Anabaenopeptins from Nostoc edaphicum CCNP1411

Citation: Konkel, R.; Grabski, M.; Cegłowska, M.; Wieczerzak, E.; W˛egrzyn, G.; Mazur-Marzec, H Anabaenopeptins from Nostoc edaphicum CCNP 1411 Int. J. Environ Res. Public Health 2022 , 19 , 12346 https://doi.org/10.3390/ ijerph 191912346 Academic Editor: Paul B. Tchounwou Received: 2 September 2022 Accepted: 26 September 2022 Published: 28 September 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations Copyright: © 2022 by the authors Licensee MDPI, Basel, Switzerland This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/) International Journal of Environmental Research and Public Health Article Anabaenopeptins from Nostoc edaphicum CCNP 1411 Robert Konkel 1 , Michał Grabski 2 , Marta Cegłowska 3 , Ewa Wieczerzak 4 , Grzegorz W˛egrzyn 2 and Hanna Mazur-Marzec 1, * 1 Division of Marine Biotechnology, Institute of Oceanography, University of Gda ´nsk, M. J. Piłsudskiego 46, PL-81378 Gdynia, Poland 2 Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, PL-80308 Gda ´nsk, Poland 3 Institute of Oceanology, Polish Academy of Sciences, Powsta ´nc ó w Warszawy 55, PL-81712 Sopot, Poland 4 Department of Biomedical Chemistry, Faculty of Chemistry, University of Gda ´nsk, Wita Stwosza 63, PL-80308 Gda ´nsk, Poland * Correspondence: hanna.mazur-marzec@ug.edu.pl Abstract: Cyanobacteria of the Nostoc genus belong to the most prolific sources of bioactive metabolites. In our previous study on Nostoc edaphicum strain CCNP 1411, the occurrence of cyanopeptolins and nostocyclopeptides was documented. In the current work, the production of anabaenopeptins (APs) by the strain was studied using genetic and chemical methods. Compatibility between the analysis of the apt gene cluster and the structure of the identified APs was found. Three of the APs, including two new variants, were isolated as pure compounds and tested against four serine proteases and carboxypeptidase A (CPA). The in vitro enzymatic assays showed a typical activity of this class of cyanopeptides, i.e., the most pronounced effects were observed in the case of CPA. The activity of the detected compounds against important metabolic enzymes confirms the pharmaceutical potential of anabaenopeptins Keywords: anabaenopeptins; cyanobacteria; apt gene cluster; nonribosomal peptide synthetase; biological activity 1. Introduction Anabaenopeptins (APs) are commonly occurring cyanobacteria metabolites assembled by nonribosomal peptide synthetase (NRPS) [ 1 , 2 ]. The peptides were identified for the first time in Anabaena flos aquae NRC-525-17 from the Saskatchewan River in Canada and named after the source organism [ 3 ]. The production of APs was also documented from Nostoc [ 4 – 6 ], Brasilonema [ 7 , 8 ], Desmonostoc [ 7 ], Aphanizomenon [ 9 ], Anabaena/Dolichospermum [ 10 – 14 ], Nodularia [ 6 , 15 – 18 ] (Nostocales order), Lyngbya/ Limnoraphis [ 19 , 20 ], Oscillatoria [ 21 – 23 ], Planktothrix [ 24 – 31 ], Tychonema [ 32 ] (Oscillatoriales order), Microcystis [ 11 , 28 , 29 , 33 – 40 ] (Chrocococales order), Woronichinia [ 41 – 43 ] (Synechococales order) and Schizothrix [ 44 ] (Pseudanabaenales order). Additionally, anabaenopeptinlike compounds, keramamides [ 45 , 46 ] and konbamide [ 47 ], were found in marine sponges Theonella and Melophlus ( Psammocinia aff bulbosa ) [ 45 – 49 ]. As the AP gene cluster was also detected in the tectomicrobial bacterium, the potential for the production of the peptides by other bacteria was suggested [ 4 ]. The structure of APs comprises a five-membered peptide ring linked through the ureido bond with one amino acid side chain. The general formula of this class of peptides is X 1 -CO-[Lys 2 -X 3 -X 4 -MeX 5 -X 6 ], where Lys 2 in D-configuration is the only conservative amino acid in the structure [ 35 , 50 , 51 ]. Positions 4 and 5 are often occupied by homoamino acids (e.g., Hph, Hty) and N -methylated amino acids (e.g., MeAla, MeHty, MeAsn or MeHph) The structural diversity of APs is determined by the organisation of the gene cluster and the corresponding modular NRPS multienzyme complex. Each module catalyses the Int. J. Environ. Res. Public Health 2022 , 19 , 12346. https://doi.org/10.3390/ijerph 191912346 https://www.mdpi.com/journal/ijerph

Int. J. Environ. Res. Public Health 2022 , 19 , 12346 2 of 15 activation and incorporation of an amino acid into a peptide chain. The modules contain several catalytic domains that are responsible for the subsequent steps of the process. These include the adenylation domain (A), responsible for the recognition and activation of a specific amino acid residue, the thiolation domain (T) that transfers the peptide between the domains, the condensation domain (C) that catalyses the formation of the peptide bond, and the thioesterase domain (Te) that is a part of the last module. Te is responsible for the release and, optionally, for the cyclisation of the free peptide [ 4 , 6 ]. Anabaenopeptin NRPS also contains the epimerase domain (E) in the Lys 2 activating module and the methyltransferase domain that catalyses N -methylation of residues at position 5 [ 4 , 6 ]. The biosynthesis of the peptides proceeds according to the collinearity rule so that the number of modules corresponds to the number of residues in the peptide chain Peptides with typical AP structural features were named arbitrarily, so their nomenclature is not systematic. Among the 155 identified APs [ 51 ], there are structural variants of the compounds named nodulapeptins [ 15 ], brunsvicamides [ 32 ], ferintoic acids [ 52 ], lyngbyaureidamides [ 20 ], nostamides [ 4 , 6 ], oscillamides [ 21 , 24 ], pompanopeptins [ 19 ], schizopeptin [ 44 ], mozamides [ 48 ], paltolides [ 53 ] and psymbamide [ 54 ]. Of these, 38 are produced by cyanobacteria of the genus Nostoc , isolated mainly from terrestrial environments (Tables 1 and S 1) Table 1. Nostoc strains that were identified as anabaenopeptins producers Species Strain Place of Isolation References Nostoc sp CENA 543 Brazilian saline–alkaline lake, Nhecol â ndia, Pantanal [ 4 ] Nostoc sp Great Hungarian Plain [ 5 ] N. punctiforme KVS 11 Blassia pusilla (lichen) Norway [ 55 ] N. insulare CBT 163 [ 56 ] Nostoc sp CBT 599 [ 56 ] Nostoc sp CENA 352 Southeastern Brazilian coastal forest [ 7 ] N. punctiforme PCC 73102 Plant symbiont [ 6 ] Nostoc sp ATCC 53789 Macrozamia sp [ 56 ] N. calcicola CB 158 Scotland, Arron Island (lichen) [ 56 ] Nostoc sp CENA 358 [ 7 ] In the peptides produced by Nostoc , amino acid residues common for other AP variants are present, e.g., in the exocyclic position, Ile/Leu, Phe, Val, Lys or Arg can be found, while position 3 is occupied by Ile/Leu or Val and aromatic amino acids are in position 6 (Figure 1 ). The unique feature of APs from Nostoc is phenylnorvaline (PNV) and phenylnorleucyne (PNL) in position 4 [ 56 ] and the presence of Cl-substituted Hty in positions 4 and 6 [ 56 ]. The structural diversity of APs results in a wide range of activities revealed in the in vitro assays. The peptides inhibit protein phosphatases [ 24 , 57 ], elastase [ 40 , 58 , 59 ], carboxypeptidase A (CPA) [ 9 , 23 , 57 ], carboxypeptidase B [ 60 ] and TAFIa (activated thrombin-activable fibrinolysis inhibitor) [ 49 , 56 , 61 ]. In our previous work, sequencing of the Nostoc edaphicum CCNP 1411 genome revealed the presence of a region similar to the anabaenopeptin synthetase gene cluster. In the current work, the organisation of the gene cluster was studied and compared with the results of structural analysis of the detected APs performed with the application of LC- MS/MS and NMR. In addition, three of the APs were isolated from the collected biomass and tested against four serine proteases and carboxypeptidase A.

Int. J. Environ. Res. Public Health 2022 , 19 , 12346 3 of 15 Figure 1. General structure of anabaenopeptins detected in cyanobacteria of the Nostoc genus. The number of variants with specific amino acids is given in the brackets 2. Materials and Methods 2.1. NRPS Alignment The alignment of anabaenopeptin synthetase gene clusters (GenBank numbers GU 174493 and HM 773422) [ 1 , 6 ] to the selected region was carried out with BLASTn [ 62 ]. Correction of the gene prediction was achieved using Prodigal [ 63 ]. Genes found within the aligned regions were subjected to the NCBI Conserved Domain Database search (CDD v 3.19) to determine the evolutionary conserved protein domains and motifs [ 64 ]. The recognition of the residue positions of amino acids in the substrate-binding pocket of adenylation domains was performed manually, according to Stachelhaus et al. [ 65 ]. The CGView Comparison Tool [ 66 ] was used to create a map of the genome fragment 2.2. Extraction and Isolation of Anabaenopeptins Nostoc edaphicum CCNP 1411 was isolated from the Gulf of Gda ´nsk and grown for biomass as previously described [ 67 ]. The lyophilised biomass of N. edaphicum CCNP 1411 (80 g) was homogenised with a mortar and pestle and extracted twice with 75% methanol (MeOH) in MilliQ water (2 × 500 mL) by vortexing for 15 min. The combined extracts were centrifuged (10,000 × g ; 15 min; 4 ◦ C) and diluted to a MeOH concentration lower than 10%. To assess the relative content of the AP variants in N. edaphicum CCNP 1411, the cells (23 mg) were additionally extracted twice with 75% MeOH (2 × 5 mL) by 15 min vortexing The supernatants were evaporated to dry residue and redissolved in 75% MeOH (1 mL) The separation of the compounds was performed with flash and preparative chromatography using the Shimadzu HPLC system (Shimadzu Corporation, Kyoto, Japan). First, the extract was loaded into a 120 g SNAP cartridge KP-C 18-HS (Biotage, Uppsala, Sweden) at a flow rate of 20 mL · min − 1 . The elution started with MilliQ water, and every 17.5 min, the content of MeOH increased by 10% until it reached 100% MeOH. The 40-L fractions were collected and analysed with an LC-MS/MS system. The fractions containing anabaenopeptins were combined, evaporated in a vacuum concentrator (MiVac, SP Scientific, Ipswich, UK) and separated in a Jupiter Proteo C 12 column (250 × 21.2 mm, 4 µ m, 90 Å) (Phenomenex, Aschaffenburg, Germany) by repeated preparative chromatography The mobile phase was composed of a mixture of 5% acetonitrile in MilliQ water (phase A) and 100% acetonitrile (phase B), both with 0.1% formic acid. In the chromatographic runs, gradients from 5% B to 99% B were used. The collected fractions (2 mL each) were analysed with LC-MS/MS 2.3. LC-MS/MS Analysis The LC-MS/MS system was composed of Agilent 1200 HPLC (Agilent Technologies, Waldbronn, Germany) and a QTRAP 5500 tandem mass spectrometer. The compounds were

Int. J. Environ. Res. Public Health 2022 , 19 , 12346 4 of 15 separated in a Zorbax Eclipse XDB-C 18 column (4.6 × 150 mm, 5 µ m) (Agilent Technologies, Santa Clara, CA, USA). Gradient elution (0.6 mL · min –1 ) was performed with the same mobile phases as in the preparative analysis. The turbo ion spray operated at 550 ◦ C; voltage, 5.5 kV; nebuliser gas pressure, 60 psi; curtain gas pressure, 20 psi. To determine the content of the samples, an IDA (information-dependent acquisition) mode was used, and ions within the m / z range 500–1250 and intensity higher than 5 × 10 5 cps were fragmented The assessment of the relative content of APs in the extract was performed in multiple reaction monitoring mode (MRM). The following transitions were monitored: 807 → 402, 231, 120 for anabaenopeptin AP 806 Ne ( m / z 807); 821 → 448, 248, 120 for AP 820 Ne ( m / z 821); 835 → 448, 248, 120 for AP SA 6 ( m / z 835); and 837 → 448, 248, 120 for AP 836 Ne ( m / z 837) The collision energy was 60 eV, and the dwell time was 100 msek 2.4. NMR Analysis 1 D 1 H-NMR and 2 D NMR (COSY, TOCSY, and ROESY) were acquired on a Varian Unity Inova 500 spectrometer (500 MHz). Spectra were recorded in DMSO-d 6 . NMR data were processed and analysed with TopSpin (Bruker) and POKY software [ 68 ]. 2.5. Enzymatic Assays The enzyme inhibitory activity of anabaenaopeptins was assayed against trypsin [ 69 ], chymotrypsin [ 70 ], thrombin [ 70 ], elastase [ 71 ] and carboxypeptidase A [ 70 ]. The samples were serially diluted (1 mg, 1:1–1:10,000 times) in 1% DMSO; the standard inhibitors were also prepared in 1% DMSO (Table S 2). The mixtures containing the anabaenopeptins or positive control (standard inhibitors) were preincubated for 5–20 min in a microplate reader (Varioskan Flash Thermo Fisher Scientific OY, Vantaa, Finland) with the addition of the enzyme and buffer (Table S 2). As a negative control, 1% DMSO, without the addition of the enzyme, was used. Then, the substrates were added (Table S 2), and mixtures were incubated for an additional 10 or 20 min (Table S 2). The absorbance was measured at 350 nm (carboxypeptidase A) or at 405 nm (other enzymes). The tests were performed in triplicates 3. Results 3.1. Anabaenopeptin Nonribosomal Peptide Synthetase (NRPS) Gene Cluster Although the anabaenopeptin synthetase gene cluster has never been studied in N. edaphicum CCNP 1411, it was suggested that nonribosomal anabaenopeptin synthetase might be encoded within its genome [ 65 ]. Nevertheless, given spans were inaccurately indicated, in which only a fragment of the gene coding for potential synthetase overlaps with some extended spans of the studied gene cluster. The core structure of the potential anabaenopeptin synthetase cluster is located between the 2,265,881 and 2,288,626 positions within the N. edaphicum CCNP 1411 chromosome, and it consists of four genes (locus tags HUN 01_12140, HUN 01_12145, HUN 01_12150 and HUN 01_12160) (Figure 2 , Table 2 ). Identified genes are described as those hypothetically encoding proteins being amino acid adenylation domain-containing proteins. The cluster was found on the complementary strand; therefore, locus tag numbers are in the descending order The first NRPS ORF, apt A (HUN 01_12160), is a 6957 bp long gene, coding for a protein comprised of two modules, one containing the adenylation domain (A) and the second identified as the peptidyl carrier protein (PCP). The first module, lacking the condensation (C) domain but containing the adenylation domain, was predicted (based on the nonribosomal consensus code [ 65 , 72 ]) to be involved in the activation of Leu, Ile or Val, which become amino acid substrates (Table 3 ). PCP is the shuttle to the nascent (C) catalytic domain of the second module. No mutations were found in the region encoding the HHXXXDG motif of this condensation domain or every other condensation domain found within this cluster, suggesting that a peptide bond between nascent peptides forming the peptide chain may occur. The epimerase domain, included in the second module,

Int. J. Environ. Res. Public Health 2022 , 19 , 12346 5 of 15 explains the stereochemistry of Lys (D-Lys), an amino acid activated by the second module adenylation domain, whose amino group was found on the right side in its Fisher projection Figure 2. Fragment of the Nostoc edaphicum CCNP 1411 chromosome, encoding the anabaenopeptin synthetase gene cluster (red) Table 2. The organisation of the anabaenopeptin synthetase gene cluster and domain organisation within the proteins encoded by these genes. Domains are abbreviated as follows: A, adenylation; C, condensation; D, docking; E, epimerisation; M, methyltransferase; PCP, peptidyl carrier protein Locus_Tag Gene Bp Domain Organisation HUN 01_12160 aptA [fragment 1] A PCP aptA [fragment 2] 6957 C A PCP E HUN 01_12150 aptB 3231 C A PCP HUN 01_12145 aptC [fragment 1] C A PCP aptC [fragment 2] 7947 C A M PCP HUN 01_12140 aptD 4215 D C A PCP Table 3. Amino acid residues in the substrate-binding pocket of the adenylation domains, encoded by the apt ABCD gene cluster, according to GrsA Phe numbering. Amino acids in brackets mark inconsistencies with references [ 65 , 72 ]; position 331 was not taken into account, as in Challis et al. [ 72 ], this position was not considered Proposed AA Activated Residue Position According to GrsA Phe Numbering 235 236 239 278 299 301 322 330 331 aptA [fragment 1] Leu/Ile/Val D A F F L G [A] T F aptA [fragment 2] Lys D [T] E [Q] I G S [I] I aptB Leu* D A [L] F L G [A] V F aptC [fragment 1] Phe D L [G] T I G [C] V I aptC [fragment 2] Asn D [A] T K V G E V G aptD Phe D A W T [V] A G V C Leu* stands for Leu or Ile.

Int. J. Environ. Res. Public Health 2022 , 19 , 12346 6 of 15 The second ORF, the apt B gene (HUN 01_12150), is a 3231 bp long DNA locus encoding a polypeptide that contains one module. The condensation domain is similar to the DCLtype protein, which catalyses bond formation between the donor D-Lys and the L-amino acid acceptor, namely Leu activated by the adenylation domain The third ORF, apt C (HUN 01_12145), encodes a protein containing two modules with methyltransferase found nascent to the C-terminus of the second module adenylation domain. The former module was found ambiguous. It appears that the signature sequence of the adenylation domain might activate Phe; nonetheless, such an assessment is based on residue positions obtained from not one but several reference domains. However, in favour of this prediction, the adenylation domain of the apt C-encoded module was found to possess Thr and Ile at positions 278 and 299, respectively, which were found exclusively in the Phe-activating reference domains. The amino acid activated by the second adenylation domain is Asn, presumably methylated by the methyltransferase and transferred further by the PCP domain found at the C-terminus of the apt C gene product The start codon of the fourth ORF, putatively encoding NRPS, was found to overlap the stop codon of apt C, suggesting that apt CD may be transcribed as an operon. The apt D gene (HUN 01_12140), composed of 4215 bp, encodes a protein, which, apart from the main module, also contains the N -terminal docking domain and the thioesterase domain at the C-terminus, required to release the peptide Upstream of the apt ABCD synthetase gene cluster, a gene (HUN 01_12135) was found, which translated a sequence that was homologous (88% identity) to HphA. As Hph is present in the produced peptide, further investigations revealed that besides HUN 01_12135, the genes HUN 01_12175 and HUN 01_12180 are putatively synonymous with the hph CD and hph B genes, respectively, which are essential for the homo-amino acid biosynthetic pathway [ 73 ]. These two genes are located downstream of the apt A gene, and their transcripts were found to be 91% identical to those derived from Nostoc punctiforme (GenBank accession number WP_012409012). The core structure of the anabaenopeptin synthetase gene cluster was aligned against three previously characterised hypothetical anabaenopeptinproducing cyanobacterial strains, namely Nodularia spumigena CCY 9414, Anabaena sp. 90 and Nostoc punctiforme PCC 73,102 [ 74 ]. Although the structures of clusters derived from Nodularia and Anabaena exhibit overall similarity, as evidenced by the percentage of identities (mean percentage identity = 78.48), the Nostoc punctiforme NRPS sequence does not resemble the structure of the anabaenopeptin synthetase gene cluster (Figure S 1) 3.2. Identification of AP Structures The nontargeted LC-MS/MS analysis of the N. edaphicum CCNP 1411 cell extract in IDA mode did not reveal the presence of any anabaenopeptin variants. Variants of four anabaenopeptins with pseudomolecular ions ([M+H] + ) at m / z 807, 821, 835 and 837 (Table 4 ) were only detected when several chromatographic fractions were combined and concentrated. MRM analyses were performed to determine the relative amount of anabaenopeptins produced by N. edaphicum . The peak area of AP 820 Ne in the MRM chromatogram was the largest (9.63 × 10 5 cps) compared with AP SA 6 (4.49 × 10 4 cps), AP 836 Ne (2.19 × 10 4 cps) and (1.84 × 10 4 cps) (Figure S 2). Structures of the peptides were elucidated based on the analysis of mass fragmentation spectra with some diagnostic ions, including immonium ions and other ions that correspond to specific fragment ions formed during collision-induced dissociation (Figures 3 and S 3–S 5). Positions 2, 3 and 6 of the peptides were found to be conserved and occupied by Lys 2 , Leu/Ile 3 and Phe 6 , respectively. The applied genetic and chemical methods (MS/MS and NMR) did not allow for the distinction between Leu and Ile; therefore, in this work, the residue is marked as Leu*. The most significant diagnostic ions were: Lys-derived ions at m/z 84, 101 and 129; immonium ions of Phe (120), Hph (134), MeAsn (101), Leu* (86) and Val (72); ions generated by the cyclic part of APs at m / z 664, 678, 678 and 694 (for AP 807 Ne, AP 820 Ne, AP SA 6 and AP 836 Ne, respectively); and [M+H-X 1-(X 3+X 4)] + ion at m / z 434 for AP 807 and at m / z 448 for AP 820 Ne, AP SA 6 and AP 836 Ne. In all MS/MS spectra of MeAsn-containing APs, the

Int. J. Environ. Res. Public Health 2022 , 19 , 12346 7 of 15 [MeAsn+Phe+H-CO] + ion at m / z 248 is present. Other ions that confirmed the structure of AP are shown in Figures 3 and S 3–S 5 Table 4. Anabaenopeptin variants identified in Nostoc edaphicum CCNP 1411 Name Calculated MW [M+H] + m / z Amino Acid Sequence 1 2 3 4 5 6 AP 806 Ne 806.43 807.43 Val CO Lys Leu* Hph Asn Phe AP 820 Ne 820.45 821.44 Val CO Lys Leu* Hph MeAsn Phe AP SA 6 834.46 835.47 Leu* CO Lys Leu* Hph MeAsn Phe AP 836 Ne 836.44 837.50 Val CO Lys Leu* Hty MeAsn Phe Leu* stands for Leu or Ile Figure 3. Enhanced product ion mass spectrum of the anabaenopeptin AP 820 Ne [Lys–Leu–Hph– MeAsn–Phe]CO–Val with precursor ion [M+H] + at m / z 821. The mass signals were assigned to the following fragments: 821 [M+H] + , 803 [M+H–H 2 O] + , 793 [M+H–CO] + , 775 [M+H–CO–H 2 O] + , 722 [M+H–Val] + , 708 [M+H–Leu] + , 704 [M+H–Val–H 2 O] + , 693 [M+H–MeAsn] + , 678 [M+H–Val–CO] + , 660 [M+H–Hph] + , 643 [M+H–Hph–H 2 O] + , 550 [M+H–(Lys+CO+Val)] + , 547 [M+H–(Leu+Hph)] + , 532 [M+H–(Lys+CO+Val)–H 2 O] + , 519 [M+H–(Leu+Hph)–CO] + , 448 [M+H–Val–(Hph+Leu)] + , 420 [M+H–Val–(Hph+Leu)–H 2 O] + , 402 [M+H–Val–(Hph+Leu)–H 2 O–CO] + , 276 [Phe+MeAsn+H] + , 248 [Phe+MeAsn+H–CO] + , 173 [Lys+CO+NH 2 +H] + , 134 Hph immonium ion, 129 [Lys+2 H] + , 120 Phe immonium ion, 86 Leu immonium ion, 84 Lys-derived ions + and 72 Val immonium ion For AP 820 Ne isolated from N. edaphicum CCNP 1411 in the highest amounts (~1 mg), structural analysis by NMR was possible. The 1 H-NMR spectra (Figures S 6–S 9) of the studied compound displayed the typical pattern of a peptide (i.e., amide protons δ H 6.62–9.02 ppm and protons α to carbonyl in amino acids δ H 3.53–5.33 ppm). The COSY and TOCSY experiments allowed to assign NMR spin systems to Val, Lys, Leu*, Hph, N -MeAsp and Phe (Figure 4 , Table 5 ). The presence of aromatic amino acid residues was recognised by the signals occurring in the aromatic region of the spectrum ( δ H 7.01–7.32 ppm). The sequence assignments based on NMR data corroborated the results of the MS experiments of the compound and are consistent with published data [ 40 ]. 3.3. Enzymatic Assays Of the four identified APs, only three were isolated as pure compounds and in sufficient amounts to perform enzyme inhibition assays. These were AP SA 6, AP 820 Ne and AP 806 Ne. In vitro experiments did not reveal the activity of the peptides against trypsin and chymotrypsin, and their activity against thrombin was only observed at the highest concentration used in the assay (45 µ g mL –1 ) (Table 6 ). The three tested APs inhibited the

Int. J. Environ. Res. Public Health 2022 , 19 , 12346 8 of 15 activity of carboxypeptidase A. The IC 50 value for AP 806 Ne was higher (21.0 µ M) than for AP 820 Ne (3.53 µ M) and AP SA 6 (4.5 µ M), indicating lower activity of AP 806 Ne. The latter two APs were also active against elastase and inhibited the enzyme with IC 50 = 5.5 µ M and 22.7 µ M, respectively Figure 4. Key ROESY correlations in anabaenopeptin AP 820 Ne identified in Nostoc edaphicum CCNP 1411 Table 5. NMR spectroscopic data for anabaenopeptin AP 820 Ne Residue Position δ H ( J in Hz) Residue Position δ H ( J in Hz) Val NH 1 2 3 4 6.62 (d, 6.7) 3.85 (m) 1.53 (m) 1.11 (d, 6.7) 1.02 (d, 6.9) Hph NH 1 2 3 4/4 0 5/5 0 6 8.82 (d, 3.9) 4.03 (t, 7.7) 2.73, 2.61 1.72 (m) 7.11 (m) 7.32 (d, 7.3) 7.27 (m) Lys NH 1 2 3 4 5 ε -NH 7.49 (d, 6.1) 3.53 (m) 1.42 (m) 1.33 (m) 1.42 (m) 2.69 (m) 6.05 (m) N -MeAsn N-CH 3 1 2 δ -NH 2 1.81 (s) 5.33 (dd, 9.8, 3.3) 2.41, 2.35 (m) 7.49, 7.03 (m) Leu* NH 1 2 3 4 5 7.06 (m) 3.96 (d, 6.6) 1.50 (m) 1.73 (m) 0.95 (m) 0.77 (dd, 6.8, 2.3) Phe NH 1 2 3/3 0 4/4 0 5 9.02 (d, 8.9) 4.32 (m) 3.30, 2.77 (m) 7.01 (m) 7.19 (d, 7.6) 7.15 (m) Leu* stands for Leu or Ile.

Int. J. Environ. Res. Public Health 2022 , 19 , 12346 9 of 15 Table 6. The activities of the peptides were assessed in serine proteases (chymotrypsin (CHY), trypsin (TRY), elastase (E), and thrombin (Thr)) and carboxypeptidase A (CPA) inhibition assays: –, not active (inhibition below 10%); *, low activity (inhibition between 10–30%); **, medium activity (inhibition between 31–70%) Name Enzyme Inhibition (IC 50 [ µ M]) TRY CHY E CPA Thr AP SA 6 – – 5.5 4.5 * AP 820 Ne – – 22.7 3.5 ** AP 806 Ne – – – 21.1 * 4. Discussion In this work, the anabaenopeptin biosynthetic gene cluster and its products were analysed. A genome of a cyanobacterial strain usually contains several NRPS gene clusters, and within one class of the gene products, numerous structural variants are produced Analysis of 184 cyanobacterial genomes deposited in the NCBI GeneBank showed a positive correlation between genome size (1.65–12.05 Mb) and the number of natural product biosynthetic gene clusters [ 75 ]. In line with this rule, cyanobacteria of the Nostocales order (including Nostoc ) belong to the most prolific sources of natural products [ 76 ]. They synthesise numerous bioactive metabolites classified as peptides, lipopeptides, fatty acids, alkaloids and terpenoids [ 35 , 77 – 80 ]. In our previous studies, two classes of nonribosomal peptides, cyanopeptolins [ 81 ] and nostocyclopeptides [ 67 ], were identified in N. edaphicum CCNP 1411 (total genome size 8.31 Mb, including five plasmids). In addition, a region similar to the anabaenopeptin gene cluster was found in the chromosome [ 67 ]. To check apt gene expression, LC-MS/MS analysis of N. edaphicum extract was performed, but APs were not detected. This result suggested either lack of apt gene expression or production of the peptides in trace amounts. The latter option was confirmed when APs were detected in the analyses of concentrated N. edaphicum biomass. Of these, AP SA 6 is the only AP that has been previously reported [ 50 , 56 ], while the other three are new structural variants Their structures were found to be characteristic of APs produced by cyanobacteria of the genus Nostoc . All positions, especially in the cyclic part of the molecule, are occupied by the residues that were most frequently reported in APs identified in Nostoc (Figure 1 ). The occurrence of MeAsn/Asn in position 5 is quite rare and unique to Nostoc [ 5 , 7 , 56 ] or Desmonostoc [ 7 ], previously classified as Nostoc [ 82 ]. To distinguish the new APs from the already known variants with the same molecular masses (and the same m / z values), we added two letters in their symbols (Ne = N. edaphicum ). However, even with these symbols, some confusion might occur when new APs are detected in other N. edaphicum strains. As has already been postulated by other authors, with the growing number of new anabaenopeptin variants, their nomenclature needs to be systematised In different taxonomic groups of cyanobacteria, the organisation of the NRPS gene clusters involved in the biosynthesis of anabaenopeptins is similar. However, even in strains of the same species, the products of the genes can vary. Namely, the specific positions in the AP structure can be occupied by different amino acid residues. The structural diversity of NRPs encoded by a single NRPS results mainly from the promiscuity of A domains responsible for the selection and incorporation of amino acids [ 1 , 6 ]. In Anabaena sp. 90, the diversity is additionally increased by the presence of two starter modules with different substrate specificity of A domains activating the first amino acid residue [ 6 ]. In N. edaphicum CCNP 141, the 22.7 kb apt gene cluster was found to be similar in size and organisation to the apt gene clusters described in other cyanobacteria [ 1 , 4 , 6 , 83 ]. The cluster is composed of four genes encoding four NRPS enzymes ( aptABCD ) containing six modules. Moreover, the predicted substrate specificity of A domains in the enzymatic complex catalysing AP synthesis supported the identification of specific amino acids in AP structures performed by MS/MS analyses. The detection of the epimerase domain in the second module corroborates the presence of D-Lys in position 2. In addition, the presence

Int. J. Environ. Res. Public Health 2022 , 19 , 12346 10 of 15 of methyltransferase at the C -terminus of the aptC gene product and the detection of genes synonymous with the hphCD and hphB genes confirmed the presence of homo-amino acid in position 4 and methylated amino acid in position 5 [ 4 , 83 ]. Homo-amino acids are also present in cyanopeptolins detected in the strain [ 81 ]. They belong to nonproteinogenic amino acids frequently detected in cyanobacterial peptides [ 50 ]. The main reasons for a wide interest in anabaenopeptins are their frequent occurrence in various taxonomic groups of cyanobacteria and their biological activity [ 23 , 24 , 40 , 51 , 56 , 77 , 84 ] The promising activity of APs with respect to pharmaceutical application was documented for TAFIa [ 56 ]. The compounds inhibit the activity of the enzyme even at a low nM range [ 56 , 59 ] TAFIa regulates the process of fibrinolysis; therefore, inhibitors of the enzymes are thought to be good candidates for the development of antithrombotic agents In our study, the effects of APs on important metabolic enzymes were also explored The enzymatic assays showed that the isolated APs were inactive against trypsin and chymotrypsin and had weak effects on thrombin, but only at the highest concentration used in the assay. In fact, with the exception of elastase, APs were reported to have mild or no effects on serine proteases such as trypsin, chymotrypsin or thrombin [ 21 , 26 , 40 , 44 , 59 ]. In contrast, APs were found to inhibit CPA, enzymes catalysing the cleavage of carboxylterminal peptide bonds in proteins [ 85 ]. Deregulation of CPA can lead to cardiovascular disease or cancer [ 86 ]. Structure–activity relationship studies revealed the importance of exocyclic amino acid for the potency of APs [ 9 , 87 ]. The presence of hydrophobic amino acids in this position significantly increased their activity compared with peptides with polar residues, such as Lys or Arg. For example, AP G (Tyr+CO[Lys+Ile+Hty+MeHty+Ile]) inhibits CPA with approximately three orders of magnitude lower IC 50 value (0.002 µ M) than AP H (IC 50 3.7 µ M), which contains Arg instead of Tyr [ 9 , 23 , 87 ]. However, the activity of the three APs tested in our study was not in line with these findings. The compounds contain hydrophobic amino acids (Leu* or Val) in the exocyclic position, but their effect on CPA was mild (IC 50 values from 3.5 µ M to 21.1 µ M). This discrepancy indicates that besides the exocyclic amino acid residue, other parts of APs’ structure also have an effect on the activity 5. Conclusions As presented in the study, nontargeted chemical analyses may not allow for the detection of compounds produced by cyanobacteria in trace amounts. Therefore, a simultaneous application of genetic and chemical methods is recommended to reveal the full metabolic profile of the organism. Like many other cyanobacteria strains, Nostoc edaphicum CCNP 1411 produces several structural variants of anabaenopeptins. The structural diversity of the peptides results in their different activities against specific biological targets. This fact raises the question of why cyanobacteria produce several structural variants of anabaenopeptins and what their natural function is Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ijerph 191912346/s 1 , Figure S 1: schematic alignment of genes coding for anabaenopeptin synthetase from N. edaphicum CCNP 1411 and three related apt regions encoding synthetases from Nodularia spumigena CCY 9414 (CP 007203.2), Anabaena sp. 90 (GU 174493.1) and Nostoc punctiforme PCC 73102 (NC_010628.1). The grey bar in the upper right corner shows the identity percentage associated with the colour of the bars connecting homologous regions. Red colour represents genes of core anabaenopeptin biosynthetic gene cluster, burgundy colour represents putative hphA gene and dark grey represents the aptE gene coding for an ATP binding cassette transporter NRPS from Nostoc punctiforme PCC 73102 is portrayed in shades of grey, as it does not resemble an apt biosynthetic gene cluster. Schematic alignment of genes was visualised by EasyFig programme ( http://mjsull.github.io/Easyfig/files.html , accessed on 23 August 2022), Table S 1: anabaenopeptin variants produced by genus Nostoc , Table S 2: conditions and solvents used in the enzyme inhibition assays, Figure S 2: MRM chromatograms of anabaenopeptins produced by N. edaphicum CCNP 1411, Figure S 3: structure and enhanced product ion mass spectrum of the anabaenopeptin AP 806 Ne [Lys–Leu*–Hph– Asn–Phe]CO–Val with precursor ion [M+H] + at m / z 807. The mass signals were assigned to the following

Int. J. Environ. Res. Public Health 2022 , 19 , 12346 11 of 15 fragments: 807 [M+H] + , 790 [M+2 H–H 2 O] + , 789 [M+H–H 2 O] + , 779 [M+H–CO] + , 762 [M+2 H–CO–H 2 O] + , 744 [M+2 H–CO–2 H 2 O] + , 708 [M+H–Val] + , 694 [M+H–Leu*] + , 690 [M+H–Val–H 2 O] + , 664 [M+H–(Val– CO)] + , 646 [M+H–Hph] + , 629 [M+2 H–Hph–H 2 O] + , 619 [M+2 H–Hph–CO] + , 601 [M+2 H–Hph–CO–H 2 O] + , 536 [M+H–(Lys–CO–Val)] + , 533 [M+H–(Leu*+Hph)] + , 518 [M+H–(Lys–CO–Val)-H 2 O] + , 515 [M+H–(Leu*– Hph)–H 2 O] + , 506 [M+2 H–(Leu*+Hph) –CO] + , 504 [M+H–(Asn+Phe)–CO] + , 434 [M+H–(Hph+Leu*)– Val] + , 420 [M+2 H–(Leu*+Hph+Asn)] + , 402 [M+2 H–(Leu*+Hph+Asn)–H 2 O] + , 390 [Asn+Phe+Lys+H] + , 362 [Asn+Phe+Lys+H–CO] + , 343 [Leu*+Phe+Lys+H–CO-H 2 O] + , 276 [Hph+Asn+H] + , 262 [Phe+Asn+H] + , 248 [Phe+Lys+H–CO] + , 234 [Phe+Asn+H–CO] + , 173 [Lys+CO+NH 2 +H] + , 134 Hph immonium ion, 129 [Lys+2 H] + , 120 Phe immonium ion, 86 Leu* immonium ion, 84 Lys-derived ions + and 72 Val immonium ion, Figure S 4: structure and enhanced product ion mass spectrum of the anabaenopeptin AP SA 6 [Lys– Leu*–Hph–MeAsn–Phe]CO–Leu* with precursor ion [M+H] + at m / z 835. The mass signals were assigned to the following fragments: 835 [M+H] + , 817 [M+H–H 2 O] + , 807 [M+H–CO] + , 800 [M+H–NH 3 –H 2 O] + , 789 [M+H–CO–H 2 O] + , 772 [M+H–CO–H 2 O–NH 3 ] + , 722 [M+H–Leu*] + , 707 [M+H–MeAsn] + , 704 [M+H– Leu*–H 2 O] + , 678 [M+H–(CO+Leu*)] + , 674 [M+H–Hph] + , 660 [M+H–(CO+Leu*)–H 2 O] + , 656 [M+H– Hph–H 2 O] + , 561 [M+H–(Leu*+Hph)] + , 550 [M+H–(Lys+CO+Leu*)] + , 533 [M+H–(Leu*+Hph)–CO] + , 448 [M+H–Leu*–(Hph+Leu*)] + , 403 [Leu*+Hph+MeAsp+H] + , 276 [Phe+MeAsn+H] + , 248 [Phe+MeAsn+H– CO] + , 173 [Lys+CO+NH 2 +H] + , 134 Hph immonium ion, 129 [Lys+2 H] + , 84 Lys-derived ions + , 120 Phe immonium ion, 101 MeAsn immonium and 86 Leu* immonium ion, Figure S 5: structure and enhanced product ion mass spectrum of the anabaenopeptin AP 836 Ne [Lys–Leu*–Hty–MeAsn–Phe]CO–Leu* with precursor ion [M+H] + at m / z 837. The mass signals were assigned to the following fragments: 837 [M+H] + , 819 [M+H–H 2 O] + , 809 [M+H–CO] + , 802 [M+H–NH 3 –H 2 O] + , 791 [M+H–CO–H 2 O] + , 738 [M+H–Val] + , 724 [M+H–Leu*] + , 720 [M+H–Val-H 2 O] + , 709 [M+H–MeAsn] + , 694 [M+H–(CO+Val)] + , 660 [M+H–Hty] + , 566 [M+H–(Lys+CO+Val)] + , 547 [M+H–(Leu*+Hty)] + , 519 [M+H–(Leu*+Hty)–CO] + , 448 [M+H–Val– (Hty+Leu*)] + , 419 [M+H–(Leu*+Hty+MeAsn)] + , 388 [Leu*+Lys+Phe+H] + , 276 [Phe+MeAsn+H] + , 248 [Phe+MeAsn+H–CO] + , 173 [Lys+CO+NH 2 +2 H] + , 150 Hty immonium ion, 129 [Lys+2 H] + , 84 Lys-derived ions + , 120 Phe immonium ion, 101 MeAsn immonium and 72 Val immonium ion, Figure S 6: 1 H NMR spectrum of anabaenopeptin AP 820 Ne in DMSO-d 6 , Figure S 7: COSY spectrum of anabaenopeptin AP 820 Ne in DMSO-d 6 , Figure S 8: TOCSY spectrum of anabaenopeptin AP 820 Ne in DMSO-d 6 , Figure S 9: ROESY spectrum of anabaenopeptin AP 820 Ne in DMSO-d 6 . Reference [ 88 ] is cited in the Supplementary Materials Author Contributions: Conceptualisation, R.K., M.G., H.M.-M. and G.W.; methodology, R.K., M.G., H.M.-M. and G.W.; software and validation, M.G.; formal analysis, R.K. (LC-MS/MS, extraction, fractionation, isolation of compounds, enzymatic assays), M.G. (genetic analysis), M.C. (enzymatic assays) and E.W. (NMR); investigation, R.K. and M.G.; original draft preparation, R.K. and M.G.; manuscript—review and editing, R.K., H.M.-M., M.G., M.C., E.W. and G.W.; visualisation, R.K. and M.G.; project administration, H.M.-M.; funding acquisition, H.M.-M. and M.C. All authors have read and agreed to the published version of the manuscript Funding: This research was funded by the National Science Centre in Poland (2019/33/B/NZ 9/02018) and by the statutory programme of the Institute of Oceanology, PAN (grant no. 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Simultaneous production, Structural analysis, Natural function, Genetic method, Cardiovascular disease, Gene expression, Biological Activity, Mass spectrometry, Mobile phase, Amino acid, IC 50 value, Bioactive properties, Mass Spectrometer, NMR spectrum, Mass spectrum, Structure-activity relationship, Bioactive metabolite, Structural diversity, Pharmaceutical Potential, Enzyme inhibition, Protease inhibitor, Liquid chromatography, Serine protease, Biologically active secondary metabolites, Metabolic profile, Genetic Analysis, MALDI-TOF, NMR analysis, Cyanobacteria, Terrestrial environment, Amino acid sequence, Enzymatic assay, Marine Sponge, Fibrinolysis, Cyclic peptides, Biosynthetic pathway, Pharmaceutical interest, Hydrophobic amino acids, Antithrombotic agent, Biological target, Gene cluster, Pharmaceutical agent, Chemical analyses, Funding, Enzyme inhibition assay, Amino Acid Residue, Aromatic amino acid, Protein tyrosine phosphatase, Bioactive constituent, Collision Induced Dissociation, Collision energy, Conceptualisation, Metabolic enzyme, Bioactive peptide, Relative content, Enzymatic inhibition assays, Protein phosphatase, Trace amount, Mass signals, Cyanobacterial metabolites, Standard inhibitors, Peptide synthetase, Peptide chain, Preparative chromatography, International Journal of Environmental Research and Public Health, Toxic cyanobacteria, Anabaena flos-aquae, Structural variant.