of rings as NAI-107.
422, 423 These enzymes (InterPro family: IPR031037) therefore signicantly expand the cyanobactin structural diversity and also represent promising tools for biocatalysis. Although the enzymology of cyanobactin prenylation has been reviewed previously, 1 more recent structural, biochemical and molecular dynamics studies have shed further light on the basis of broad substrate tolerance of the TruF family of PTases and are summarized here. Co-crystal structures of the Tyr O-prenylating enzyme PagF from the prenylagaramide biosynthetic pathway from Oscillatoria agardhii in complex with linear (PDB: 5TU4 and 5TU5) and macrocyclic (PDB: 5TU6) peptide substrates have highlighted a number of key features that explain the high substrate promiscuity of TruF-type PTases, despite the absence of obvious recognition sequences.
416 tructural data revealed that PagF (PDB: 5TTY) comprises an a/b PT barrel fold like those observed in bacterial ABBA enzymes and fungal indole PTases. 424 However, in PagF, the barrel fold is truncated, resulting in a solvent-exposed channel that must be occluded by the peptide substrate to prevent nonproductive quenching of the dimethylallyl pyrophosphate-derived carbocation intermediate, a role performed by structural elements in characterized smallmolecule PTases. Binding of peptide substrate in PagF create a deep, encapsulated hydrophobic cavity that effectively shields the allylic carbocation from solvent, as observed in cocrystal structures with a dimethylallyl S-thiolodiphosphate analog. 416 A series of hydrophobic residues line the circumference of the active-site tunnel that further protect reactive intermediates.
93A ). In some cases, such as the polytheonamides (Fig. 47 ; Section 3. 12), this structure is further methylated by rSAM enzymes (Section 5. 3. 2), whereas for other compounds the ketone is reduced to the corresponding alcohol, either by a dehydrogenase in the BGC, or by an unknown enzyme (e. g. Fig. 2 ). In the case of the biosynthesis of the lanthipeptide epilancin 15X the reduction is catalyzed by the NADHdependent oxidoreductase ElxO encoded in the BGC and has been reconstituted in vitro. 312 1] [432]  Incubation of a biosynthetic intermediate containing a C-terminal Dha with recombinant NosA resulted in enamide hydrolysis to afford a Cterminal amide (Fig. 93B ). A crystal structure of the enzyme (PDB: 4ZA1) provided insights into the mechanism of amide View Article Online formation. 431 NosA analogs are encoded in the BGCs of a number of other amidated thiopeptides such as nocathiacin, 433 GE2270A,  367  and berninamycin  434  suggesting a common mechanism of C-terminal amide formation.
412 For duramycin the crystal structure of this protein (DurN) bound to its product or biosynthetic intermediates (PDB: 6C0G, 6C0H, 6C0Y) demonstrated that the enzyme does not seem to make any contacts with the substrate near the site of the chemical reaction. Instead the enzyme binds the substrate to hold it in a reactive conformation and a hydroxylated Asp in the substrate appears to act as the base that deprotonates the Lys nucleophile. Biochemical and structural studies showed that the hydroxylation of this Asp is critical for lysinoalanine formation. 412 Thus, prior PTM of an Asp in the substrate, as discussed in Section 5. 7. 4. 1, is required for the formation of the next PTM. Such a strict order of PTMs is not uncommon in RiPP biosynthesis, but the substrate-assisted nature of lysinoalanine formation is at present unique.
g. contains Cys and Thr/Ser). BAGEL requires prokaryotic nucleotide sequence (FASTA format) as input. The workow of BAGEL4 (ref. 445) is as follows: rst, the program performs a six-frame translation. Second, the hypothetical proteins are screened for the presence of motifs found in modication enzymes for the dened RiPP classes. The third step entails dening the so-called areas of interest in the genome, based on the hits of step 2. In the nal step, these areas are analyzed in detail, i. e. rst genes are called using GLIMMER  446  and annotated based on Basic Local Alignment Search Tool (BLAST)  447  with the UniRef90 database. Modi-cation enzymes are annotated based on protein domains they entail. Subsequently, all possible small open reading frames (ORFs) are called up to a set maximum size.
RODEO Rapid ORF Description & Evaluation Online, RODEO (http:// rodeo. scs. illinois. edu/), a genome-mining tool reported in 2017, was the rst to use machine learning to predict RiPP precursor peptides (Table  2 ). 154 The workow of RODEO is as follows: when a protein or a list of proteins are used as input queries, the algorithm retrieves the relevant genomic records from GenBank. RODEO then uses prole Hidden Markov Model (pHMM)-based analysis to annotate the function of genes anking the query protein(s). To do so, RODEO utilizes the HMMER toolkit  451  and compares proteins to Pfam, TIGRFAM, or customized HMM databases. 452, 453 Since RiPP precursor peptides are oen small and hypervariable, they frequently are not predicted as protein-coding sequences. Therefore, RODEO performs a six-frame nucleotide translation of the intergenic regions in search for possible precursor peptides.
RiPPER RiPP Precursor Peptide Enhanced Recognition (RiPPER; https://github. com/streptomyces/ripper) is a tool meant for the class-independent discovery of RiPPs. 14 RiPPER leverages RODEO to fetch nucleotide data corresponding to a query protein(s) and its local genomic neighborhood. Unlike RODEO that performs translation on all six frames in the intergenic regions of the query,  154  RiPPER employs a modi-ed version of Prodigal (PROkaryotic Dynamic programming Gene-nding Algorithm) to identify candidate precursor peptides (20-120 amino acids). 14 Prodigal is an algorithm that uses features such as ribosomal binding sites, GC content and codon bias to identify bacterial coding sequences. 458 Since RiPP precursors tend to be in the same direction as that of the tailoring enzymes, Prodigal scores are enhanced if the identied precursor peptide is encoded as such.
The top three high-scoring gene products within 8 kb from the gene encoding the query protein, along with additional genes scoring beyond a threshold Prodigal score, are listed in a text le with associated information such as conserved domains and Prodigal score, among others. 14 ultiple peptides are retrieved per query, which may not all be plausible precursor peptides as shown by test analyses on lasso peptides, graspetides, and thiopeptides. Therefore, peptide similarity networks and BGC analysis must be done on multiple queries in parallel to identify possible precursors. This approach was employed to analyze 229 TfuAcontaining BGCs in Actinobacteria (Section 3. 5), revealing the diversity of thioamidated RiPPs. This dataset was leveraged to isolate thioamidated tripeptides, termed thiovarsolins, from Streptomyces varsoviensis via heterologous expression.
NeuRiPP NeuRiPP is another addition to the suite of tools for RiPP precursor peptide prediction (https://github. com/emzodls/ neuripp). Aimed at tailoring enzyme-independent prediction of RiPP precursor peptides, NeuRiPP employs deep neural networks trained on experimentally validated RiPPs, and precursors predicted with a high degree of condence by existing RiPP genome mining tools. 459 In addition to identifying precursor peptides predicted by RODEO  which hit HMMs developed for precursor peptides of known RiPP classes. 459 NeuRiPP awaits validation as new compounds are yet to be isolated from NeuRiPP-predicted precursor peptides. Like RiPPER, NeuRiPP also lacks a web-based interface and thus is currently restricted to users comfortable with a command-line interface.
RiPPMiner RiPPMiner is an online resource (http://www. nii. ac. in/ rippminer. html) that offers tools, based on a curated database,  460  allowing users to assess if a protein sequence is RiPPrelated and to which known class it belongs. Additionally, RiPPMiner attempts to predict the LP cleavage site for lanthipeptides, cyanobactins, lasso peptides, and thiopeptides. The curated database of RiPPs was generated from previously published databases and currently entails 513 records. This database was used for machine learning to generate a support vector machine able to discriminate RiPPs from other small proteins. To predict LP cleavage sites, a random-forest approach is employed. RiPPMiner also predicts cross-links within RiPP structures based on a support vector machine classication. Recently a option was added that allows the user to upload genomic data (FASTA format) to mine for RiPP BGCs.
RiPPQuest RiPPQuest is an online program (https://gnps. ucsd. edu/) that matches tandem MS data to RiPP BGCs encoded by a particular organism. 461 The search space is limited to the vicinity of the encoded RiPP biosynthesis genes identied in the genome. The generated database for the proteomic search includes an extensive number of peptide modications to enable RiPP discovery. Although it is desirable to couple genetic potential with experimental validation, this also means that detection depends on nding the producing culture conditions and sample preparation. This platform can help to further automate RiPP discovery and enables researchers to go beyond genomebased identication in a more automated way.
Tailoring reactions based on domains identied in the BGC are then virtually performed on potential sites in the predicted core peptide, to generate a combinatorial library of hypothetical structures for each identied RiPP BGC. 456, 463 One caveat, also common to many other RiPP genome mining tools,  154, 454, 457  is that all predictions are based on homology to proteins associated with known RiPP classes. As a result, RiPP-PRISM cannot identify novel RiPP families and also cannot predict novel PTMs. Nevertheless, RiPP-PRISM is unique among other RiPP genome mining tools by facilitating comprehensive analysis of genomes, beginning with the identication of potential RiPP BGCs and providing a library of possible structures, thus enhancing the prospects of RiPP discovery. 456, 463 RiPP-PRISM was used to perform a global analysis on 65 421 prokaryotic genomes in the database of the National Center for Biotechnology Information, which predicted 30 261 RiPP BGCs that would encode 2231 potentially unique RiPP products.
289 Searches on IMG-ABC can be performed using attributes associated with the secondary metabolite or the BGC of interest. Alternatively, IMG-ABC can also be browsed for secondary metabolites and BGCs by taxonomy, ecosystem, natural product/BGC type, gene count, and Pfam information for functional assignment of neighboring genes. IMG-ABC provides the most comprehensive information on secondary metabolites and BGCs in genomic and metagenomic data in IMG, with links to external databases and tools within IMG allowing users to perform large-scale analyses with any desired level of detail. 289, 471, 472 10 DeepRiPP DeepRiPP (http://deepripp. magarveylab. ca/) is a recent addition to the genome mining tools specic for RiPPs, and similar to RODEO, the tool employs a machine learning based approach.
RRE-Finder RRE-Finder (http://rodeo. scs. illinois. edu/), another recent addition to the RiPP genome mining toolkit, facilitates detection of RRE domains (Section 4. 1. 1) in known and potentially novel RRE-dependent RiPP classes. 482 The tool is available to the users in two modes of operation: precision mode and exploratory mode. Precision mode utilizes 35 custom HMMs built using RRE domains from largely known RiPP classes but includes a few high-condence RREs from predicted new RiPP classes. The intent is for precision mode to enable classdependent discovery of RRE domain-containing proteins. Exploratory mode uses a truncated version of the HHPred 483 workow and a custom database of $2400 sequence-divergent RREs to facilitate discovery of novel RRE-dependent RiPP classes. Using RRE-Finder working in precision mode, $35 000 RRE domain-containing proteins were identied in the Uni-ProtKB protein database,  484  revealing many novel, yet-to-be explored, RRE-dependent RiPP classes.
RRE-Finder is available to the user as a downloadable command-line tool. The precision mode of RRE-Finder has been integrated into RODEO (Section 6. 2) and will be integrated into the future release of antiSMASH (Section 6. 8). 6. 12 decRiPPter decRiPPter (https://github. com/Alexamk/decRiPPter) is yet another recent addition to the growing list of class-independent RiPP genome mining tools. 56 This algorithm also utilizes machine learning, specically, a support vector approach, in combination with a pan-genomic analysis to identify genes associated with RiPP biosynthesis within operon-like contexts. Two unique features consider whether these genes are distinct from the core genome (i. e. part of the accessory genome) and whether they encode sequences that overlap with known biosynthetic proteins already in the antiSMASH and MIBiG databases.
3). 490  The addition of tailoring enzymes from different BGCs to existing pathways has also been accomplished by combining dehydro amino acid reductases (Section 5. 1. 2) or C-terminal decarboxylases (Section 5. 7. 3. 3) with the nisin biosynthetic machinery to form hybrids in vivo. 498  These additional secondary post-translational tailoring modications do not rely on LP recognition. Similarly, HcaDF-ProcM modied peptides were expressed together with either the dehydrogenase NpnJ (InterPro IPR013149) or the decarboxylase MibD (InterPro family: IPR003382) in E. coli. 496  Further modication using other LP-independent enzymes or PTM enzymes activated by covalently attached cognate LPs (vide supra) might further enlarge the repertoire of available PTMs in the future. A different approach to producing non-cognate RiPPs uses chimeric substrates composed of leader and core peptides from different members of a certain class.
Chemical oxidation of selenocysteine derivatives produces the dehydroalanines that serve as a substrate for pyridine formation catalyzed by TclM (InterPro family: IPR023809) from the thiocillin gene cluster (Section 3. 2). FIT also was used to test different arrangements of the LP recognition motifs for LynD and TclM in order to obtain the correct PTMs. 490 ore recently, the FIT system has been also combined with recombinantly produced lactazole biosynthetic enzymes, achieving in vitro biosynthesis of the thiopeptide lactazole A and its analogs. 512 Stepwise addition of the enzymes to the precursor peptide resulted in either under-or overdehydrated thiopeptides while selective production of the native lactazole A occurred only when the full enzyme set was present from the beginning, implying orchestrated actions of the enzymes during cooperative maturation of the thiopeptide.