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Decoding the Papain Inhibitor from Streptomyces mobaraensis as Being Hydroxylated Chymostatin Derivatives: Purification, Structure Analysis, and Putative Biosynthetic Pathway Norbert E. Juettner, Jan P. Bogen, Tobias A. Bauer, Stefan Knapp, Felicitas Pfeifer, Stefan H. Huettenhain, Reinhard Meusinger, Andreas Kraemer, and Hans-Lothar Fuchsbauer*

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ABSTRACT: Streptomyces mobaraensis produces the papain inhibitor SPI consisting of a 12 kDa protein and small active compounds (SPIac). Purification of the papain inhibitory compounds resulted in four diverse chymostatin derivatives that were characterized by NMR and MS analysis. Chymostatins are hydrophobic tetrapeptide aldehydes from streptomycetes, e.g., S. lavendulae and S. hygroscopicus, that reverse chymosin-mediated angiotensin activation and inhibit other serine and cysteine proteases. Chymotrypsin and papain were both inhibited by the SPIac compounds in the low nanomolar range. SPIac differs from the characterized chymostatins by the exchange of phenylalanine for tyrosine. The crystal structure of one of these chymostatin variants confirmed its molecular structure and revealed a S-configured hemithioacetal bond with the catalytic Cys25 thiolate as well as close interactions with hydrophobic S1 and S2 subsite amino acids. A model for chymostatin biosynthesis is provided based on the discovery of clustered genes encoding several putative nonribosomal peptide synthetases; among them, there is the unusual CstF enzyme that accommodates two canonical amino acid activation domains as well as three peptide carrier protein domains. treptomycetes are filamentous eubacteria inhabiting soil, marine, and fresh-water ecosystems.1 Large G+C-rich linear genomes empower them to live as independent microorganisms, but evidence is growing that they communicate and exert symbiotic partnership with prokaryotes and eukaryotes by means of secondary metabolites.1,2 Streptomycetes are a wealthy source of natural products, especially of antibiotic, anticancer, and immunosuppressive agents of admirable structure and complexity that have been used for pharmaceutical purposes for decades.3 With the development of antibiotic-resistant bacterial strains, causing global health crises,4 new antibiotics are urgently needed. Gene mining is considered a promising strategy to uncover novel antibiotics and lead structures, albeit silent gene clusters do not always encode active compounds.5,6 Thirty years ago, Streptomyces mobaraensis was discovered as a producer of transglutaminase that is used by the food and pharmaceutical industries to cross-link and modify proteins.7 Study of the bacterial secretome further revealed substrate proteins of transglutaminase acting against serine proteases, cysteine proteases, and metalloproteases.8−10 While structure and function of the Streptomyces subtilisin and TAMP (transglutaminase-activating metalloprotease) inhibitor (SSTI) and the Dispase autolysis-inducing protein (DAIP) have been characterized,11−13 the Streptomyces papain inhibitor (SPI, UniProt P86242, PDB 5NTB) remained elusive. Sitedirected mutagenesis revealed the glutamine cross-linking site of transglutaminase in the N-terminal peptide of an expansin

S

© XXXX American Chemical Society and American Society of Pharmacognosy

D1 domainlike double-ψ-β-barrel fold.14 Cysteine proteases such as gingipain from Porphyromonas gingivalis were inhibited by SPI,15 but the production in E. coli showed that the 12 kDa protein is inactive. Moreover, inhibitory activity is lost when SPI from S. mobaraensis was dialyzed or purified by size exclusion chromatography.14 In view of possible therapeutic applications, inhibition of cysteine proteases is highly relevant due to their involvement in many diseases. Papain and papain-like proteases (papain family C1A) are distributed in all kingdoms acting as defensive and offensive molecular weapons.16 They are considered to be attractive targets for controlling and treating infectious diseases caused by many viral, bacterial, and protozoal agents such as SARS corona virus,17 Salmonella enterica,18 or Leishmania mexicana.19 Papain is related to human cathepsins and calpains. Cathepsins are responsible for protein degradation and enzyme processing in the lysosomes, thus maintaining intracellular homeostasis.20 Calpains are involved in cytoskeleton remodeling, cell motility, cell cycle control, proliferation, inflammation, autophagy, apoptosis, signal transduction, and neuronal Received: May 11, 2020

A

https://dx.doi.org/10.1021/acs.jnatprod.0c00201 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Separation of the papain inhibitor (SPI) from Streptomyces mobaraensis. To estimate inhibitory activity, residual activity of 1 mg/mL papain (crude powder) was measured by hydrolysis of 0.4 mM cbzFRpNA at pH 6.5 upon preincubation with diluted fractions. (A) Fractogel EMD SO3− chromatography (CEX) of culture supernatant, heated at pH 4.0 to 70 °C. (B) Superdex 75 chromatography (SEC) of concentrated CEX fractions at pH 8.0. Conductivity, protein, and inhibitor concentrations are depicted by red, blue, and gray lines, respectively. (C) Comparison of CEX (gray) and SEC-purified SPI (red) by analytical HPLC−ESI−MS. (D) Fractogel EMD SO3− chromatography of CEX-purified SPI at pH 4.0 using alternative NaCl gradient.

plasticity.21 Overexpression of these enzymes may evoke cancer; myopathies; cardiovascular and kidney disorders; and ophthalmic, autoimmune, and neurodegenerative diseases.20,21 Consequently, novel papain inhibitors may have not only great impact on infection but also on cathepsin- and calpain-caused diseases.21,22 The aim of the present study was to define the molecular structure and activity of the papain inhibitor from S. mobaraensis assuming that SPI consists of the already characterized 12 kDa protein (SPIp) and additional small active compound(s) (SPIac). As S. mobaraensis exports a plethora of proteins and small molecules during submerged culture, an elaborate purification procedure including several steps was established to obtain pure substances. Six SPIac isolates were analyzed by NMR and mass spectrometry, and the closely related structures were verified by X-ray analysis ligated with papain. Complete reduction of proteolytic activity in the low nanomolar range demonstrated that the SPIac molecules are effective inhibitors of papain and chymotrypsin. Discovery of the gene cluster encoding putative nonribosomal peptide synthetases in S. mobaraensis allowed for the prediction of chymostatin biosynthesis.

1A). Under these conditions, papain inhibitory activity clearly coincided with the eluting protein. Further purification by size exclusion chromatography (SEC) separated SPIac from SPIp, as was expected (Figure 1B). Interestingly, most of the SPIac molecules (gray line) eluted after the salt-containing fractions (red line) indicate that there is some interaction with the SEC material. Analytical HPLC−ESI−MS showed that, contrary to SEC-purified SPIp, a broad spectrum of substances, depicted by unresolved small peaks, preceded elution of CEX-purified holo-SPI protein (Figure 1C). These compounds, showing low molecular masses, clearly differed from the SPIp protein (not shown). The result prompted us to improve the CEX chromatography procedure and to separate holo-SPI by refined NaCl gradients (Figure 1D). Purification of Papain-Inhibiting Compounds. The purification procedure started by the complete removal of proteins via both heat denaturation of culture filtrates at pH 4.0 and absorption of heat-resistant proteins, mostly SPIp and SSTI, by sulfonate resins (Figures 1, 2). Most inhibitory compounds emerged in the flow-through fraction of CEX chromatography or were eluted by low amounts of NaCl. Two out of several examined materials were then chosen for the next purification steps due to their capacity of binding small hydrophobic and cationic compounds. Amberlite XAD-4 allowed strong accumulation of active substance and elimination of contaminating compounds by washing with 20% methanol. Anhydrous methanol eluted the papain inhibitory compounds, SPIac, that were concentrated under reduced pressure. It is important to note that this procedure requires the continuous addition of water to prevent the formation of yellow-brownish, insoluble residues.

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RESULTS Partition of the Papain Inhibitor. To evaluate the assumption that the Streptomyces papain inhibitor (SPI) consists of 12 kDa SPIp protein and one or several small active SPIac compounds, supernatant from a S. mobaraensis culture was heated, which removed the vast majority of secreted proteins, and cation exchange chromatography (CEX) of the residue separated SPI from the heat-resistant Streptomyces subtilisin and TAMP inhibitor (SSTI) (Figure B



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Figure 2. Purification of papain inhibitory compounds. The purification scheme is shown on the left side. (A) Amberlite IR-120 chromatography showing UV absorption (blue line), conductivity (red line), and papain inhibitory activity (gray dots). (B) Silica-60 chromatography showing papain inhibitory activity (gray dots). Combined fractions, indicated as pools A−C, are framed by green vertical lines. (C) Preparative RP18 chromatography. The UV absorption lines are colored green (SiO2 pool A), red (pool B), and blue (pool C), respectively. The acetonitrile gradient is shown by a gray line. (D) Papain inhibitory compounds of pool C for structure analyses are marked by green (AC1.1, AC1.2), red (AC2.1AC2.3), and blue (AC3.1) vertical lines.

referred to as pools A−C in Figure 2 (panel B). The following preparative RP18 chromatography proved that these mixtures differed little from each other in substance composition (panel C). Six SPIac fractions, AC1.1, AC1.2, AC2.1−AC2.3, and AC3.1, were collected and used for structure analyses (panel D). Mass Analysis of the Papain Inhibitory Compounds. The inhibitory compounds were then characterized by high resolution mass spectrometry (MS−MS) and fragmentation by collision-induced dissociation (CID) and higher energy collision dissociation (HCD). Study under various CID and HCD conditions revealed few major fragments, which are

The attachment of the XAD-4 extracts to Amberlite IR-120 was a second milestone to remove uncharged hydrophobic impurities by several elaborated washing steps, showing that SPIac has a positive charge (Figure 2, panel A). SPIac eluted at pH 12 yielded a soluble powder upon reconcentration by XAD-4 and freeze-drying of the watery residue. Starting with culture supernatants of about one liter, preparation of this powder was repeated many times to obtain sufficient amounts of active substance. The final purification procedure required eight IR-120/XAD-4 lyophilizate batches. The penultimate silica chromatography separated SPIac into three broad and unresolved peaks of active compounds that are C



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Figure 3. Molecular structure of the papain inhibitory compounds from S. mobaraensis. The chymostatins A−C are shown for comparison.

summarized in Table S1. Molecular ions (M+, [M+1]+) and fragment patterns gave rise to the assumption that all purified SPIac isolates have a similar molecule structure. The mass analyses further suggested that AC1.1/AC1.2 and AC2.1/ AC2.3 are identical substances, although they are eluted at various times. Probably they adopt different conformations in solution. The four remaining tetrapeptides differed slightly in sequence and molecular mass by m/z of 16 (oxygen atom) or m/z of 14 (methylene group) (Table S1 and Figure S1). These isolates are herein referred to as inhibitory compounds 1−4 (Figure 3). In accordance with the mass spectra, the largest SPIac compound (1, isolates AC1.1/AC1.2, m/z of 626) consists of two tyrosines, one arginine, and one valine, provided that a single carboxyl function is reduced to an aldehyde (the electrophilic warhead reacting with the catalytic cysteine of papain), and one residue is oxidized. Frequently occurring fragments such as those with m/z of 445 (−181, -Tyr) and 419 (−207, -TyrNCO) further depicted the breakdown of a carbamide (ureido) function and the preferred removal of tyrosine and tyrosine isocyanate. Smaller fragments, showing m/z of 252 (−374, -Tyr/-OHCTyral) and m/z of 226 (−400, -TyrNCO/-OHCTyral), suggested the additional cleavage of formyl tyrosinal from the opposite peptide terminus. Thus, compound 1 is flanked by two tyrosines, one of which is reduced to exert the warhead function. Independent elimination of formylated Val-Tyral dipeptide leaves a positively charged, decarboxylated arginine fragment with m/ z of 127 (−499, -TyrNCO/-OHCVal-Tyral) that lacks two hydrogen atoms. Although fragments with m/z of 98, emerging in all SPIac MS−MS spectra and yielding the base peak under harsh breakdown conditions, pointed to 2-aminodihydropyrimidinium ions, side-chain oxidation of arginine and formation of capreomycidine (Cam) were supported only by NMR.

Furthermore, compound 2 (isolates AC2.1 and AC2.3) had the same fragmentation pattern as that of 1 and differed only by the tyrosinal warhead that is replaced by phenylalaninal. The molecular mass (m/z of 610) was correspondingly reduced by the missing oxygen atom. Interestingly, the modified fragmentation pattern of 3 (AC2.2) suggested that, compared to that of 2, tyrosine has changed the position with phenylalanine, thus forming the same warhead as that of compound 1. Compound 4 (AC3.1) has a similar molecular structure to that of 2, but valine should be replaced by leucine or isoleucine, which is supported by the larger molecular ion (m/z of 624), an increase explained by the added methylene group. NMR Analysis of the Papain Inhibitory Compounds. Sufficient amounts of the inhibitors 1 (AC1.1) and 2 (AC2.3) permitted analysis by 1H NMR, 13C NMR, and 15N NMR spectroscopies (Table 1, Figure S2). The tetrapeptidic backbone of both molecules harboring the supposed ureido function was indicated by hydrogen atoms H-3 and H-6 at both peptide bonds (above 8 ppm), at the ureido nitrogens H-25 and H-27 (below 7 ppm), and by couplings between the amide hydrogens and the carboxyl carbon atoms, detected in HMBC spectra. The observed aldehyde at C-1 (the warhead) and the carboxylic acid at C-13 revealed that an ureido function reverses the sequence of both tetrapeptides. All 1H, 13C, and 15N chemical shifts, multiplicities, homonuclear and heteronuclear couplings, and nuclear Overhauser enhancements (NOEs) were consistent with tyrosine, phenylalanine, and valine residues. Oxidation of arginine and the formation of a diazinanimine ring were detected by the intensity of the H-25 hydrogen signal and by the positive signal of C-24 in the DEPT_135 spectrum (Figure S2), which yields the unusual amino acid capreomycidine. 1H and 13C couplings were observed between H-8 and H-9 and between H-8 and D



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Table 1. 1H NMR (500 MHz), 13C NMR (125 MHz), and 15N NMR (50 MHz) Spectroscopic Dataa 1 δ, 13C

δ, 1H

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

200.1 60.0

9.44 s 4.30 m 8.48 d (7.0)

16 17, 17′ 18, 18′ 19 20 21 22 23 24 25 26 27 28

127.0 130.0 114.9 155.8

no.

171.5 57.7 169.5 54.3

54.2 173.4 32.6

30.3 17.5 19.2 50.8

20.4

30 31

36.6 127.0 130.0 114.9 155.8

δ, 1H

200.1 59.8

9.51 s 4.40 ddd (10.3, 7.6, 4.3) 8.54 d (7.6)

171.5 57.8

4.23 dd (8.4, 5.8) 8.07 d (8.3)

−263.4 169.5 54.3

4.50 dd (8.5, 6.9) 6.71 d (8.6)

−300.0

4.21 dd (8.3, 6.2) 8.03 d (8.2) 4.50 dd (8.5, 6.8) 6.72 d (8.4)

157.2 −294.0

6.43 d (7.9) 4.26 m

57.8 173.5

12.6 s (broad) 2.71 dd (14.3, 9.1) 3.03 dd (14.3, 5.0)

33.2 137.3 129.1 128.1 126.3

7.01 d (8.5) 6.65 d (8.4) 9.2 s (broad) 1.99 oct (6.5) 0.84 d (6.9) 0.86 d (6.8) 3.51 m 7.42 s (broad)

30.8 17.3 19.0 50.8 −297.0

153.7 35.9

δ, 13C

−256.2

157.2

29

32 33, 33′ 34, 34′ 35 36

2 δ, 15N

6.42 d (7.9) 4.24 dt (7.6, 6.3) 12.6 s (broad) 2.73 dd (14.1, 10.8) 3.03 dd (14.1, 4.2) 7.23 m 7.26 m 7.20 m 1.88 0.66 0.73 3.54 7.46

oct (6.6) d (6.8) d (6.8) m s (broad)

7.88 3.15 3.30 1.61 1.68 6.77 2.78 2.89

s (broad) m (broad) m (broad) m m s (broad) dd (13.9, 7.9) dd (13.9, 5.3)

151.0 7.86 3.13 3.29 1.62

−302.0

s (broad) m m m (broad)

36.0 20.5 −310.0

6.73 s (broad) 2.76 dd (13.9, 7.3) 2.88 dd (13.9, 5.2)

36.6 127.3 130.1 114.9 155.9

6.95 d (8.5) 6.64 d (8.4) 9.2 s (broad)

6.93 d (8.0) 6.63 d (8.0) 9.2 s (broad)

a

Spectroscopic data is for the inhibitory compounds 1 (AC1.1) and 2 (AC2.3) from S. mobaraensis (δ in ppm, J in Hz) in DMSO-D6. b Abbreviations are d, doublet; dd, doublet of doublets; t, triplet; q, quartet; oct, octet; and m, multiplet.

H-24 in COSY spectra and also between H-8 and C-7, C-10 and C-24 in HMBC spectra. Another indicator for the ring closure is the coupling between H-24 and H-25, also seen in the COSY spectrum. Furthermore, NOE effects between hydrogen atom H-8 and the neighboring atoms H-6, H-9, and H-24 prove their close spatial relationship. In summary, the NMR spectra indicate that the papaininhibiting compounds are amphiphilic tetrapeptide aldehydes, which consist of two aromatic (Tyr or Phe), one aliphatic (Val), and one charged (oxidized Arg) amino acid. A CAS search revealed that S. mobaraensis produces novel inhibitory compounds differing from chymostatin by tyrosines in place of phenylalanines. Crystal Structure of the Phenylalaninal-Containing Tetrapeptide 2 in Complex with Papain. Attempts were then made to verify the structures of the inhibitors 1 (AC1.1)

and 2 (AC2.3) by X-ray analysis. While papain crystals harboring 1 were insufficient to determine the exact conformation of the inhibitor, compound 2 in the complex could be determined at 1.65 Å resolution (Table S2). Using papain (PDB 9PAP) as the molecular replacement model, structures of active and inhibited papain were largely identical. The overlay with an overall root-mean-square deviation (RMSD) of 0.276 Å suggested covalent binding of 2 scarcely changes the papain structure. Like leupeptin-inhibited papain (PDB 1POP, see Figure 4C), the aldehyde function of 2 was covalently bound by the catalytic Cys25, thus forming the tetrahedral hemithioacetal with the negatively charged oxygen directed to the papain oxyanion hole (Figure 4). It seems remarkable that the nucleophilic attack of the cysteine thiolate occurred from the si-side, as found in similar structures of aldehyde-inhibited proteases.23 While orientation of the E



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Figure 4. Binding of AC2.3 to papain. (A) Binding of AC2.3 to papain shown as a cartoon representation (gray). The inhibitory peptide is shown as a stick representation (wheat). Hydrogen bonds are indicated as yellow dashed lines. (B) Sliced view of the binding pocket with a 2Fo-Fc electron density map for the ligand at a contour level of 1 σ. The ligand is colored according to b-factors, where blue represents low b-factors and red represents high b-factors, indicating a higher flexibility of the peptide around the terminal tyrosine. (C) Overlay of the binding modes of AC2.3 and leupeptin. The binding pocket is shown as a surface representation. (D) Interaction network of AC2.3 and papain analyzed by ligplot+,24 highlighting hydrogen bonds and specific hydrophobic interactions.

hydrophobic phenyl to Asn64 and Gly65 filled the S1 pocket of papain, the valine of 2 occupied the papain S2 subsite, shaped by Tyr67, Pro68, Val133, Val157, and Phe207. Interestingly, the backbone of the P1/P2 tetrapeptide residues (Phe-al, αNH; Val, α-CO, α-NH) showed mainly dipolar interactions with papain Asp158 (α-CO) and Gly66 (α-CO; α-NH) (Figure 4). Although the carboxyl group of the terminal tyrosine forms a salt bridge with the Trp69 ring nitrogen of papain, flexibility of the tetrapeptide near the ureido function seems to be increasing as depicted by reduced electron density and increasing b-factors (Figure 4B). The oxidized arginine may adhere only by weak hydrophobic forces to the papain Tyr61 and Tyr67 (Figure 4D) due to the absence of other residues in contact with the dipolar or charged guanidine function. Low electron density further suggested that the terminal AC2.3 tyrosine interacts with papain to a lesser extent. Thus, X-ray analysis of inhibited papain clearly confirmed the chymostatin-like molecular structure of inhibitor 2 as proposed by MS and NMR, including the S-configured L-amino acids. Interaction of the Papain Inhibitory Compounds with Proteases and the SPI Protein. The thermal solidification of papain and chymotrypsin by the inhibitory compounds 1 (AC1.1) and 2 (AC2.3) was next evaluated (Table 2). Leupeptin (acetyl Leu-Leu-Arg-H), antipain (HO-PheNHCONH-Arg-Val-Arg-H), and a commercial mixture of

the chymostatins A−C (Figure 3) were used as controls. The unfolding of the inhibited enzymes was examined by changes in tryptophan fluorescence at 330 and 350 nm using differential scanning fluorimetry. As may be expected from the crystal structure, binding of the inhibitory peptides hardly influenced thermoresistance of papain. The melting point was only enhanced by one degree (Table 2). Similarly, leupeptin and antipain could not improve the integrity of chymotrypsin structure, most likely due to weak interactions with the enzyme. However, binding of the chymostatin mixtures A−C and the related inhibitors 1 and 2 considerably enhanced thermoresistance of chymotrypsin, suggesting that conformational changes in protein structure might be induced by the inhibitory tetrapeptides. Inhibition experiments with papain and chymotrypsin revealed that compounds 1 and 2 are as efficacious inhibitors as the chymostatins are, affecting equally both proteases in the low nanomolar range (Table 2, Figure S3). Compared to that of AC1.1, the potency of leupeptin and antipain against papain seemed to be increased, but neither substance affected chymotrypsin. Despite minor differences in IC50 values, the oxidation of phenylalanine to tyrosine is likely to promote inhibition power of the compound 1. The interaction of the SPI protein with the protease inhibitors 1 and 2 was further investigated. Holo-SPI was F



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Table 2. Effect of Inhibitors on Papain and Chymotrypsin

alanine and valine, leucine, or isoleucine (Figure 6B). Moreover, arginine oxidation might occur at the CstD synthetase in the activated (thioester) state, as cstD is followed by the gene cstE that encodes a putative dehydrogenase enzyme. The Tyr/Phe-binding module 1 of the bimodular CstF is separated from the Val/Leu/Ile-binding module 2 by a third peptide carrier protein (PCP) or thiolation (T) domain in close vicinity to the PCP (T) of module 1. We assume that tyrosine carbamic acid intermediate and ureido bond formation by nucleophilic attack of CstD-activated capreomycidine are established at both adjacent CstF thiolation domains according to a mechanism described for syringolin A (Figure 6C).28 The ureido dipeptide, HO-Tyr-NH-CO-NH-Cam-COS-CstD, may be fused to CstD as a result, which then causes the return transfer to CstF by the inherent condensation (C) domain supporting the Cam-Val peptide bond formation under release of CstD. His-134 of the conserved CstD 133-HHxxxDG-139 motif is likely the general base in this reaction.29 A following shift to the middle thiolation position may clear the CstF module 2 binding place and may support condensation and transfer reaction to CstG by the close spatial relationship to the terminal tyrosine. CstG accommodates an NAD-dependent oxidoreductase moiety that may reduce the tyrosine thioester bond, thus yielding the aldehyde function and chymostatin in the final step (Figure 6C). CstC is composed of two chloramphenicol acetyl transferase domains (CAT), as is described for many C domains.29 The need for a third condensation domain cannot be answered conclusively. Bacterial chloramphenicol resistance is caused by CAT-mediated acetylation that compromises the inhibitory effect on ribosomal peptidyltransferase in prokaryotic protein biosynthesis.30 CstC may influence the protease inhibition by a similar chymostatin modification.

protease papain

chymotrypsin

inhibitor none antipain leupeptin chymostatins A−C 1 (AC1.1) 2 (AC2.3) none antipain leupeptin chymostatins A−C 1 (AC1.1) 2 (AC2.3)

melting point/ °Ca

Δ/°C

IC50 / nM (±SD)b

75.5 76.4 76.5 76.8

0.9 1.0 1.3

3.4 ± 0.2 2.2 ± 0.1 8.9 ± 0.3

76.4 76.4 54.4 56.1 54.6 69.5

0.9 0.9

6.3 ± 0.3 17.8 ± 1.7

1.7 0.2 15.1

[>30 μM] [>30 μM] 6.7 ± 0.5

69.5 67.9

15.1 13.5

4.8 ± 0.2 7.0 ± 0.3

Unfolding of 20 μM enzyme, incubated on ice with 200 μM inhibitor in 50 mM phosphate pH 6.8, 10 mM EDTA, 2 mM DTT (papain) or 100 mM Tris pH 7.8, 10 mM CaCl2 (chymotrypsin) for 15 min, occurred in duplicates by 1 °C/min heating and measuring changes in fluorescence at 330 and 350 nm by differential scanning fluorimetry. b Residual activity was monitored in triplicates by 1.5 nM papainmediated hydrolysis of 50 μM cbzFRamc in 50 mM phosphate pH 6.8, 10 mM EDTA or 1.5 nM chymotrypsin-mediated hydrolysis of sucAAPFamc in 100 mM Tris pH 7.8, 10 mM CaCl2, respectively, and the increase in AMC fluorescence at 450 nm (λexc of 350 nm). a

purified from culture broth of S. mobaraensis according to the described procedure10 and proved to be active. The protein was then removed by precipitation with 90% methanol, equally solubilizing the inhibitory compounds. HPLC−ESI−QTOF of the supernatant separated several compounds showing m/z of 610, 624, and 626 (Figure 5). Base peaks with m/z of 644 and 628 further revealed that these molecules form hydrates under the conditions used. Obviously, purified holo-SPI includes the characterized tetrapeptide aldehydes 1−4. The SPI protein interaction with the inhibitory tetrapeptides was estimated by isothermal titration calorimetry. Compounds 1 (AC1.1) and 2 (AC2.3) were titrated at pH 8.0 to SPI, expressed, and purified from E. coli.15 The absence of significant heat release indicated that the recombinant SPI protein failed to bind either inhibitors. Predicting Chymostatin Biosynthesis. After we took advantage of recent progress in understanding nonribosomal peptide synthetases (NRPSs), the putative gene cluster and biochemical pathway for chymostatin biosynthesis was investigated. Previously, the S. albulus antipain gene cluster, anpA-H, was used for surveying related NRPS encoding genes in the genomes of other bacteria, among them S. mobaraensis.25 The predicted S. mobaraensis genes, albeit incomplete, were found on contig 84 of the shotgun sequenced genome.26 The missing gene for AnpE-like synthetase suggested fusion with the adjacent anpF-like gene in S. mobaraensis, thus resulting in the seven ORFs’ cstA-G (Figure 6A). While the starting genes, cstA and cstB, encode a MarR family transcriptional regulator and a major facilitator superfamily (MFS) transporter, cstCDEFG is most likely involved in chymostatin biosynthesis. The occurrence of at least two condensation (C) and three adenylation (A) domains is indicative for the formation of two peptide bonds, presumably Cam-Val and Val-Tyr, and the consecutive linkage of three different amino acids. Signature of the adenylation domains27 suggests that the CstD and CstF modules specifically activate arginine, tyrosine, or phenyl-

Article

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DISCUSSION The active ingredient (SPIac) of the papain inhibitor from S. mobaraensis (SPI) was characterized as a mixture of novel chymostatins, tetrapeptide aldehydes that interact with catalytic serine and cysteine residues of many endopeptidases and exopeptidases. The established purification procedure from culture supernatants allowed for the isolation of four major compounds that differed from the characterized chymostatins by the exchange of phenylalanine for tyrosine. Chymostatins were originally discovered in S. hygroscopicus MC521-C8 and S. lavendulae MC524-C1.31 They are composed of two flanking phenylalanines, one arginine (oxidized to cyclic capreomycidine, Cam), and one hydrophobic amino acid, either leucine (chymostatin A), valine (B), or isoleucine (C). Moreover, the common sequence of chymostatins, HO-Phe-NH-CO-NH-Cam-Leu(Val/Ile)-PheH, is characterized by special features encompassing the ureido group that reverses the peptide direction, the unusual amino acid Cam and the reduced phenylalanine that forms the warhead aldehyde.32 NMR and MS analyses of the novel papain inhibitors revealed tyrosine substitution of one or both phenylalanines yielding HO-Tyr···Tyr-H 1 (AC1.1, AC1.2), HO-Tyr···Phe-H 2 (AC2.1, AC2.3), and HO-Phe···Tyr-H 3 (AC2.2) chymostatin analogues. Purification of another compound, HO-Tyr···Leu/Ile-Phe-H 4 (AC3.1), further suggested that the P2 position may be occupied by the same aliphatic amino acids as those in the characterized chymostatins. Hence, although only six isolates, including two identical compounds, were examined, S. mobaraensis is G



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Figure 5. Separation and MS analysis of inhibitory compounds from holo-SPI. Active SPI was purified from culture supernatants of S. mobaraensis, and proteins were precipitated by 90 vol % methanol. The residue of the concentrated methanol supernatant was separated by a Multospher 120 RP 18-AQ 5 μ column and analyzed by quadrupole time-of-flight (QTOF)−MS. (Insets) MS spectra of the indicated compounds.

solely on the second-order rate association step (kon), defined by (i) encounter complex formation between protease and chymostatin and (ii) nucleophilic addition of the catalytic serine to the inhibitor’s aldehyde carbonyl function. The basecatalyzed development of the hemiacetal, mimicking the oxyanion intermediate, further provokes conformational changes prior to forming the final enzyme inhibitor complex.35 Our data, revealing IC50 values of 6.7 ± 0.5 (chymostatins A− C), 4.8 ± 0.2 (1), and 7.0 ± 0.3 (2) nM, are in agreement with the reported inhibitor constants (Table 2). Inhibition of papain by 1 likewise occurred at low inhibitor concentrations. Interestingly, enhanced thermoresistance of chymotrypsin in complex with 1 and 2 substantiates the former result35 that chymostatins may stabilize the protein structure by conformational changes. In contrast, binding of inhibitory compounds, among them leupeptin and antipain, did not alter the “melting point” of papain (Table 2).

likely to synthesize 12 inhibitor variants, among them the chymostatins A−C. The hydrophobic character of the novel compounds, assessed on the basis of the final RP 18 chromatography (Figure 2), seems to increase in the order of 1 < 2 ∼ 3 < 4. More hydrophobic variants, eluting beyond compound 4 (HO-Tyr···Leu/Ile-Phe-H), were depicted by papain inhibitory activity, but the small amounts isolated did not allow characterization (Figure 2). The most hydrophobic compounds, chymostatins A−C, were probably lost during the applied purification process. S. mobaraensis seems to prefer valine in the P2 position, while leucine-accommodating chymostatin A may account for 78% in commercial inhibitor mixtures from other sources.33 Chymostatins inhibit a series of serine and cysteine proteases competitively. Binding kinetics show that chymostatin belongs to the “slow-binding inhibitors”,34 yielding Ki values of 0.4 and 150 nM for chymotrypsin and cathepsin G, respectively.35 The inhibitory strength appears to depend H



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Article

Figure 6. Predicted gene cluster from S. mobaraensis and chymostatin biosynthesis. (A) The putative gene cluster on contig 84 of the S. mobaraensis genome includes genes for the MarR family transcriptional regulator (TR); major facilitator superfamily (MFS) transporter; chloramphenicol acetyltransferase (CAT); dehydrogenase (DH); and the nonribosomal peptide synthetases CstD, CstF, and CstG; accommodating condensation domains (C), ATP-dependent adenylation domains (A), phosphopantetheine-dependent thiolation or protein carrier protein domains (T), and an NAD-dependent reductase domain (R). (B) Signature of the adenylation domains: Allocation of adenylation domain specificity follows the gramicidin S synthetase code system.27 The specificity-conferring code for antipain biosynthesis is shown for comparison.25 (C) Predicted biosynthesis pathway of chymostatin.

commercial chymostatin mixtures, solely occupies the active site in the resolved enzyme structures. The Streptomyces griseus proteinase A (SGPA, PDB 1SGC), a structure homologue of

The mode of chymostatin inhibition in complex with several proteases and amidases was examined by X-ray analysis showing that chymostatin A, the predominant compound in I



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mammalian pancreatic serine proteases, interacts with the chymostatin A backbone by three hydrogen bonds.33 The S1 pocket and S2 subsite, lined by hydrophobic amino acids, further contribute to strong inhibitor binding by inserting the chymostatin phenylalanine and leucine side chains. In contrast, the Cam pyrimidine ring, sticking out of the protein surface, is surrounded by water and forms an intramolecular salt bridge with the terminal Phe carboxylic group. An oxyanion hole seems to be missing, as the chymostatin hemiacetal adopts the R and S configurations in equal proportions.33 Obvious stereospecificity was observed in Pseudomonas (sedolisin, PDB 6M9D) and wheat carboxypeptidases (CPD-WII, PDB 1BCS), revealing S (sedolisin) or R antipodes (CPD-WII), while the peptide amidase from Stenotrophomonas maltophilia (PDB 1M21) interacts with chymostatin like a substrate without developing the hemiacetal.23,36,37 It may be of some interest that the sedolisin S1 subsite (Glu154, Ser190) forms excellent hydrogen bonds with the phenolic tyrosinal hydroxy group of tyrostatin (PDB 1KDZ).23 We analyzed the first chymostatin papain complex, primarily to confirm the inhibitor structure of compound 2. Covalent inhibition and strong interactions were displayed by the S-configured hemithioacetal and the hydrophobic S1/S2 subsites filled by the benzyl and isopropyl residues of Val-Phe-H of compound 2. Flexibility of Cam and Tyr, indicated by lower electron density and increased b-factors, showed that S3/S4 subsites are not welldeveloped in papain (as they are in SGPA33) to support inhibition. Nevertheless, the P3/P4 chymostatin amino acids may be important for inhibitor strength in other proteases. For instance, the subsite S3 of the peptide amidase is lined by two negatively charged amino acid clusters (Asp224, Glu231, Asp359, Glu471 and Gly464, Asp465, Asp466, Glu467) enclosing capreomycidine in a sandwichlike manner. 37 Unfortunately, we failed to resolve the complex with tyrosinal-containing compound 1 which inhibits papain stronger than variant 2 inhibits it. We assume that the S1 pocket of papain reinforces the inhibitor interaction in a sedolisin-like manner,23 as it even accommodates the basic argininal of antipain. Noticeably, unlike inhibition of chymotrypsin,35 SGPA33, or the peptide amidase,37 chymostatin hardly changes the papain structure, thus confirming the results of our unfolding experiments. Chymostatin and synthetic analogues have been investigated for therapeutic potency. Control of enzyme activation by inhibition of the processing proteases38−40 as well as prevention of protein degradation, as may occur with liver41 and skeleton muscle proteins,42 may be beneficial in certain diseases. The synthesis of tyrosine-containing chymostatin derivatives has not been reported, and dipeptides and tripeptides did not show better activity than that of the bacterial chymostatins.43 The therapeutic benefit of chymostatin was recognized in tackling the hepatitis C virus,44 Clostridium diff icile,45 Plasmodium falciparum,46 and Porphyromonas gingivalis infections,47 or tumorigenesis,48−50 but inhibition of chymase, a chymotrypsin-like serine protease in the mast cell secretory granules,51 attracts more attention in recent work. Chymase may be involved in the formation of angiotensin II (ANG II), independent of the angiotensinconverting enzymes (ACEs) under inflammatory conditions,52 cytokines, including tissue growth factor beta (TGF-β), antibacterial peptides, matrix metalloproteinases activation, and extracellular matrix degradation.53 Conversion of ANG I by chymase may account for 80−90% in the heart and human

arteries, and chymostatin was reported to diminish blood pressure in hypertensive rats.52,54 Enhanced chymase activity further contributes to diabetic nephropathy,55−57 angiogenesis,51 alarmins degradation,58 lung injury,59 periodontitis,60 inflammation during wound healing,53 and the recruitment of white blood cells in wounded skin.61 These pathogenic changes, caused by chymase, can be reversed by chymostatin. The poor solubility of chymostatin has been blamed for the dearth of in vivo studies targeting chymase or other non-ACE proteases.54 The isolated inhibitor 1 from S. mobaraensis is obviously more hydrophilic than the chymostatins A−C due to the phenolic tyrosine hydroxy groups and has the potency to interact more strongly with appropriate amino acids in the protease S1 pocket as observed in sedolisin tyrostatin complexes.23 Moreover, the discovery of a putative chymostatin gene cluster in S. mobaraensis opens avenues to more soluble, tailor-made drugs by bioengineering. Interestingly, we could not localize S. mobaraensis-like orthologues in the genomes of S. lavendulae subsp. lavendulae and S. hygroscopicus that should be closely related to the early characterized chymostatin-producing strains.31,62−64 The most related genes that we found are available in transglutaminase-producing streptoverticillia, S. olivoreticuli and S. rimosus subsp. rimosus (Table S3).

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were conducted on a Bruker DRX 500 MHz spectrometer (Bruker Biospin, Karlsruhe, Germany) using solvent signals and nitromethane for referencing the 1H, 13C, and 15N NMR spectra. ESI−MS analyses were obtained on an Agilent ESI−QTOF MS spectrometer (X500r QTOF AB Sciex) and an Agilent Series-1200 6110 Quadrupole LC/ MS (Agilent Technologies, Waldbronn, Germany). Papain inhibitor separation was carried out on BioLogic low-pressure (BioRad, Dreieich, Germany) or Agilent 1266 Infinity (Agilent) chromatography systems. Protein unfolding measurements were performed on a Prometheus NT.48 (NanoTemper Technologies, München, Germany). Isothermal titration calorimetry was performed on a PEAK ITC (Malvern Instruments, Malvern, U.K.), and protease activity readings were performed on a GENios Spectra Fluor plus (Tecan, Männedorf, Switzerland) and a PHERAstar Plus (BMG LABTECH, Ortenberg, Germany). Protein content determination by A280 and bicinchoninic acid, SDS polyacrylamide gel electrophoresis, and Coomassie Brilliant Blue staining were carried out as described elsewhere. Production and Purification of SPIac. Streptomyces mobaraensis DSM 40847 was grown in 110 mL of mineral salt starch medium (10 g/L starch, 10 g/L glucose hydrate, 20 g/L peptone, 2 g/L yeast extract, 1g/l K2HPO4, 2 g/L (NH4)2SO4, 1 g/L MgSO4 × 7 H2O, 1 g/L NaCl, 2 g/L CaCO3), with pH 7.0, containing 0.5 mL of trace elements (4 g/L CaCl2, 1 g/L Fe(III) citrate × H2O, 0.2 g/L MnSO4, 0.1 g/L ZnCl2, 40 mg/L CuSO4 × 5 H2O, 30 mg/L CoCl2 × 6 H2O, 30 mg/L Na2MoO4 × 2 H2O, 60 mg/L Na2B4O7) under shaking (100 rpm) in 1 L Erlenmeyer flasks at 42 °C. After 65 h of culture and separation of cell aggregates by suction through Büchner funnels, the filtrate of 9 flasks was adjusted to pH 4.0, heated to 70 °C for 30 min, and centrifuged. The supernatant was pumped through a 30 mL Fractogel EMD SO3− column at a flow rate of 2 mL/min, thus removing residual proteins, rolled with 100 g of Amberlite XAD-4 at 4 °C overnight and filtrated. Upon 4-fold washings with CH3OH/ H2O 2:8 (v/v), the Amberlite XAD-4 resin was extracted with CH3OH. The extract was concentrated in vacuo by occasional addition of H2O and pumped through 200 mL Amberlite IR-120 columns at pH 4.0 and a flow rate of 5 mL/min. Upon several washings at pH 4.0 with 20 mM acetate (until the A280 baseline was reached), 200 mL of 3 M NaCl in 20 mM acetate, and 300 mL of 70% CH3OH in 20 mM acetate, elution occurred by 500 mL of 20 mM J



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KCl in 0.01 N NaOH of pH 12.0. Extra washings were performed with 20 mM acetate of pH 4.0 after the last two washing steps. The papain-inhibiting fractions were combined and concentrated in vacuo upon the second Amberlite XAD-4 adsorption and CH3OH extraction as described before. The watery residue was diluted 4:1 by H2O to obtain about 40 mL, and it was lyophilized, thus yielding a pale-yellow powder that was stored at −20 °C. This procedure, starting with culture supernatants, was repeated many times. Final purification was performed by separation of lyophilized residues from 1.3 L of culture broth using a 600 mm × 30 mm column filled with silica-60 (Macherey-Nagel, Düren, Germany) and CH3OH/CH2Cl2 7:3 (v/v) as eluent. The fractions obtained were combined as shown in Figure 2, evaporated to dryness, and appointed as pools A−C. Baseline separation of the pools A−C was performed by VarioPrep VP250/21 Nucleosil 300-5 C18 (Macherey-Nagel) chromatography using Agilent 1266 Infinity, the eluents 0.1% TFA in H2O and 0.1% TFA in CH3CN, and the gradient of Figure 2, thus obtaining six papain-inhibiting fractions referred to as AC1.1 (5−10 mg), AC1.2 (1−2 mg), AC2.1 (1−2 mg), AC2.2 (1−2 mg), AC2.3 (5−10 mg), and AC3.1 (1−2 mg). Purity of the isolated compounds was verified by analytical HPLC−ESI−MS using a Waters Sunfire C18 column (Waters, Eschborn, Germany) and a CH3CN gradient in 0.1% HCO2H. (2S)-2-[[(1S)-1-(2-Amino-1,4,5,6-tetrahydropyrimidin-6-yl)-2[[(2S)-3-methyl-1-oxo-1-[[(2S)-1-oxo-3-(4-hydroxyphenyl)propan2-yl]amino]butan-2-yl]amino]-2-oxoethyl]carbamoyl amino]-3-(4hydroxyphenyl)propanoic Acid (AC1.1, AC1.2, 1). This compound is colorless. For 1H, 13C, and 15N NMR data of AC1.1, see Table 1. For MS data, see Table S1 and Figure S1. For AC1.1, m/z is 626.2931 [M + 1]+ (calcd for C30H40N7O8, 626.2938). For AC1.2, m/z is 627.2945 [M + 2]+ (calcd for C30H41N7O8, 627.3017). (2S)-2-[[(1S)-1-(2-Amino-1,4,5,6-tetrahydropyrimidin-6-yl)-2[[(2S)-3-methyl-1-oxo-1-[[(2S)-1-oxo-3-phenylpropan-2-yl]amino]bu t a n - 2 - y l ] a mi n o ] - 2 - o x o e t hy l ] c a r b a m o y la mi n o ] - 3 - ( 4 hydroxyphenyl)propanoic Acid (AC2.1, AC2.3, 2). This compound is colorless. For 1H and 13C NMR data of AC2.3, see Table 1. For MS data, see Table S1 and Figure S1. For AC2.1, m/z is 610.3000 [M + 1]+. For AC2.3, m/z is 610.2975 [M + 1]+ (calcd for C30H40N7O7, 610.2989). (2S)-2-[[(1S)-1-(2-Amino-1,4,5,6-tetrahydropyrimidin-6-yl)-2[[(2S)-3-methyl-1-oxo-1-[[(2S)-1-oxo-3-(4-hydroxyphenyl)propan2-yl]amino]butan-2-yl]amino]-2-oxoethyl]carbamoyl amino]-3phenylpropanoic Acid (AC2.2, 3). This compound is colorless.; MS data see Table S1 and Figure S1; m/z 610.2974 [M+1]+ (calcd for C30H39N7O7, 610.2989). (2S)-2-[[(1S)-1-(2-Amino-1,4,5,6-tetrahydropyrimidin-6-yl)-2[[(2S)-4-methyl-1-oxo-1-[[(2S)-1-oxo-3-phenylpropan-2-yl]amino]pentan-2-yl]amino]-2-oxoethyl]carbamoylamino]-3-(4hydroxyphenyl)propanoic acid or (2S)-2-[[(1S)-1-(2-amino-1,4,5,6tetrahydropyrimidin-6-yl)-2-[[(2S)-3-methyl-1-oxo-1-[[(2S)-1-oxo3-phenylpropan-2-yl]amino]pentan-2-yl]amino]-2-oxoethyl]carbamoylamino]-3-(4-hydroxyphenyl)propanoic Acid (AC3.1, 4). This compound is colorless. For MS data, see Table S1 and Figure S1; m/z is 624.3132 [M + 1]+ (calcd for C31H42N7O7, 624.3146). Protease Assays. General proteolytic activity was determined by degradation of alkali-soluble casein and A280 measurement of supernatants upon spinning at 10 000 g.10 Papain/chymotrypsin activity was monitored by hydrolysis of cbzFRpNA or cbzFRamc/ sucAAPFamc (Bachem, Bubendorf, Switzerland) and A405 or E450 (λexc of 350 nm) readings. For inhibitor screening, 20 μL of 2 mM cbzFRpNA in ethanol, 50 μL of 0.1 M citrate of pH 6.5, 10 μL of the sample, and 20 μL of 5 mg/mL papain (crude powder P3375, SigmaAldrich, St. Louis, Missouri, USA) were incubated at 37 °C for 10 min. Hydrolysis was terminated by the addition of 100 μL of 2 mM iodoacetamide. For kinetic analyses, 50−70 μL of 50 mM phosphate/10 mM EDTA/2 mM DTT of pH 6.8 (papain) or 100 mM Tris/10 mM CaCl2 of pH 7.8 (chymotrypsin), 0−20 μL of inhibitor in the concentrations of Figure S3, and 10 μL of 15 nM papain (P4762, Sigma-Aldrich) or 15 nM chymotrypsin (C4129, Sigma-Aldrich) were preincubated for 10 min at 30 °C. Upon addition of 20 μL of 50 μM cbzFRamc or 20 μL of 15 μM

sucAAPFamc, release of 4-amino-7-methylcoumarin was continuously monitored every 30 s over 60 min. Chymostatin, antipain (Bachem), and leupeptin (Sigma-Aldrich) were used as controls. The obtained data were fitted using GraphPad PRISM 6.0. Peptide Structure Analysis. High resolution mass spectrometry was performed by Proteome Factory (Berlin, Germany). To determine holo-SPIac by mass analysis, proteins of culture supernatants were precipitated by 90 vol % methanol at 72 °C (8 min). The residue of the concentrated supernatant was applied onto a 250 mm × 4.6 mm Multospher 120 RP18-AQ 5 μ column (CS-Chromatographie-Service, Langerwehe, Germany) using 0.1% TFA, eluted by 50% CH3CN in 0.1% TFA, and detected by ESI−QTOF MS. All NMR spectra were measured on a Bruker DRX 500 MHz spectrometer equipped with a room temperature probe at 303 K using DMSO-d6 (99.9% D from Sigma) as solvent. The concentrations of the samples were ∼10−3 mol/L. Chemical shift assignment was achieved with 1D 1H and 13C spectra, 2D 1H−1H COSY (correlated spectroscopy), 2D 1H−1H TOCSY (total correlated spectroscopy, mixing times of 80 ms), 2D NOESY (nuclear Overhauser enhancement spectroscopy), 2D 1H−13C HSQC (heteronuclear single quantum correlation), and 2D 1H−13C and 1H−15N HMBC (heteronuclear multiple-bond correlation) using the Bruker pulse sequences zg30, cosygpmfqf, mlevesgpph, noesygptp, invietgpsi, inv4gplrl2ndqf, and hmbcgpndqf, respectively. The 1D 1H spectra were recorded using an excitation pulse of 30 ° and a repetition time of 4.5 s. 128 scans were added and Fourier-transformed with a final digital resolution of 0.09 Hz. The heteronuclear long-range correlation spectrum (HMBC) was recorded by a matrix of 1 k data points (f2, 1H dimension) and 512 increments (data points in f1 13 C dimension). The spectral width was 10 × 206 ppm. For every increment, 80 (120 for 15N) scans were added. Raw data were processed with Topspin 3.2 (Bruker Biospin, Karlsruhe, Germany), and 2D data were analyzed using the MestReNova software (Mestrelab Research, Santiago de Compostela, Spain). Protein Crystallization and Data Collection. Papain was purchased from Sigma-Aldrich and dissolved to 30 mg/mL in buffer containing 100 mM ethanolamine and 20% DMSO. The protein was mixed with the inhibitor 2 (AC2.3) in a 1:5 molar ratio and incubated for 1 h on ice. The inhibited protein was crystallized using the sittingdrop vapor diffusion method by mixing equal volumes of protein and precipitant solution (final drop volume 400 nL). The precipitant solution contained 100 mM HEPES of pH 7.5 and 80% 2-methyl-2,4pentanediol (MPD). Crystals grew within 3−10 days to full size. Diffraction data were collected at beamline ID23-1 (ESRF, Grenoble, France) at a wavelength of 1.0 Å at 100 K. No cryoprotectant was added. Data were processed using XDS65 and scaled with aimless.66 The papain structure with the PDB code 9PAP67 was used as an initial search MR model using the program MOLREP.68 The final model was built manually using Coot69 and refined with REFMAC5.70 Data and refinement statistics are summarized in Table S2. Protein structure was deposited in the Protein Data Bank (PDB) with the ID 6TCX.

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ASSOCIATED CONTENT

sı Supporting Information *

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00201. CID-/HCD-induced chymostatin fragments, MS and NMR spectra of the chymostatin isolates, crystallographic data of compound 2 (isolate AC2.3) in complex with papain (6TCX), chymostatin and papain inhibition curves, and putative Streptomyces genes for chymostatin biosynthesis (PDF) K



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AUTHOR INFORMATION

Corresponding Author

Hans-Lothar Fuchsbauer − The Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, 64295 Darmstadt, Germany; Phone: +49 6151 1638181; Email: [email protected]

Authors

Norbert E. Juettner − The Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, 64295 Darmstadt, Germany; The Department of Biology, Technische Universität Darmstadt, 64287 Darmstadt, Germany Jan P. Bogen − The Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, 64295 Darmstadt, Germany Tobias A. Bauer − The Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, 64295 Darmstadt, Germany Stefan Knapp − Institute of Pharmaceutical Chemistry and Structural Genomics Consortium, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, 60438 Frankfurt am Main, Germany; orcid.org/00000001-5995-6494 Felicitas Pfeifer − The Department of Biology, Technische Universität Darmstadt, 64287 Darmstadt, Germany Stefan H. Huettenhain − The Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, 64295 Darmstadt, Germany Reinhard Meusinger − The Department of Chemistry, Technische Universität Darmstadt, 64287 Darmstadt, Germany Andreas Kraemer − Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany

Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jnatprod.0c00201 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the beamline scientists at ESRF for their support during data collection, and the authors thank Marius Muth, Dominik Happel, and Marina Ehret for technical assistance and valuable preliminary work. H.-L.F. gratefully acknowledges the financial support of N.E.J. and the experimental work by the Centre for R&D (ZFE) at the University of Applied Sciences of Darmstadt. S.K. is grateful for support by the SGC, a registered charity that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative [ULTRA-DD], Janssen, Merck KGaA Darmstadt Germany, MSD, Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and the Wellcome Trust.

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REFERENCES

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