dataesr/scientific-paragraphs-categorization huggingface dataset:
33. 118 pproximately 10% of methanogens lack an identiable tfuA in their genome. TfuA-independent YcaOs from Methanocaldococcus jannaschii (Mj-YcaO) and Methanopyrus kandleri (Mk-YcaO) efficiently catalyzed thioamide installation on a synthetic 11-mer McrA substrate that contained ve residues upstream and downstream of the modied Gly. 118 Nucleotidebound crystal structures were obtained for Mk-YcaO (PDB: 6CI7), which displayed an overall architecture similar to LynD (azoline-forming YcaO; PDB: 4V1T) and Ec-YcaO (orphan YcaO; PDB: 4Q86) (Section 3. 4). While the nucleotide-binding residues are largely conserved across these proteins, the active site region of Mk-YcaO is different from the azoline-forming YcaOs, hinting towards a divergent substrate-binding site or reorganization upon substrate binding.
118 More recently, Mj-YcaO was co-crystallized with a synthetic 11-mer peptide substrate derived from the MCR a-subunit (Mj-McrA). 119 Mj-YcaO bound to Mj-McrA (PDB: 6PEU) (Fig. 34 ) represents the rst reported structure of any YcaO protein bound to its peptide substrate and permitted denitive identication of residues involved in peptide binding. This study also revealed that when presented with an 11-mer peptide variant containing a Cys residue near the site of modication, Mj-YcaO was capable of forming a thiazoline. 119 8] [119] [120] [121]  Mj-YcaO, however, lacks a basic residue for initial Cys deprotonation and cyclodehydration activity was observed only when a surrogate base was supplied in the assay. This cyclodehydration activity further supports the idea that all YcaO proteins modify their substrates via a shared mechanism and that it is the identity of the nucleophile attacking the amide carbonyl carbon that determines the chemical outcome.
8 and 5. 9). 135 PatA homologs are the only biosynthetic enzymes common to all predicted cyanobactin BGCs, as exemplied by genome mining studies. 133 This renders PatA and its orthologs as the class-dening enzyme for cyanobactins. 3. 6. 1 Class-dening enzyme: N-terminal protease, PatA. PatA is composed of an N-terminal protease domain and a Cterminal domain of unknown function. The crystal structures of the protease domains of PatA (PatA Pro ; PDB: 4H6V) and a related ortholog, PagA (PagA Pro ; PDB: 4H6W) have been determined. 136, 137 Both PatA Pro and PagA Pro (Fig. 36 ) feature the classic subtilisin-like fold but lack the high affinity metalbinding site conserved across the subtilisin protease family that is necessary for activation of the propeptide. Although substrate-bound PatA Pro structures are not available, the GLEASVT motif comprising RSII has been modeled into PatA Pro by comparison to the structure of thermitase complexed with its substrate eglin-C in order to understand the substrate speci-city determinants.
Sactipeptides: introduction and biosynthesis Sactipeptides (Fig. 39 ) are RiPPs typied by sulfur-to-a-carbon thioether (i. e. sactionine) linkages between a donor Cys and an acceptor residue. Thus far, six sactipeptides, namely subtilosin A, thurincin H, thuricin CD (two peptide products), sporulation killing factor (SKF), and huazacin have been characterized from various Bacillus species, most of which exhibit narrow-spectrum growth inhibitory activity towards other Firmicutes. 1, 43 Aer ribosomal synthesis of the sactipeptide precursor, sactionine linkage(s) are installed by a radical S-adenosylmethionine (rSAM) enzyme (InterPro family: IPR007197). The initial demonstration of this activity was with AlbA, which acts on the subtilosin precursor, SboA, in a LP-dependent manner. 172 The subtilosin BGC also encodes proteases and transporters, which are predicted to convert the thioether(s)-bearing precursor peptide into mature subtilosin.
For instance, SkfB installs one sactionine linkage during the maturation of the SKF, which is produced by Bacillus subtilis 168 under nutrient limiting conditions to delay sporulation. 176 In addition to the canonical [4Fe-4S] cluster, sactisynthases oen display auxiliary iron-sulfur clusters (AISC) which are found in a C-terminal domain. When there are two additional [Fe-S] clusters, the domain has been called a SPASM domain (InterPro family: IPR023885, dened in Section 2. 14). 48 ith the exception of anaerobic sulfatase-maturing enzyme,  177  the other founding members of 'SPASM' are RiPP maturases. Some sactisynthases, such as SkfB, feature a truncated SPASM domain, dubbed a "twitch" domain, which binds one auxiliary [Fe-S] cluster. 178 Numerous RiPP-related rSAM proteins contain AISC domains; however, these domains are not always retrieved by the SPASM/twitch model as the location and number of Cys is highly variable.
181 This nding, along with studies using AlbA variants that lack the AISC, points towards a role in thioether installation, but the precise role played by the auxiliary clusters remains unclear. 172, 176, 179, 181 he only sactionine-forming rSAM enzyme that has been structurally characterized thus far is SkfB. SAM-bound SkfB (PDB: 6EFN) displays an overall modular architecture composed of a core rSAM domain anked by an N-terminal RRE (Section 4. 1. 1) and the C-terminal AISC (twitch) domains (Fig. 41 ). 178 The rSAM domain is folded into a partial (b/a) 6 triose phosphate isomerase (TIM) barrel, characteristic of rSAM enzymes where the b-sheets line the inside of the barrel and a-helices ank the exterior. The SAM-binding [4Fe-4S] cluster resides in the loop region following the b1 strand in the core domain.
Ranthipeptides: introduction and biosynthesis A bioinformatic investigation of the rSAM superfamily identi-ed a group of Cys-rich peptides, primarily from the Clostridium genus, called SCIFFs (six Cys in forty-five residues). 42 These peptides were encoded next to a gene encoding a rSAM enzyme and thus were predicted to be substrates for post-translational modication. Although no mature SCIFF-derived natural product has ever been isolated, puried rSAM enzymes from predicted SCIFF-containing BGCs, CteB and Tte1186, installed a single thioether linkage between a donor Cys and an acceptor Thr on cognate precursor peptides, CteA and Tte1186a, in vitro. 183, 184 A more recent bioinformatic analysis conducted on rSAM maturases predicted to install thioether linkages, including sactisynthases and SCIFF maturases, revealed that CteB (and Tte1186) is more closely related to QhpD (Interpro family: IPR023886) than to any sactisynthases.
43 QhpD is involved in the maturation of quinohemoprotein amine dehydrogenase (QHNDH), an enzyme that catalyzes the oxidative deamination of aliphatic and aromatic amines. 185 QhpD installs two b-thioethers and one g-thioether linkage between Cys and Asp/Glu residues in the small subunit of QHNDH, QhpC (Interpro family: IPR015084). 185 In contrast to sactionines, which readily fragment under standard collision-induced dissociation (CID) conditions, no inter-ring fragmentation by CID was reported for the SCIFF-derived products,  183, 184  which is reminiscent of lanthipeptide-like behavior. Quantum mechanical calculations performed on model, isomeric peptides that were linked by either a sactionine or a b-thioether revealed that the b-thioether linked isomer was more stable, providing a possible explanation for the differential behavior of SCIFFderived products vs.
1), and sactipeptides that include sactionines installed by rSAM enzymes (Section 3. 9). 43, 44. 10. 1 Class-dening enzyme: rSAM enzyme installing non-a thioether linkages. All characterized ranthipeptide maturases [i. e. CteB, Tte1186, NxxcB (the NxxC peptide maturase, Fig. 14 ), and PapB (freyrasin maturase)] are bioinformatically predicted to be SPASM domain (InterPro family: IPR023885)-containing rSAM enzymes. 44, 183, 184, 186 hile Tte1186 and CteB are known to possess two AISCs,  178, 184  NxxcB and PapB have been shown to harbor at least one AISC and they have additional conserved Cys residues, which suggests they possess two AISCs. 44, 186 Two mechanisms have been proposed for the installation of b-thioethers in the NxxC peptide. 44 The proposed mechanism begins with the generation of a Cbcentered radical by the 5 0 -dAc produced as a result of reductive cleavage of SAM by the primary [4Fe-4S] + cluster.
44 his observation is suggestive of ligation of the substrate Cys thiolate with the [4Fe-4S] cluster also observed in sactionine synthesis,  172  consistent with the proposed radical mechanism. When the reaction was carried out in deuterated water, deuterium was not incorporated into the product, suggesting that the polar mechanism is not likely operative. Although not conclusive, this study provides a working model for studying the mechanism of non-a thioether installation in ribosomal peptides. CteB, originally proposed to be a sactisynthase, is the rst ranthipeptide maturase and thioether-forming rSAM enzyme to be structurally characterized. The crystal structure of CteB (PDB: 5WGG) was obtained with bound SAM and a truncated version of the substrate CteA comprising residues 1-21 (CteA1-9 and 21 were resolved in the structure).
188 owever, investigation of orthologous rSAM proteins from Streptococcus suis and Streptococcus agalactiae (SuiB and AgaB, respectively) pointed to the proteins having two AISCs. 192 This conclusion was later veried by X-ray crystal structures of SuiB bound to SAM and the SuiA precursor peptide (PDB: 5V1S and 5V1T). 193 Both radical addition and electrophilic aromatic substitution (EAS) mechanisms have been proposed for C-C bond formation in streptides (Fig. 45 ). 188, 194 Each pathway begins with the abstraction of a H-atom from the b-methylene of Lys by 5 0 -dAc to generate a substrate radical. In the radical addition mechanism (A, Fig. 45 ), this C-centered radical reacts with the indole side chain of Trp to generate an indolyl radical. Deprotonation, rearomatization, and  In the EAS mechanism, the lysyl radical undergoes deprotonation at the a-position, and electron transfer to the AISC, leading to the generation of an a,b-unsaturated amide.
Graspetides including microviridins: introduction and biosynthesis Microviridins (Fig. 51 ) are compounds characterized by a cagelike structure, resulting from two lactone linkages between the side chain carboxyl group of Asp/Glu and the hydroxyl group of Ser/Thr, and a lactam linkage between the d-carboxyl group of Glu and the 3-amino group of Lys. Microviridins inhibit serine proteases such as trypsin, chymotrypsin, subtilase, elastase, and the 20S proteasome, and have been recognized for their potential use in the treatment of pulmonary emphysema and some cancers. 1  Their biosynthesis was elucidated using in vitro and in vivo studies by two independent groups. 206, 207 The lactones and lactams are sequentially installed on the precursor peptide by two dedicated ATP-grasp ligases, MvdD/MdnC and MvdC/MdnB (InterPro families: IPR026446 and IPR026439), that are homologous to the RimK ATP-grasp ligases involved in the maturation of the ribosomal protein S6.
3. 15. 1 Class-dening enzyme: macrolact(one/am)installing ATP-grasp ligase. The crystal structures of MdnB and MdnC (PDB: 5IG8 and 5IG9), involved in the biosynthesis of microviridin J have been determined, which aided in understanding the basis for precursor peptide recognition by this class of RiPP modifying enzymes. 212 MdnB and MdnC display highly similar structures overall, with the three characteristic domains observed across the ATP-grasp superfamily (Fig. 52 ). The lactone-forming MdnC was crystallized with bound MdnA precursor peptide (and variants). These structures revealed that the strictly conserved "PFFARFL" motif in the precursor peptide, previously shown to be important for biosynthesis through bioinformatic and mutational studies,  213, 214  binds as an a-helix to the a7-helical region in the central domain of MdnC (Section 4.
55B ). 225 In addition to the precursor peptide, the BGC contains 14 genes (Fig. 55A ). Gene disruption experiments supported the association of many of these genes with ustiloxin production, with the two proteins that have no characterized homologs, UstYa and UstYb (Protein family: PF11807; DUF3328; InterPro family: IPR021765), being required for production. 17, 226 equence alignment of more than 200 UstY homologs that were all found in aspergilli suggested N-terminal transmembrane helices. 17 sing an algorithm developed by the authors to detect highly repeated peptide sequences, other examples of genes encoding a repeated core peptide with an N-terminal signal peptide were detected in Aspergillus and Fusarium as well as the rice pathogen Ustilaginoidea virens, the producer of ustiloxins A and B.
Amatoxin biosynthesis and class-dening enzyme Amatoxins such as a-amanitin are N-to-C cyclized peptides produced by mushrooms (Fig. 57 ). They also contain Cys-Trp crosslinks (tryptathionine) and various hydroxylations. Amatoxins are produced as a linear precursor peptide that contains a leader peptide, core peptide and C-terminal recognition sequence. 1 Several recent studies have provided new insights into the biosynthesis of the amatoxins (Fig. 57 ). Given that the core peptides for a-amanitin are anked by Pro residues, the involvement of a prolyl oligopeptidase (POP; InterPro family: IPR001375, IPR002470) had been previously proposed in amatoxin biosynthesis, but cyclization had not been experimentally veried. In vitro activity of a POP from Galerina marginata showed the enzyme catalyzes two reactions during the biosynthesis of a-amanitin.
Biosynthesis of N-to-C cyclized peptides including cyclotides and orbitides Head-to-tail cyclized RiPPs include large peptides  236, 237  and also many cyanobactins from cyanobacteria (Section 3. 6), the fungal amatoxins (Section 3. 18), and the plant-derived cyclotides and orbitides. 1 In recent years, several of the enzymes that catalyze the cyclization step have been experimentally characterized. The Pro oligopeptidase involved in amatoxin cyclization was discussed in Section 3. 18, and structures of the cyanobactin macrocylase PatG (Section 3. 6) were solved in 2012. 238 This section will discuss the newly discovered or newly characterized members of the plant asparaginyl endoprotease family (AEP, C13 protease family; InterPro family: IPR001096) including butelase 1 (ref. 239) and OaAEP1b, 240 a cyanobactin macrocyclase from Oscillatoria sp.
58 ). 243 Mutagenesis experiments demonstrated that substrate recognition is governed by an 11-16-residue follower peptide that is removed in the cyclization step. 243 The crystal structure of PCY1 (PDB: 5UW3) showed that the enzyme utilizes the cleaved C-terminal follower peptide of the substrate to attain a conformation that promotes macrocyclization. 248 This nding explained why the enzyme produces nine different orbitides of different sequence and ring size from substrates that have highly conserved follower sequences with especially high conservation in the C-terminal six residues that were visualized in the structure. Unlike the mostly hydrophobic interactions used for LP recognition in the biosynthesis of class I lanthipeptides, cyanobactins, lasso peptides and PQQ (see Section 4. 1.
59A ). 19 One of these genes encodes the novel peptide ligase, PGM1, that was shown to catalyze amide bond formation between (S)-2-(3,5-dihydroxy-4methoxyphenyl)-2-guanidinoacetic acid and a peptide derived from the precursor peptide encoded by pgm2 (Fig. 59B ). PGM1 is an ATP-grasp enzyme (dened in Section 3. 15) that connects a ribosomally synthesized peptide with the a-guanidino acid. The enzyme was demonstrated to be tolerant to changes in both the amino acid and the peptide allowing preparation of pheganomycin analogs. This substrate tolerance was analyzed by site-directed mutagenesis and structure determination of the enzyme (PDB: 3WVQ and 3WVR). 19 PGM4, PGM7, PGM8 and PGM12 were hypothesized to convert 2-(3,5-dihydroxyphenyl)-2oxoacetic acid to the N-terminal guanidino acid, whereas PGM3, a rSAM methyltransferase, was proposed to C-methylate Val to tert-Leu (see Section 5.
271 While the N-terminal sequence was shown to be essential for PaaA catalysis, removal of the follower still allows the rst condensation reaction to occur (by either route described previously). Structural studies have also been conducted for PaaA (PDB: 5FF5) and revealed the presence of an RRE domain (Section 4. 1. 1) that interacts with PaaP and is believed to direct the core peptide into the enzyme active site. More recently, mRNA display was employed to gain further insights into enzyme-substrate interactions, revealing broad enzyme promiscuity for PaaA outside of its core and predicted binding epitope. 272 This work highlights the potential of mRNA display-based techniques for studying RREs, in addition to creating new possibilities for introducing the indolizidinone moiety into synthetic peptide libraries.
MbnB, a previously uncharacterized protein belonging to DUF692 encoded within methanobactin BGCs, was identied as a potential candidate for this transformation based on low sequence similarity to members of a TIM barrel family that include representatives bearing divalent metal cofactors. 275 Heterologous co-expression experiments revealed that MbnB forms a heterotrimeric complex with MbnC, a protein of unknown function, and the precursor peptide MbnA, suggesting a role of MbnBC in biosynthesis. Spectroscopic studies further revealed the presence of a multinuclear Fe (di/tri) cofactor within MbnB,  274  consistent with the as-yet unpublished crystal structure from a homolog from Haemophilus somnus 129Pt (PDB: 3BWW) that possesses a diiron cluster for which no activity has yet been reported.
coli of the pallidocin S-glycosyltransferase and export protein with His 6 -tagged leaderless core peptides that were identied in two other Bacillus genomes, provided two additional examples of antibacterial glycocins. 288. 25. 1 Class-dening enzyme: combination of glycosyl transferase family 2 and peptidase C39. A recent bioinformatic survey using the Integrated Microbial Genomes-Atlas of Biosynthetic gene Clusters (Section 6. 9) for the combination of a SunS-like glycosyltransferase (Protein family PF00535) and a PCAT bifunctional peptidase-transporter (Section 4. 4. 1) 289  suggests that the glycocins are more phylogenetically widespread and structurally more diverse than previously anticipated. 279.
In addition, Glu(À11) and Glu(À8) of the LP made salt bridges and hydrogen bonds with LynD (Fig. 66 ). Using a combination of crystallographic data from these cocrystal structures and bioinformatic approaches, it was demonstrated that biosynthetic pathways from many different RiPP classes, including cyanobactins, LAPs, class I lanthipeptides, thiopeptides, microcins, lasso peptides, sactipeptides, PQQ/mycofactocin, proteusins, and bottromycins use the same architectural domain to recognize the LP. 66 This RiPP precursor Fig. 65  (A) Co-crystal structure of NisB with the dehydrated NisA peptide; only the LP is resolved (PDB: 4WD9). 49 The LP binds in antiparallel fashion to b3 of the RRE with further interactions to a3. (B) Interactions between a highly conserved motif on the LP of NisA (the FNLD motif) with pockets and amino acids of the RRE of NisB.
Hydrogen-deuterium exchange MS performed on LP-bound RRE showed decreased hydrogen-deuterium exchange at the ahelices and b3 strand, suggestive of an interaction with the LP. The b1 and b2 strands showed increased exchange consistent with exibility/exposure aer binding. These data, along with mutational studies, support a model for substrate recognition in line with that observed for other RREs, where the LP lies along the a3 helix and interacts with b3 to extend into a four-stranded antiparallel b-sheet. 66, 157 he recent crystal structures of discrete RRE proteins TfuB1 (also referred to as FusE) and close orthologs (PDB: 6JX3, 5V1V, and 5V1U) bound to cognate LPs support this model. 160, 161 LP binding was shown to be primarily mediated by hydrophobic interactions with the RRE (Fig. 68 ).
1. 3). Microcin C (McC, Fig. 73A ) is an unusual RiPP because it lacks a LP on its precursor peptide. Therefore, biosynthetic enzymes must use an alternative method for substrate recognition. The N-formylated precursor peptide was shown to be a better substrate than the desformyl analog for MccB, the adenylyltransferase responsible for forming the phosphoramidate linkage in McC. 297 Analysis of MccB in complex with Nformyl MccA depicted the N-formyl moiety in a pocket formed by two adjacent monomers of an MccB homodimer (Fig. 73B ; PDB: 6OM4) explaining the importance of N-formylation. 298 hereas the heptapeptide microcin C from E. coli is biosynthesized from a precursor lacking a LP, related compounds are made from longer precursor peptides with an N-terminal extension that is released proteolytically.
Leader peptide removal Removal of the LP is a critical step in RiPP biosynthesis and is oen coupled to export of the mature compound. The LP cleaving enzyme varies between different RiPP classes, but common strategies have been recognized as discussed below. 4. 4. 1 AMS/PCAT bifunctional transporters. Genes encoding peptidase-containing ATP-binding cassette (ABC) transporters [PCAT; also called ABC-transporter maturation and secretion (AMS) proteins] are oen found clustered with the core biosynthetic genes in RiPP BGCs. This family of enzymes serves a dual function being responsible for coupling the hydrolysis of the LP through an N-terminal papain-like C39 peptidase domain (InterPro family: IPR005074) with export of the cleaved core peptide by the C-terminal ABC-type transporter (InterPro family: IPR027417).
309 imilarly, bifunctional AMS proteins are found in all four classes of lanthipeptides, 62 but in some instances, the transport and proteolysis activities are encoded in two different polypeptides. To distinguish the two types of transporters (with and without a protease domain), LanT is used for ABC transporters that lack the protease domain and LanT p was introduced for transporters that contain the N-terminal C39 protease domain. 310. 4. 2 Subtilisin-type Ser proteases. LP removal for many class I/II lanthipeptides (Section 3. 1) relies on a subtilisin-like serine protease LanP (peptidase S8 family; InterPro family: IPR008357). NisP,  311, 312  ElxP,  76  LicP,  313  and CylA 314 responsible for LP removal for nisin, epilancin 15X, lichenicidin, and enterococcal cytolysin, respectively, have been characterized in vitro, and cleave both modied and unmodied precursor peptides.
NisP (PDB: 4MZD),  315  EpiP (PDB: 3QFH),  316  and LicP (PDB: 4ZOQ)  313  have also been structurally characterized, but without bound substrate. Therefore, the mechanism by which substrate recognition is achieved remains incomplete. The efficiency of NisP activity has been investigated with NisA substrate analogs containing different numbers of (methyl) lanthionines. 317  These experiments revealed that NisP can cleave substrate without any rings but enzymatic activity is signicantly increased following installation of the rst ring. In the case of epilancin 15X, LP removal results in hydrolysis of the newly exposed N-terminal Dha residue to a ketone that is subsequently reduced by the dehydrogenase ElxO to yield the nal product (Section 5. 9). 312  Unique substrate RSs for different LanP enzymes greatly expand the toolbox of specic proteases for use in biotechnology.
A similar mechanism is governing LP removal for class IV lanthipeptides,  85  lipolanthines,  88  and possibly other RiPPs. 321 g. 76  Crystal structure of the protease domain of the PCAT LahT (teal) covalently linked via a Cys in its active site to a peptide aldehyde corresponding to the C-terminal sequence of the ProcA2. 8 LP (gold) (PDB: 6MPZ). 308 The double Gly residues (positions À1 and À2) are indicated as are two key Leu residues (positions À7 and À12) on an ahelix that insert into hydrophobic pockets to mediate substrate engagement. This journal is © The Royal Society of Chemistry 2021 Nat. Prod. Rep. , 2021, 38, 130-239 | 187 4. 4. 5 Pro oligopeptidases. In a subset of class III lanthipeptide BGCs, a conserved protein with homology to prolyl oligopeptidases (POPs), was identied.