4-Methylproline Guided Natural Product Discovery: Co-Occurrence of

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4‑Methylproline Guided Natural Product Discovery: Co-Occurrence of 4‑Hydroxy- and 4‑Methylprolines in Nostoweipeptins and Nostopeptolides Liwei Liu,† Jouni Jokela,† Lars Herfindal,‡ Matti Wahlsten,† Jari Sinkkonen,§ Perttu Permi,∥ David P Fewer,† Stein Ove Døskeland,‡ and Kaarina Sivonen*,† †

Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, P.O. Box 56, Viikki Biocenter, Viikinkaari 9, FI-00014 Helsinki, Finland ‡ Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway § Department of Chemistry, University of Turku, FI-20014 Turku, Finland ∥ Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, P.O. Box 65, FI-00014 Helsinki, Finland S Supporting Information *

ABSTRACT: 4-methylproline (4-mPro) is a rare nonproteinogenic amino acid produced by cyanobacteria through the action of a zinc-dependent long-chain dehydrogenase and a Δ1-pyrroline-5-carboxylic acid (P5C) reductase homologue. Here, we used the presence of 4-mPro biosynthetic genes to discover new bioactive compounds from cyanobacteria. Eight biosynthetic gene clusters containing the 4-mPro biosynthetic genes nosE and nosF were found from publicly available cyanobacteria genomes, showing that 4-mPro is a good marker to discover previously unknown nonribosomal peptides. A combination of polymerase chain reaction (PCR) and liquid chromatography−mass spectroscopy (LC-MS) methods was used to screen 116 cyanobacteria strains from 8 genera. The 4mPro biosynthetic genes were detected in 30 of the 116 cyanobacteria strains, 12 which were confirmed to produce 4-mPro by amino acid analysis. Species from the genus Nostoc were responsible for 80% of the positive results. Altogether, 11 new nonribosomal cyclic peptides, nostoweipeptin W1−W7 and nostopeptolide L1−L4, were identified from Nostoc sp. XPORK 5A and Nostoc sp. UK2aImI, respectively, and their chemical structure was elucidated. Interestingly, screening with 4-mPro genes resulted in the detection of peptides that do not contain just one 4-mPro but also 4-hydroxylproline (nostopeptolides) and, in case of nostoweipeptins, two 4-mPros and two 4-hydroxyprolines. Peptides from both groups inhibit microcystin-induced apoptosis of hepatocytes HEK293. The cell experiments indicated that these cyclic peptides inhibit the uptake of microcystin by blocking the organic anion-transporters OATP1B1/B3. This study enriches the drug library of microcystin antitoxin.

as heterocylization, glycosylation, acylation, and methylation and many others.5 Methylprolines are nonproteinogenic amino acids with a methyl group connected to the 3, 4, or 5 carbon or nitrogen atom of proline.6−9 Some methylprolines have a hydroxyl group on the 3rd or 4th carbon atom (Supporting Information (SI) Figure S1).7,8,10 In cyanobacteria, 4-mPro was first discovered from spumigin A produced by Nodularia spumigena AV1 strain.11 4-mPro amino acids have been found in compounds from cyanobacteria such as nostopeptolide A1, thrombin inhibitor spumigin J, trypsin inhibitor spumigin E, protein

Cyanobacteria are well-known producers of a variety of secondary metabolites with diverse chemical structures and biological properties.1,2 Many of the bioactive secondary metabolites from cyanobacteria are nonribosomal peptides,3 which are the end-products of nonribosomal peptide synthetase (NRPS) pathways.4 The pathways comprise of modular NRPS enzyme complexes each module of which is responsible for the recognition and activation of amino acid substrates. Their biosynthetic gene clusters are often colinear with the structures of the peptides.5 The nonribosomal peptides show diverse chemical structures with linear, cyclic, and branched conformations and are produced on mixed complexes of peptide synthetases and modifiying enzymes. Nonribosomal peptides are composed of not only proteinogenic amino acids but also nonproteinogenic amino acids, which carry modifications such © 2014 American Chemical Society

Received: June 2, 2014 Accepted: September 9, 2014 Published: September 9, 2014 2646

dx.doi.org/10.1021/cb500436p | ACS Chem. Biol. 2014, 9, 2646−2655

ACS Chemical Biology

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Figure 1. Gene clusters of 4-methylproline encoding biosynthetic genes in cyanobacteria identified with BLAST from NCBI.

biosynthetic genes of a rare but not unique amino acid 4-mPro turned out to be productive method. In this study, we screened 116 cyanobacteria strains with PCR and LC-MS. Eleven new 4mPro containing peptides were described from two cyanobacteria strains. The structures were solved with MS, NMR, and amino acid analysis. The five main variants are new antitoxins against microcystin.

kinase inhibitor bisebromoamide, and microcystin antitoxin nostocyclopeptide M1.9,12−15 In addition, methylproline amino acids have also been found in bioactive secondary metabolites from plants, fungi, actinobacteria.8,16,17 The synthesis of 4-mPro has been determined by Luesch et al.18 at genetic and enzymatic levels. The enzymes of a zincdependent long-chain dehydrogenase and a Δ1-pyrroline-5carboxylic acid (P5C) reductase were proven to catalyze the synthesis of 4-mPro from L-leucine, and the coding genes of the enzymes were named as nosE and nosF.18,19 The homologues of nosE and nosF were also found in the nonribosomal biosynthetic pathways of nostocyclopeptide A and spumigin E.12,20 Surprisingly methylproline has also been found from ribosomally synthesized peptides, highly post translationally modified bottromycins, produced by marine ascidian-derived Streptomyces spp. strain. 21 However, the radical SAM methyltransferase believed to be responsible for the methylation of proline is β-methylating it and so the product is 3methylproline.21 The diverse synthesis mechanisms reveal multiple strategies for the inclusion of the rare amino acids in the natural products produced by cyanobacteria and other organisms. In this work, we used 4-mPro to find previously unidentified bioactive compounds from cyanobacteria. 4-mPro has been found from just a few cyanobacterial bioactive compounds.9,11−14,22,23 We hypothesized that screening cyanobacteria for the presence of the 4-mPro biosynthetic genes, nosE and nosF, could lead to the identification of novel 4-mPro containing peptides, which could be purified and studied further. The idea to discover previously unknown bioactive nonribosomal peptides from cyanobacteria by screening with

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RESULTS AND DISCUSSION Gene Clusters Containing 4-mPro Synthetizing Genes. The nosE and nosF genes that code zinc-dependent long-chain alcohol dehydrogenase (nosE) and a Δ1-pyrroline-5carboxylic acid (P5C) reductase homologue (nosF) have been proven to be involved in the biosynthesis of 4-mPro amino acid.18 We therefore used nosE and nosF to screen 283 cyanobacterial genomes and draft genomes in NCBI database. Eight gene clusters encoding nosE and nosF homologues were identified: nostocyclopeptide A cluster from Nostoc sp. ATCC 53789, nostopeptolide A cluster from Nostoc sp. GSV 224, spumigin cluster from Nodularia spumigena CCY9414, nostopeptolide A cluster from Nostoc punctiforme PCC 73102, and unknown clusters from Anabaena variabilis ATCC 29413, unknown sp. cyanobiont, Fischerella sp. PCC 9339, and Anabaena sp. PCC 7108 (Figure 1). They range in length from 21 kb to 41 kb. Four of these gene clusters originating from Nodularia and Nostoc have been characterized to genetic and enzymatic level.12,19,20,24 Two of these gene cluster products turned out to be especially relevant with respect to this study: Nostopeptolides A1, A2, and A3 because of their similar structure with nostopeptolides L1−L4 described in this 2647



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To identify strains that produce large amounts of 4-mPro containing peptides in the PCR positive strains, the methanol extracted material of those 30 strains and one control strain was acid hydrolyzed. 4-mPro together with other amino acids of the hydrolyzates were Marfey derivatized, and the derivatives were separated with reversed phase chromatography and detected with MS. Marfey method allows the identification of the stereochemistry of the amino acids. Only 12 of the 30 screened strains were shown to produce 4-mPro (Figure 2, SI Table S1). One reason could be that the 4-mPro containing compounds were not methanol soluble although cyanobacterial nonribosomal peptides are more or less methanol soluble. Perhaps production levels were too low to be detected with the amino acid analysis method used or production conditions were not favorable. One third of the PCR positive strains of Anabaena and Nostoc contained 4-mPro. From nine Nostoc, one Anabaena, Fischerella, and Scytonema strains, from which 4-mPro was found (SI Tables S1 and S2), four extracts from Nostoc sp. XPORK 5A, Nostoc sp. UK2aImI, Anabaena sp. PCC 7108, and Fischerella sp. PCC 9339 had a high 4-mPro content. In addition, 4-mPro containing compounds were found from Nostoc sp. XPORK 5A and Nostoc sp. UK2aImI, by collecting the major peaks present in the total ion current chromatograms (TICC) and by analyzing the 4-mPro content of the peaks (SI Figures S2 and S3). Several previously unidentified 4-mPro containing cyclic peptides were identified from the aforementioned strains, seven nostoweipeptins from Nostoc sp. XPORK 5A and four nostopeptolides from Nostoc sp. UK2aImI, a strain originating from lichen symbiosis. Structures of Nostopeptolides and Nostoweipeptins. The major nostoweipeptin (Nwp) W1 with minor variants W2, 3, 4, 5, 6, and 7 was identified from Nostoc sp. XPORK 5A using liquid chromatography−ion trap mass spectrometry (LCITMS; SI Figure S2). The product ion spectrum of sodiated Nwp W1 (m/z 1237.6) produced several ions giving an indication of the preliminary amino acid content and sequence of the peptide (Figure 3). The spectrum from protonated Nwp W1 (m/z 1215.6) gave a strong indication that the peptide is cyclic with a side chain of 203 Da (SI Figure S2). It is typical for peptides that have a side chain composed of amino acids that in product ion spectrum there is a strong base peak representing the cyclic part of the peptide and a neutral loss, which represents the side chain. This was exactly the situation with protonated Nwp W1 showing a neutral loss of 203 Da. In methanol extract, 32 Da larger products were formed, which were linearized Nwp’s containing a 4-mPro methyl ester. Protonated linear Nwp W1 produced a simple spectrum, which further confirmed the amino acid sequence (Figure 3). Acid hydrolysis and amino acid analysis of Nwp W1 produced NCH3-L-Phe, L-Ser, D-Ser, (2S,4S)-4-CH3-Pro, D-Leu, L-Ile, L-Tyr, and (2S,4R)-4-OH-Hyp. 15N labeling showed that Nwp’s contained 10 nitrogen atoms, indicating a presence of 10 amino acids. All these results together with the elemental composition data presented in Table 1 and accurate product ion masses presented in Figure 3 made it possible to draw the structures of the nostoweipeptins W1−W7 (Figure 4). The last missing elements of the Nwp W1 structure were obtained with NMR analysis. Spectra and shift data from 1H NMR, 1H-1H DQF-COSY, 1H-1H TOCSY and NOESY, 13C HSQC and HMBC, and 15N HSQC are presented in SI Table S3 and in SI Figure S4A−H. In NMR spectra, many groups, especially the peptide bond amides, showed two signals with ratios of 1:1,

study and nostocyclopeptides A1, A2, and M1 because of the similar bioactivity as that described in this study.9,22,23 All of the eight detected gene clusters have relatively conserved gene structures with respect of alcohol dehydrogenase, MeP5C reductase, ABC transporter, and ORF, which locate together and form a characteristic group (Figure 1). The second most prominent group consists of NRPS and PKS genes, which are responsible for the loading and incorporating of amino and carboxylic acids into the synthesized molecules. In addition, among of these gene clusters are some genes coding for modifying enzymes such as oxidoreductase, sulfotransferase, epimerase, D-peptidase, and JmjC domaincontaining protein. Among these 4-mPro encoding gene clusters, the one from Anabaena sp. PCC 7108 was more diverse than the others (Figure 1). Screening of 4-mPro Producers. In cyanobacteria, little research has been carried out concerning 4-mPro containing compounds and their synthetic pathways; therefore, the 4-mPro biosynthesis gene clusters are considered rare. In order to find new 4-mPro containing compounds from cyanobacteria, we designed a pair of specific primers based on six 4-mPro gene sequences from A. varabilis ATCC 29413, Nostoc sp. GSV 224, Nostoc sp. ATCC 53789, N. punctiforme PCC 73102, N. spumigena CCY9414, and Anabaena sp. PCC 7108. The designed primers could detect the fragment covering alcohol dehydrogenase and MeP5C reductase genes. One control strain (SI Table S1) and 30 (26%) of the 116 screened cyanobacterial strains were identified to be positive in the PCR screening (Figure 2, SI Table S1). This 26% is a high amount compared

Figure 2. PCR screening of 4-methylproline biosynthetic genes from 116 cyanobacterial strains and 8 genera, and LC-MS analysis of the precence of 4-methylproline in the PCR positive strains.

to the claim that 4-mPro is uncommon in cyanobacteria (Figure 2, SI Table S2). However, 4-mPro genes were indeed uncommon in all other genera but in Nostoc sp. (SI Table S2). Eighty percent of Nostoc sp. were PCR positive. In Anabaena, 4-mPro genes were rare. Only 3 strains of the 64 tested were PCR positive, which is 5% of the tested strains. From the other genera, only Fischerella sp. PCC 9339 and Scytonema PCC 7110 were PCR positive. No positive strains were found from Calothrix (6 strains), Tolypothrix (4), Rivularia (1), Planktothrix (1), and one unknown genus. The number of screened strains in these genera was too low to get a picture of the abundance of 4-mPro biosynthetic gene frequencies. 2648



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Figure 3. Product ion spectra of sodiated (m/z 1237.6) and protonated (m/z 1215.6) nostoweipeptin W1 (Nwp W1) and protonated (m/z 1247.9) linear Nwp W1 methyl ester. Ac = acetyl; N-mPhe = N-methyl-Phe; Hyp = hydroxyproline.

Table 1. Nostoweipeptins (Nwp) and Nostopeptolides (Np) Characterized from Two Nostoc Strainsa [M + H]+ (m/z) variant

formula

expt.

calc.

Δ ppm

Rt (min)

RA (%)

Nwp W1 Nwp W2 Nwp W3 Nwp W4 Nwp W5 Nwp W6 Nwp W7 Np L1 Np L2 Np L3 Np L4

C61H87N10O16 C61H87N10O15 C61H87N10O15 C60H85N10O16 C60H85N10O16 C60H85N10O16 C59H85N10O15 C53H78N9O14 C53H78N9O13 C52H76N9O14 C52H76N9O13

1215.6337 1199.6366 1199.6366 1201.6150 1201.6150 1201.6150 1173.68c 1064.5661 1048.5712 1050.5512 1034.5560

1215.6296 1199.6347 1199.6347 1201.6140 1201.6140 1201.6140 1173.6190 1064.5663 1048.5714 1050.5506 1034.5557

3.4 1.6 1.6 0.87 0.87 0.87 52 −0.16 −0.15 0.55 0.28

19.7 20.0 20.0 19.1 19.1 19.1 14.9 22.6 24.0 22.0 23.4

80 11b 8b

1 55 30 10 5

a Variant name abbreviation, molecular formula, experimental and calculated mass (m/z) of protonated molecule [M + H]+, mass error (Δ) in ppm, retention time (Rt) in mins, and relative amount (RA) of nostoweipeptins (Nwp) W1−W7 and nostopeptolides (Np) L1−L4. Relative amounts of Nwp’s were calculated from peak areas of protonated and sodiated ions and of Np’s from protonated ions. b11% for Nwp W2 and W3 together and 8% for Nwp W4, W5, and W6 together. cMass from ion trap MS because intensity was too low to get signal from QTOF.

bond between 4-mPro11 and Ser3 (SI Figure S4H). NOESY correlations connected all the other residues together, but between 4-Hyp10 and 4-mPro11, the connective signals were not clear enough. However, product ion spectra presented in Figure 3 showed clearly that 4-Hyp10 was connected to 4-mPro11. The HMBC (SI Figure S4H) and NOESY (SI Figure S4E) signals proving the subunit sequence are collectively shown in SI Figure S5. These signals ultimately proved that 4-mPro11 closed the ring to the side chain hydroxyl of Ser3 and that Phe2 formed a side chain. Product ion spectra presented in SI Figure S2 showed that in minor Nwp’s W2 and W3 4-hydroxyproline in position 9 and 10 have been replaced by proline. In Nwp W4, the N-terminal methyl group is missing from amino acid N-Ac-N-CH3-Phe2 and in Nwp’s W5 and W6 4-mPro11 and 4-mPro4 have been

which indicate that the purified Nwp W1 contained more than one conformation of the Nwp W1 peptide. CH3 protons (δH 1.71, 1.84) of the N-terminal acetyl group and the N−CH3 (δH 2.79, 2.85) protons of the amino acid NCH3-Phe1 showed a HMBC correlations to a common carbonyl (δC 170.3, 170.7) (SI Figure S4G). So, the N-terminus of the side chain amino acid Phe1 is doubly protected with an acetyl and a methyl groups. 4-Hyp9 and 4-Hyp10 protons of the 4-OH and C4 showed COSY correlations, which together with the other δH and δC values proved the positions of the hydroxyl groups. Signals from 4-mPro4,11, Leu5, Ile6, Tyr7, and Ser8 were without any special features. HMBC correlations from 4mPro11 (H2a,b: δH 4.29, 4.38; H3a,b: δH 1.59, 1.64) and from Ser3 (H3a,b: δH 4.07) to a common carbonyl (4-mPro11-C1, δC 170.5) showed that macrocyclic ring was closed with an ester 2649



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Figure 4. Chemical structures of nostoweipeptins W1−W7 from Nostoc sp. XPORK 5A, nostopeptolides L1−L4 from Nostoc sp. UK2aImI, and nostocyclopeptide M1 from Nostoc sp. XSPORK 13A.9

also to a carbonyl group δC 168.0. A hydrocarbon chain C3−C6 connected to each other with COSY correlations (SI Figure S10 and S14) and to the C2 via a HMBC correlation of H3 (δH 6.18) formed the rest of the fatty acid structure. In O-mSer3, the connection of O-methyl group (δH 3.29) to C3O‑mSer (δC 71.9) is seen as a strong signal in the HMBC spectrum (SI Figure S9). In PhePr4, the characteristic proton signals of a phenyl ring were present at δH 7.17, 7.20, and 7.22. COSY and HMBC correlations (SI Figures S9, S10, and S11), which are collectively displayed in SI Figure S14, made up a phenylalanine kind of structure so that a ketone carbonyl (δC 201.8) was present in the structure. Two methine protons δH 3.95 (H2) and 4.75 (H4, weak) had correlations to the ketone carbonyl (SI Figure S11). H2 also showed HMBC correlation to another carbonyl C1 (δC 170.5). A COSY correlation of a methyl group (δH 1.17) with the C2 methine finalized the structure. In Dhb6, both methine H3 (δH 4.70) and methyl H4 (δH 1.36) protons showed mutual COSY correlations and HMBC correlations to quaternary alkenic C2 (δC 130.0) with no signal in 13C HSQC (SI Figure S9). Amide proton (δH 9.37) HMBC correlation to C2 was absent, but correlation to carbonyl C1

replaced with proline. In Nwp W7 the N-terminal acetyl group was missing from the amino acid N-CH3-Phe2. No other variation in the chemical structure of Nwp was detected. The major nostopeptolide (Np) L1 with three other variants L2, L3, and L4 were identified from Nostoc sp. UK2aImI strain using LC-ITMS. Ion chromatograms and product ion spectra are presented in SI Figure S3. The spectra showed that the found compounds were clearly peptides. We purified 3.91 mg of Np L1 from nine grams of freeze-dried cells. Np L1 was colorless powder, which λmax was 197 nm. NMR spectra (SI Figures S6−S13) and shift data from Np L1 have been summed in SI Table S4. 1 H NMR spectrum contained six secondary amide type doublet proton signals at δ = 9.37, 8.98, 8.60, 8.45, 7.34, 6.07 from the amino acids Dhb6, PhePr4, O-mSer3, Gln8, Thr2, and Ile7 (SI Figure S6). These signals correlated with amide nitrogens in 15N HSQC (SI Figure S7). The first subunit, short chain fatty acid 2-methylhex-2-enoic acid (MHEA1) contained a quaternary alkenic C2 (δC 130.2) with no signal in 13C HSQC (SI Figure S8). A HMBC correlation connected a methyl group (δH 1.70; δC 12.3) to the C2 (SI Figure S9). The 2-CH3 protons had HMBC correlation 2650



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Figure 5. Product ion spectrum of protonated (m/z 1064.6) nostopeptolide (Np L1). PhePr = phenylalanylpropionic acid; Dhb = dehydrobutyric acid; Hyp =4-OH-proline.

described from nostopeptolides A1, A2, and A3, which also are depsipeptides.22 Because of these structural similarities, the second peptide group described in this study was named as nostopeptolides. Variation (Np L2−L4) was again found in respect of 4-mPro and 4-Hyp (Figure 4). The 4-mPro screening method resulted in peptides that contained a considerable number of Pro derivatives. We found 4-Hyp, an even rarer amino acid in cyanobacteria than 4-mPro. Also, to our knowledge O-mSer is not commonly encountered in cyanobacterial peptides. O-mSer is situated next to the L-Thr, which leads to an intriguing idea that O-methylation could prevent the macrocyclic ring closure to Ser hydroxyl. Then, these peptides would contain one amino acid (L-Thr) side chain with N-acyl group (MHEA). This is a much more common structure in this peptide group than the structure in which only N-terminal acyl group forms the side chain.25,26 If this idea could be proven, it would be, to our knowledge, a new mechanism to direct the closure of the macrocyclic ring in these depsipeptides. Inhibition on Hepatocyte Apoptosis Induced by Microcystin. Nostocyclopeptide M1 (Ncp M1, Figure 4) belongs to the same peptide group as Ncp A1 and A2.9,22 Ncp M1 is a potent antitoxin against microcystin (MC)9 by inhibition of the liver transporters OATP1B1 and B3.27 We therefore expected a similar effect of the 4-mPro containing cyclic peptides and tested first Np L1 for ability to inhibit nodularin (Nod) induced apoptosis in freshly isolated mouse hepatocytes. Np L1 provided substantial inhibition at concentrations below 9.4 μM; thus, we concluded that Np L1 is a potent antitoxin against the cyanobacterial cyclic peptide toxins Nod and MC (Figure 6).

having a typical shift value (δC 162.6) for Dhb proved the structure (SI Figure S11). In Gln8, 5-NH2 protons (δH 6.71, 7.20) with no signals in 13C HSQC showed 15N HSQC correlation to a nitrogen atom δN 108.7 and HMBC correlation to C4 (δC 30.2) (SI Figures S7 and S9). C4 protons (δH 2.16) showed HMBC correlation to a carbonyl C5 (δC 173.0). These results show that this amino acid residue was Gln, not Glu, which is the product after acid hydrolysis. In Hyp9, protons of the 4-OH and C4 showed a COSY correlation, which together of the other δH and δC values proved the position of the hydroxyl group. Signals from Thr2, 4-mPro5, and Ile7 were without any special features. HMBC correlations from Hyp9-H2 (δH 4.13) and from Thr2-H3 (δH 4.82) to Hyp9 carbonyl C1 (δC 170.7) showed that macrocyclic ring was closed with an ester bond between Hyp9 and Thr2 (SI Figure S11). The HMBC (SI Figure S11) and NOESY (SI Figure S12) signals proving the subunit sequence are collectively shown in SI Figure S14. In amino acid analysis, the following amino acids were found from all Np’s: L-Thr, L-Ser, L-Ile, D-Glu (originate from D-Gln). In acid hydrolysis, the commercial O-mSer produced Ser, and hence, it was deduced that the native amino acid in Np was Om-L-Ser. (2S,4S)-4-mPro was found from Np L1 and L2, L-Pro from L2, L3, and L4, and (2S,4R)-4-OH-Hyp from L1 and L3 (Figure 4). Annotated product ion spectra of Np L1, L2, L3, and L4 and elemental compositions were in full agreement with NMR and amino acid analysis results (Figure 5, SI Figure S3, Table 1, and SI Table S5). The structures of the four nostopeptolide variants are presented in Figure 4 and Table 1. The final result was that both peptide families belong to the abundant cyclic depsipeptide category25,26 in which the carboxyl group of the C-terminal amino acid forms an ester bond with a hydroxyl group of usually Ser or Thr, which are situating near the N-terminal so that there is one or more subunits forming a side chain. The major variant Nwp W1 is complex, containing two 4mPro, two 4-OH-Hyp, D -Leu, and D -Ser, as well as proteinogenic amino acids. The N-terminal was double protected both with acetyl and methyl groups, which is one of the special features in nostoweipeptins. Normally, just a short chain carboxylic acid with a versatile structure is present in the N-terminal of depsipeptides.25,26 Variations (Nwp W2− W7) were found with respect to 4-mPro, 4-Hyp, and Nterminal protection (Figure 4). The second peptide group, nostopeptolides, contained even more modifications than nostoweipeptins. Additionally to the proteinogenic amino acids, L-Thr, and L-Ile, Np L1 contained one 4-mPro, one 4-Hyp, D-Gln, dehydrobutyric acid (Dhb), Omethylated Ser (O-mSer), phenylalanylpropanoic acid (PhePrOH), and 2-methylhex-2-enoic acid (MHEA). A PhePrOH analogous amino acid leucylacetic acid (LeuAcOH) has been

Figure 6. Nostopeptolide L1 is an antitoxin against nodularin in hepatocytes. Freshly isolated rat hepatocytes were treated with nostopeptolide L1 alone (A) or in combination with 70 nM nodularin (B) and further incubated for 60 min before fixation and assessment of apoptosis by microscopic evaluation of cell morphology. Note the difference in scale on the Y-axis between A and B. 2651



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Figure 7. 4-Methylproline-containing nostopeptolides (Np) L1−L4 and nostoweipeptin (Nwp) W1 inhibit OATP1B1/1B3 mediated transport of nodularin (Nod) into transfected HEK293T cells. (A) Fluorescent images of cells cotransfected with GFP and either OATP1B1 or OATP1B3. Note the round appearance and polarized blebbing of Nod-treated cells compared to control. Np L2 and Nwp W1 show complete protection whereas Np L3 and Np L1 have intermediate protection and the cells were proapoptotic. (B) HEK293T cells transfected with OATP1B1 or 1B3 were treated with increasing concentrations of peptides before addition of Nod and further incubations as in panel A. IC50 values were determined based on the ratio of GFP-positive cells with apoptotic or normal morphology. The data are average of three independent experiments and SEM. The curves are based on the IC50 values from the regression analyses.

We next screened the main five cyclic peptides that could be purified, Nwp W1 and Np L1−L4, on their ability to inhibit OATP1B1 and 1B3 in transfected HEK293T cells. Such cells respond similarly to hepatocytes upon Nod-treatment, whereas nontransfected cells are resistant to Nod or MC at above 1.5 μM even after several hours of incubation.27 Inhibitors of OATP1B1 or 1B3 such as Ncp M127 or rifamycin SV28 can block the Nod-induced morphology of OATP1B1 or 1B3 transfected cells. All peptides inhibited Nod-induced apoptosis in cells transfected with either transporter and showed a sigmoid dose−effect relationship (Figure 7). We noted that the different Np’s had different potency of inhibition of the two liver channels (Figure 7). Np L2 showed equal potency toward both transporters (Figure 7C), Np L4 was most potent against OATP1B1 (Figure 7D), and Nwp W1 showed highest potency against OAPT1B3 (Figure 7A). Np L1 was the least potent of the peptides, whereas Np L2 seemed to have the highest overall activity. We conclude that the peptides inhibit microcystininduced apoptosis by preventing uptake of the toxin through the liver-specific channels OATP1B1 and 1B3. Although structurally different, both Ncp M1 and the 10 of the 11 cyclic peptides presented in this study contain 4methylated or 4-hydroxylated proline, as well as one or more

aromatic amino acids (Figure 4). It is noteworthy that all-L analogs of Ncp M1 with proline instead of 4-mPro had significantly lower activity than the original compound27 suggesting that 4-mPro can be a determining factor for a cyclic peptide’s ability to inhibit OATP1B1/1B3. Moreover, a cyclic structure appears to be important for peptides to potently inhibit OATP1B1/1B3.27 A later study also showed that cyclic nucleotide analogs with aromatic ring-substituents are potent inhibitors of transport of Nod, MC and other substrates through OATP1B1 and 1B3.28 The cyclic peptides presented in this study, thus, seem to fulfill some requirements to be able to inhibit OATP1B1/1B3, they have (1) a cyclic peptide structure, (2) one or more methylated or hydroxylated prolines, and (3) aromatic ring moieties. However, due to the complexity of the structures, one cannot predict the activity of the peptides with respect to preference toward either of the transporter polypeptide OATP1B1 or 1B3. Conclusion. Herein, we developed a method to detect bioactive peptides in cyanobacteria through 4-mPro biosynthetic genes, combined with 4-mPro LC-MS screening. As a result, four new biosynthetic gene clusters of 4-mPro containing compounds were identified. Among the 116 screened cyanobacterial strains, 4-mPro genes are rare in all 2652



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(Heidolph Instruments GmbH & Co., Germany) homogenizer. After centrifugation at 8000g for 10 min, the supernatant was removed and added to water and dichloromethane at a ratio 1:1:1. The mixture was homogenized and centrifuged at 8000g for 10 min. The dichloromethane layer was collected and evaporated under a stream of air. The dry residue was suspended in 25 mL of 32.5% aqueous acetonitrile with 0.1% HCOOH. The solution was centrifuged at 20 000g for 5 min, and the supernatant was injected into a Luna C8 column (150 × 10 mm, 5 μm, Phenomenex, Torrance, CA, U.S.A.) in batches of 1 mL. Nwp W1 was purified by eluting the sample isocratically with 32.5% aqueous acetonitrile (ACN) with 0.1% HCOOH at 3.5 mL min−1 at 30 °C. The column was washed with 85% aqueous ACN between injections. Because of insufficient purity of Nwp W1, the collected and pooled fractions were injected into a Zorbax SB-C18 column (250 × 4.6 mm, 3.5 μm, Agilent Technologies, U.S.A.) in batches of 100 μL. Nwp W1 was eluted with 37.5% aqueous ACN with 0.1% HCOOH at 1 mL min−1 at 30 °C. The collected and pooled fractions were dried. Yield of Nwp W1 was 11 mg. For the purification of Np L1−L4, 9 g of freeze-dried cells of Nostoc sp. UK2aImI were extracted with 2 × 315 mL methanol, centrifuged, dissolved, and evaporatyed as described in previous paragraph. The dry residue was suspended in 45% aqueous ACN and centrifuged at 20 000g for 5 min, and the supernatant was injected into a Luna C8 column (150 × 10 mm, 5 μm, Phenomenex) in batches of 1 mL. Np L1−L4 were purified by eluting the column with a solvent gradient from 20% to 85% of aqueous ACN at 3.5 mL min−1 at 30 °C. Np peaks were collected and pooled together. Because of the low purity of Np, pooled fractions were reinjected into the C8 column in batches of 100 μL. The column was eluted with a gradient of aqueous ACN from 45% to 85%. Pooled fractions were dried, and yields of the Np’s were 3.91 mg (L1), 1.88 mg (L2), 0.51 mg (L3), and 0.64 mg (L4). All chromatographic runs were performed on a HP 1100 Series modular HPLC containing autosampler, manual injection valve (Rheodyne), column oven, and diode array detector (Agilent Technologies, Palo Alto, U.S.A.). Amino Acid Analysis. L-Thr, D-Thr, D-allo-Thr, L-Ile, D-Ile, L-alloIle, D-allo-Ile, L-Leu, D-Leu, L-Ser, D-Ser, L-Pro, D-Pro, L-Glu, D-Glu, LTyr, D-Tyr, (2S,4S)-4-Hyp, (2S,4R)-4-Hyp, (2R,4R)-4-Hyp, and (2R,4S)-4-Hyp from Sigma-Aldrich (Switzerland), L-allo-Thr from ICN Biomedicals Inc. (U.S.A.), (2S,4S)-4-mPro from ABCR GmbH & Co. (Germany), N-methyl-L-Phe from Fluka (U.S.A.) and N-methyl-DPhe from Iris Biotech GmbH (Germany), were used as amino acid standards. Roughly 50 μg of dried cell extracts, purified Nwp W1, and Np L1 preparates were hydrolyzed in 200 μL 6 M HCl at 110 °C for 18 h. After vacuum drying, the residues and amino acid standards (20 μg) were dissolved in water (50 μL), and 20 μL 1 M NaHCO3 and 100 μL 1% L-FDAA (N-α-(5-Fluoro-2,4-dinitrophenyl)-L-alaninamide, ABCR GmbH & Co., Germany) or L-FDLA (N-α-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide, ABCR GmbH & Co., Germany) in acetone were added. The reaction was terminated with 20 μL of 1 M HCl after incubation for 1 h at 37 °C. After centrifuging at 20 000g for 5 min, the amino acid derivatives in the supernatants were analyzed (amino acid standards after 1:100 dilution) with LC-ESI-ITMS using a Kinetex C18 column (150 × 2.1 mm, 5 μm, 100 Å, Phenomenex), which was eluted 0.2 mL min−1 with various 0.1% aqueous formic acid and acetonitrile gradients at 40 °C. LC-MS. The LC-ESI-ITMS was Agilent 1100 Series LC/MSD TRAP System HPLC (Agilent Technologies, Palo Alto, U.S.A.) with XCT Plus model ion trap mass detector. Accurate mass measurements were performed on a UPLC-ESI-QTOF mass spectrometer Synapt G2 HDMS (Waters, MA, U.S.A.). NMR. For NMR data collection, the Nwp and Np peptides were dissolved in d6-DMSO. All NMR spectra were measured at 25 °C on the Varian INOVA 500 NMR spectrometer, equipped with a 5 mm ⌀ triple-resonance {1H}/13C/15N z-axis pulsed field gradient probehead. For structural elucidation of Nwp, the 1H spectrum as well as a set of two-dimensional homonuclear and heteronuclear NMR experiments was measured. 1H spectrum was collected using 64 transients and acquisition time of 2 s. Homonuclear double quantum filtered

other genera but Nostoc in which 69% of the strains contained 4-mPro synthetic genes. Eleven new 4-mPro containing cyclic depsipeptides, named as nostoweipeptins and nostopeptolides, were found and characterized from two Nostoc strains. Both of these compound groups were confirmed to be antitoxins of the potent hepatotoxins, nodularin and microcystin. In the future, it would be worthwhile to identify and analyze the gene clusters of nostoweipeptins and the new nostopeptolides, as they contained many uncommon amino acids, especially 4-Hyp and PhePr and modifications such as O-methylation of Ser.

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Strains and Cultivation. The cyanobacteria strains used for PCR and LC-MS screening (SI Table S1) were cultivated in 40 mL of Z8 media in a continuous light at a photon irradiance of 10−25 μmol m−2 s−1 at 20−25 °C for 12−20 days. The strain XPORK 5A and UK2aImII (isolated from lichen) were grown in 3 L Z8X media at a photo irradiance of 8−20 μmol m−2 s−1 at 20−25 °C for 30 days. The cells were collected by centrifugation for 10 min at 8000g. The biomass was lyophilized for the purification of compounds Nwp W1 and Np L1−L4. Stable Isotope Labeling. 15N labeling was performed by growing the XPORK 5A strain in modified Z8 growth medium (buffered with 10 mM HEPES pH 8) containing 15N-urea as a nitrogen source in a nitrogen free atmosphere maintaining by bubbling the medium with nitrogen-free argon with 20.9% O2 and 0.45% CO2 (quality 5.7; AGA Gas Ab, Sweden). The XPORK 5A strain was grown under a photon irradiance of 15 mmol m−2 s−1 at 20 °C. After 4 weeks, the biomass was collected and lyophilized. 15N labeled freeze-dried cells was extracted with MeOH and the extracts were analyzed with LC-ESIITMS. Genome Mining. The 4-mPro biosynthetic genes, nosE and nosF, form Nostoc18,19 were used to screen cyanobacterial genome sequences at NCBI. The candidate methylproline gene clusters were found by BLASTp and BLASTx to identify cyanobacterial strains carrying the 4mPro biosynthetic genes nosE and nosF. PCR Screening. Fresh cyanobacteria cells (50 mg) were used for extraction of Genome DNA with DNeasy Plant mini kit (Qiagen). Two different sizes of glass beads (180 μm and 425−600 μm, SigmaAldrich) were added and the cells were disrupted by shaking at 6.5 m s1 for 60 s using a FastPrep homogenizer (M.P. Biomedicals). After the extraction, the concentration of Genome DNA was detected with a Nanodrop Spectrophotometer. The fragment of biosynthetic gene cluster of 4-mPro was amplified with PCR with forward primer nosEf and reverse primer nosFr. The oligonucleotide sequences were as follow nosEf, 5′-GAAACCTGTTACAACTGCTGGTATTG; nosFr, 5′-TGAACVCCAGCATCAATCAT. The primers were designed according to the conservative regions of 4-mPro genes from Anabeana sp. PCC 7108, Anabaena variabilis ATCC 29413, Nostoc punctiforme PCC 73102, Nostoc sp. GSV 224, Nostoc sp. ATCC 53789, and Nodularia spumigena CCY9414. The PCR amplified as follows: initial denaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 30 s; annealing at 51 °C for 30 s; extension at 72 °C for 1 min; final extension at 72 °C for 10 min. The PCR enzyme (Dream Tag) and dNTP mixture were purchased from Thermo Scientific. The PCR products were verified in 1% (w/v) of agarose gel (Molecular grade, BioTop). The PCR fragments of 4-mPro genes from UK2aImI and XPORK 5A were sequenced with service from Institute of Biotechnology, University of Helsinki. Extraction. Freeze-dried cells (20−50 mg) were extracted with 1 mL MeOH by homogenizing with FastPrep (M.P. Biomedicals) 6.5 m s−1 for 30 s. Suspensions were centrifuged at 20 000g for 5 min, and supernatants were collected, which were used for the LC-ITMS and UPLC-ESI-QTOF analyses. Fifty microliters of extracts were evaporated to dryness for amino acid analysis. Purification of Nwp W1 and Np L1−L4. For the purification of Nwp W1, 5 g of freeze-dried cells of Nostoc sp. XPORK 5A were extracted with 2 × 200 mL methanol for 30 s using SilentCrusher M 2653



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(DQF) COSY and TOCSY were collected using 256 (1792) complex points in t1 (t2) domain, corresponding to acquisition time of 37 ms (256 ms), respectively. Isotropic mixing time in TOCSY was 60 ms. Homonuclear NOESY was collected with a mixing time of 500 ms and using 800 (4096) complex points in t 1 (t 2 ) time domain, corresponding to acquisition time of 100 ms (512 ms). Heteronuclear Single Quantum Coherence (13C-HSQC) experiment for aliphatics and CH2-edited 13C-HSQC were measured using 128 (896) complex points in t1 (t2) domain, corresponding to acquisition time of 11.3 ms (128 ms). The signal was accumulated using 64 transients per FID. 13 C-HSQC for aromatics was collected using 64 transients and 128 (896) complex points in t1 (t2), which corresponds to acquisition time of 25.6 ms (128 ms). To establish long-range 1H−13C connectivities, the heteronuclear multiple-bond correlation (13C-HMBC) experiment was measured using two different 1H−13C transfer delays, 71.4 and 100 ms. First-order J filter with 1JHC set to 140 Hz was used to remove one-bond H−C connectivities. The spectra were collected using 256 transients and 128 (896) data points in t1 (t2) domain, translating to acquisition time of 4.3 ms (128 ms). The 15N-HSQC spectrum was collected using 2048 transients and 32 (896) time points in t1 (t2), corresponding to acquisition time of 16.5 ms (128 ms). For structural characterization of Np, a one-dimensional 1H experiment was measured using acquisition time of 2 s and accumulating signal with 8 transients. DQF-COSY and TOCSY were collected using 256 (896) complex points in t1 (t2), translating to acquisition time of 42.7 ms (128 ms). Two-dimensional NOESY experiment was measured using 500 ms mixing time and 8 transients. There were 512 (3072) complex points in t1 (t2) collected, corresponding to acquisition time of 85.3 ms (512) ms. 13C-HSQCs for aliphatics and for aromatics were collected with 16 transients using 128 (896) complex points in t1 (t2), translating to acquisition time of 7.3 ms (128 ms). 13C-HMBC was measured using 160 transients per FID, and 256 (896) data points in t1 (t2), translating to acquisition time of 4.3 ms (128 ms). The long-range 1H−13C transfer delay was set to 100 ms. For 15N-HSQC, 32 (896) complex points in t1 (t2) was collected using 768 transients per FID, resulting in acquisition time of 16.5 ms (128 ms). Cell Experiment with New Discovered Cyclic Peptides. Primary rat hepatocytes were isolated from male Wistar rats (80− 120 g) by in vitro collagen perfusion, as previously described.29,30 The experimental conditions were as described in Jokela et al.9 The use of rats for isolation of primary hepatocytes were approved by the Norwegian Animal Research Authority and conducted according to the European Convention for the Protection of Vertebrates Used for Scientific Purposes. Protocols for the culturing of human embryonic kidney cells (HEK293T, ATTC No. CRL-11268) as well as enforced expression of green fluorescent protein (GFP) alone, or with OATP1B3 or OATP1B1 are described in detail in Herfindal et al.27 Vectors for OATP1B131 and 1B332 were a gift from Dietrich Keppler, German Cancer Research Center, Division of Tumor Biochemistry, Heidelberg, Germany. Experiments on OATP1B1 or 1B3-transfected HEK293T with the different Nwp W1 and Np L1−L4 peptides as well as the regression analysis of the data to estimate IC50 values for inhibition of nodularin-induced apoptosis were conducted as described in Herfindal et al.27 Every peptide was tested for ability to inhibit nodularin-induced cell death at concentrations from 1 to 100 μM. HEK293T cells were incubated with the peptides for 15 min before addition of nodularin (0.25 μM for OATP1B1 and 1.0 μM for OATP1B3) and further coincubated for 90 min before fixation and assessment of GFP-positive cells with apoptotic or normal morphology.27

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AUTHOR INFORMATION

Corresponding Author

*Email: kaarina.sivonen@helsinki.fi. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank L. Saari for helping to isolate and cultivate the collected cyanobacteria strains. Ing. N. L. Larsen assisted in maintenance of cell lines and transfection of HEK293T cells. This work was supported by the grants (118637 and 258827) from the Academy of Finland to K. Sivonen, the Norwegian Western Regional Health Authorities, and the Norwegian Research Council (project no. 205793) to L. Herfindal and S.O. Døskeland. L.W. Liu is a matching fund student at the Doctoral Programme in Microbiology and Biotechnology and partially supported by China Scholarship Council.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Detailed data from PCR, LC-MS, and NMR analyses as indicated in the text. This material is available free of charge via the Internet at http://pubs.acs.org. 2654



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4-Methylproline Guided Natural Product Discovery: Co-Occurrence of

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