Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Radical S‑Adenosylmethionine Protein NosN Forms the Side Ring System of Nosiheptide by Functionalizing the Polythiazolyl Peptide S‑Conjugated Indolic Moiety Yanping Qiu,†,∥ Yanan Du,†,∥ Shoufeng Wang,† Shuaixiang Zhou,† Yinlong Guo,‡ and Wen Liu*,†,§
Org. Lett. Downloaded from pubs.acs.org by MIDWESTERN UNIV on 02/20/19. For personal use only.
†
State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China ‡ State Key Laboratory of Organometallic Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China § Huzhou Center of Bio-Synthetic Innovation, 1366 Hongfeng Road, Huzhou 313000, China S Supporting Information *
ABSTRACT: NosN is a radical S-adenosylmethionine protein observed in the biosynthesis of the bicyclic thiopeptide nosiheptide. Insights are provided in terms of the timing of NosN action, its catalytic mechanism, and its role in side ring formation. Beyond being a methyltransferase, NosN transforms a polythiazolyl peptide intermediate by functionalizing the Sconjugated indolic moiety to selectively build a C1 unit, form an ester linkage to the thiopeptide framework, and establish the side ring system specific for nosiheptide.
T
Nosiheptide (NOS, Figure 1A), produced by Streptomyces actuosus ATCC 25421, is an archetypal bicyclic thiopeptide.6,7 It possesses a 2,4-dimethylindolic acid (DMIA) moiety, which conjugates the residues Glu6 and Cys8 of the thiopeptide framework through ester and thioester linkages, respectively, to form a 19-membered compact side ring system. DMIA is biologically important and can interact with A1067, a nucleobase of 23S rRNA that contributes to mutation-induced bacteria resistance.2,8,9 The selective 5′-fluorination of this moiety led to the generation of a halogenated NOS derivative, which displays improved antibacterial activity compared to the parent compound.10 In S. actuosus, DMIA formation starts with processing a precursor peptide-independent L-Trp residue by NosL, a radical S-adenosylmethionine (SAM)-dependent protein. NosL catalyzes a complex arrangement of the carbon side chain of L-Trp to produce 3-methyl-2-indolic acid (MIA) as a key intermediate (Figure 1B).10 Recently, we reported that the incorporation of this intermediate follows the common
hiopeptide antibiotics are a growing family of sulfur-rich and highly modified peptide natural products that have long been appreciated because of their potent antibacterial activity, complex architecture, and unusual mode of action.1 Distinct from other chemotherapeutics that target bacterial ribosome, many members of this family inhibit bacterial protein synthesis by binding within a cleft located between the L11 protein and 23S rRNA of the 50S large ribosomal subunit.2 Each thiopeptide arises from complex post-translational modifications (PTMs) of a ribosomally synthesized precursor peptide that is composed of an N-terminal leader peptide (LP) sequence and a C-terminal core peptide (CP) sequence.3,4 Common PTMs transform the CP sequence, which is Ser/Thr and Cys-rich, to a characteristic thiopeptide framework that features a 6-membered nitrogen-containing domain central to multiple azoles and dehydroamino acids.5 In contrast, specific PTMs are highly diverse for individualized treatment, thereby underlying the thiopeptide family that contains over 100 individual members in addition to sequence permutation of precursor peptides. © XXXX American Chemical Society
Received: January 23, 2019
A
DOI: 10.1021/acs.orglett.9b00293 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
based on analysis by high-performance liquid chromatography with high-resolution mass spectrometric detection (HPLC− MS) (Figures 2A,B). To determine whether they are derived
Figure 1. Nosiheptide (NOS) and biosynthetic reactions related to side ring formation. The L-Trp-derived indolic moiety and the thiolation protein NosJ are indicated in blue and red, respectively. (A) Structure of NOS, in which the DIMA moiety is indicated in a dashed rectangle. (B) MIA formation and activation. (C) Transformation of the precursor peptide NosM into the MIA-S-Cys8 pentathiazolyl peptide intermediate 3 through peptide intermediate 2.
Figure 2. Assays of NosN activity using peptide intermediate 3. (A) HPLC−MS analysis of the conversions of 3 in the absence (blue) and presence (red) of NosN. EIC m/z = 1017.5. (B) HR-MS spectra of 6 (left) and 7 (right). (C) Fragmentation patterns of 6 (top) and 7 (bottom) by HR-MS/MS.
PTMs of the precursor peptide (NosM, 1) to the pentathiazolyl peptide intermediate (2).11 With the adenylation protein NosI, MIA is activated and loaded on the discrete thiolation protein NosJ, yielding NosJ-S-MIA (5). NosK, an αhydrolase-fold protein, then transfers MIA to the unmodified residue Cys8 of 2, leading to the production of the MIA-SCys8 peptide intermediate 3 (Figure 1C).11 We have previously indicated that the functionalization of MIA to DMIA within the side ring system requires the second radical SAM protein NosN because the S. actuosus mutant strain in which nosN was inactivated accumulates 4,12 a MIA-Sconjugated, side-ring-opening NOS derivative. The radical SAM-dependent activity of NosN was recently validated in vitro;13,14 however, related experimental evidence appeared to be contradictory in terms of the timing of NosN action, its associated catalytic mechanism, and its role in the establishment of the DMIA-containing side ring system. Using 3, 4, and 5 as substrates, we here evidenced the flexibility of NosN activity and thereby provide insights into NosN-catalyzed PTM in NOS biosynthesis. We expressed C-terminally 8 × His tagged NosN in Escherichia coli. This recombinant protein was purified to >90% homogeneity using Ni2+ affinity chromatography under strict anaerobic conditions. After the reconstitution of the [4Fe-4S] cluster, purified NosN appeared gray and exhibited an absorbance spectrum characteristic of radical SAM-dependent enzymes (Figure S1). We first assayed NosN activity using the peptide intermediate 3 (Figure S2). In the presence of saturated SAM, the anaerobic incubation of NosN with 3 resulted in two distinct products, 6 and 7 (with a ratio ∼2.5:1),
from 3, HR-MS/MS analysis was conducted with variable collision energy (Figures 2C and S3,4). Consequently, 6 and 7 share several b+ fragments with 3, indicating that both products are identical to 3 at the N-terminus and possess an unmodified LP sequence. The difference in 6 and 7 from 3 was narrowed to their CP sequences. Specifically, compared with the fragmentation of 3, which produced the y+ fragments y6+ 1102.1918 and y8+ 1373.2532, fragmentation of the major product 6 yielded the y+ fragments y6+ 1114.1929 and y8+ 1385.2565 (as well as y9+ 1456.2960 and y10+ 1543.3248), indicating that the 8-aa C-terminal sequence was subjected to NosN-catalyzed PTM. These findings led to a hypothesis that 6 possesses a NOS-like side ring, in which Cys8-S-conjugated MIA might be functionalized at C4′ by a C1 unit that links Glu6 through an ester bond to account for a +12 Da increase in molecular weight (MW). In particular, 6 fragmentation resulted in a daughter ion with m/z at 188.0703, which is different from that specific for MIA (m/z at 158.0592) by 3 fragmentation, a finding consistent with the prediction that the cleavage of both the ester and thioester linkages of 6 produces a hydroxymethylated MIA derivative (HOMMIA). By contrast, 7, the minor product with a +14 Da increase in MW, is likely a shunt product with an unlinked DMIA moiety. It could be generated by C4′ methylation of 3, according to the distinct daughter ion with m/z at 172.0756 after fragmentation. These findings supported the notion that NosN is a side ringforming enzyme for radical SAM-dependent MIA functionalization rather than an MIA C4′-methyltransferase as previously proposed during NOS biosynthesis.13 B
DOI: 10.1021/acs.orglett.9b00293 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Next, to determine whether NosN can function after pyridine formation, we tested its activity using the NOS derivative 4. This derivative, which was previously assigned on the basis of MS/MS analysis, was scaled up by fermenting the ΔnosN S. actuosus strain.12 After isolation and purification, the structural identity of 4 was confirmed by nuclear magnetic resonance (NMR) in this study (Figure S5). With NosN, ∼40% of 4 ([M + H] + m/z calcd 1248.1713 for C53H45N13O12S6, obsd 1248.1669) was converted into a new product, 8 ([M + H] + m/z calcd 1262.1869 for C54H47N13O12S6, obsd 1262.1842) (Figures 3B and S6), over
Figure 4. Assays of NosN activity using the peptide substrate 5. (A) NosN-catalyzed conversion of 5 into 9 and 10. (B) HPLC−HR-MS analysis of the conversions of 5 in the absence (top) and presence (bottom) of NosN. (C) Fragmentation pattern of 9 by HR-MS/MS.
S-DMIA (Figures 4C and S9). Similar results were observed in the conversion of SNAC-S-MIA to SNAC-S-DMIA (Figure S7).Notably, the conversion of 5 also produced a trace of a previously uncharacterized +30 Da product, 10 (Figure 4B), allowing for the prediction that NosN activity confers a hydroxymethyl group at C4′ of MIA, yielding NosJ-SHOMMIA. In the biosynthetic pathway of NOS, it is unlikely that NosN plays the role for NosJ-S-MIA conversion, which could be a shunt reaction, given that no unannotated biosynthetic genes within the nos cluster remain to be responsible for further PTMs to form an ester linkage with Glu6 and complete the side ring system. However, the finding of the product NosJ-S-HOMMIA is important and supports the hypothesis that NosN functions beyond being an MIA C4′-methyltransferase. Mechanistically similar to radical SAM homologues that were characterized recently (Figure 5),17,18 NosN could reductively cleave the first SAM molecule, yielding 5′-dA•, which then abstracts a hydrogen atom from the second SAM molecule,14 rather than 5′-methylthioadenosine as Zhang et al. indicated,13 to produce a SAM methylene radical. This SAM radical can be added to C4′ of the MIA moiety shared by 3−5, and subsequent S-adenosyl-L-homocysteine (SAH) elimination might lead to the production of a substrate radical containing an exocyclic methylene group at C4′. The observation of SAH as a coproduct in the tested reactions (Figure S10), all of which were 5′-methylthioadenosine independent, supported this conclusion. Methylated products could be derived from the substrate methylene radical intermediate by electron transfer and subsequent protonation. Alternatively, branching may occur after electron loss to produce a reactive electrophilic species and depends on the MIA-S-conjugated peptide nature of substrates. Only by using 3, which appears to be the native substrate of NosN, this species can approach Glu6 to complete the side ring formation. Conducting the NosN-catalyzed 3 transformation in H218O resulted in the unlabeled cyclized
Figure 3. Assays of NosN activity using the NOS derivative 4. (A) NosN-catalyzed conversion of 4 into 8. (B) HPLC analysis of the conversions of 4 in the absence (blue) and presence (red) of NosN (λ at 330 nm).
a 1 h incubation period. Although the yield of 8 was insufficient for NMR-based structural elucidation, HR-MS/ MS analysis suggests that it bears an uncyclized DMIA moiety, consistent with a +14 Da increase in MW compared with 4. These results indicate that NosN exhibits methyltransferase activity in the presence of 4, in contrast to the previous reports by Zhang et al. showing that NosN cannot recognize 4 as a substrate.16 Compound 4 shares with 3 an MIA-S-conjugated polythiazolyl skeleton, and a linear peptide mimic of this skeleton was recently used by Booker et al. to test NosN activity for the formation of the DMIA-containing side ring system.14 The strained cyclic structure of 4 could hamper the residue Glu6 approaching the active site of NosN in the functionalization process of MIA, thereby leading to C4′methylation of MIA as the only result. Most likely, NosNcatalyzed PTM proceeds before pyridine formation, in line with the previous finding of an NOS derivative that possesses a complete side ring system but lacks the thiopeptide-characteristic, 6-membered nitrogen-containing central domain.15 Finally, we examined NosN activity using NosJ-S-MIA (5). This peptide substrate (Figure S8), along with the mimic S-(Nacetyl)cysteamine (SNAC)-S-MIA (Figure S7), was used by Zhang et al. to assign NosN function as an MIA C4′methyltransferase;13,16 however, it did not work in assays conducted by Booker et al. Consistent with previous findings, the incubation of NosN with 5 primarily produced a +14 Da methylated product, 9 (Figures 4B), albeit with a yield much lower than 6 in NosN-catalyzed 3 transformation. Further HRMS/MS analysis supported the structural identity of 9 to NosJC
DOI: 10.1021/acs.orglett.9b00293 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Author Contributions ∥
Y.Q. and Y.D. contributed equally.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank Prof. Heinz G. Floss at the University of Washington for providing of the TSR-producing strain S. laurentii ATCC 31255. This work was supported in part by grants from NSFC (31430005, 21750004, 21520102004, 21621002, and 21472231), CAS (QYZDJ-SSW-SLH037 and XDB20020200), K. C. Wang Education Foundation, and Chang-Jiang Scholars Program of China.
■
Figure 5. Proposed mechanisms for NosN-catalyzed conversions using various substrates.
product 6 (Figure S11), consistent with the hypothesis that the ester bond linkage comes from Glu6. Otherwise, the species would be quenched by a hydroxyl ion in solution to afford a hydroxymethyl group. To examine this hypothesis, we conducted the NosN-catalyzed conversion of 5 in H218O with d3-SAM (Figure S12). As anticipated, this conversion produced the +2 Da NosJ-S-DMIA derivative and, particularly, the +4 Da NosJ-S-HOMMIA derivative. In conclusion, based on the examination of NosN activity using various substrates, we provide insight into the role of this unusual radical SAM protein in the biosynthesis of the bicyclic thiopeptide member NOS. Beyond being an methyltransferase only, NosN likely functions before pyridine formation and transforms a polythiazolyl peptide intermediate by functionalizing the Cys8-S-conjugated MIA moiety at C4′ to build a C1 unit and link Glu6 through an ester bond during the formation of the side ring system specific for NOS. This study highlights the unusual logic in the development of peptide-based structural complexity and diversity through the interdependence of common and specific PTMs. Following NosN activity, common PTMs may proceed to furnish multiple dehydroamino acids and the central heterocyclic domain to establish the characteristic thiopeptide framework.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00293. Supplementary methods, results, figures and tables (PDF)
■
REFERENCES
(1) Bagley, M. C.; Dale, J. W.; Merritt, E. A.; Xiong, A. Chem. Rev. 2005, 105, 685. (2) Harms, J. M.; Wilson, D. N.; Schluenzen, F.; Connell, S. R.; Stachelhaus, T.; Zaborowska, Z.; Spahn, C. M.; Fucini, P. Mol. Cell 2008, 30, 26. (3) Chen, M.; Liu, J.; Duan, P.; Li, M.; Liu, W. Natl. Sci. Rev. 2017, 4, 553. (4) Arnison, P. G.; Bibb, M. J.; Bierbaum, G.; Bowers, A. A.; Bugni, T. S.; Bulaj, G.; Camarero, J. A.; Campopiano, D. J.; Challis, G. L.; Clardy, J.; Cotter, P. D.; Craik, D. J.; Dawson, M.; Dittmann, E.; Donadio, S.; Dorrestein, P. C.; Entian, K. D.; Fischbach, M. A.; Garavelli, J. S.; Goransson, U.; Gruber, C. W.; Haft, D. H.; Hemscheidt, T. K.; Hertweck, C.; Hill, C.; Horswill, A. R.; Jaspars, M.; Kelly, W. L.; Klinman, J. P.; Kuipers, O. P.; Link, A. J.; Liu, W.; Marahiel, M. A.; Mitchell, D. A.; Moll, G. N.; Moore, B. S.; Muller, R.; Nair, S. K.; Nes, I. F.; Norris, G. E.; Olivera, B. M.; Onaka, H.; Patchett, M. L.; Piel, J.; Reaney, M. J.; Rebuffat, S.; Ross, R. P.; Sahl, H. G.; Schmidt, E. W.; Selsted, M. E.; Severinov, K.; Shen, B.; Sivonen, K.; Smith, L.; Stein, T.; Sussmuth, R. D.; Tagg, J. R.; Tang, G. L.; Truman, A. W.; Vederas, J. C.; Walsh, C. T.; Walton, J. D.; Wenzel, S. C.; Willey, J. M.; van der Donk, W. A. Nat. Prod. Rep. 2013, 30, 108. (5) Burkhart, B. J.; Schwalen, C. J.; Mann, G.; Naismith, J. H.; Mitchell, D. A. Chem. Rev. 2017, 117, 5389. (6) Benazet, F.; Cartier, M.; Florent, J.; Godard, C.; Jung, G.; Lunel, J.; Mancy, D.; Pascal, C.; Renaut, J.; Tarridec, P.; Theilleux, J.; Tissier, R.; Dubost, M.; Ninet, L. Experientia 1980, 36, 414. (7) Wang, S.; Zhou, S.; Liu, W. Curr. Opin. Chem. Biol. 2013, 17, 626. (8) Lin, Z.; He, Q.; Liu, W. Curr. Opin. Biotechnol. 2017, 48, 210. (9) Baumann, S.; Schoof, S.; Bolten, M.; Haering, C.; Takagi, M.; Shin-ya, K.; Arndt, H. D. J. Am. Chem. Soc. 2010, 132, 6973. (10) Zhang, Q.; Li, Y.; Chen, D.; Yu, Y.; Duan, L.; Shen, B.; Liu, W. Nat. Chem. Biol. 2011, 7, 154. (11) Qiu, Y.; Du, Y.; Zhang, F.; Liao, R.; Zhou, S.; Peng, C.; Guo, Y.; Liu, W. J. Am. Chem. Soc. 2017, 139, 18186. (12) Yu, Y.; Duan, L.; Zhang, Q.; Liao, R.; Ding, Y.; Pan, H. ACS Chem. Biol. 2009, 4, 855. (13) Ding, W.; Ji, W.; Wu, Y.; Wu, R.; Liu, W. Q.; Mo, T.; Zhao, J.; Ma, X.; Zhang, W.; Xu, P.; Deng, Z.; Tang, B.; Yu, Y.; Zhang, Q. Nat. Commun. 2017, 8, 437. (14) LaMattina, J. W.; Wang, B.; Badding, E. D.; Gadsby, L. K.; Grove, T.; Booker, S. J. J. Am. Chem. Soc. 2017, 139, 17438. (15) Guo, H.; Wang, J.; Li, Y.; Yu, Y.; Zheng, Q.; Wu, J.; Liu, W. Chem. Sci. 2014, 5, 240−246. (16) Ding, W.; Li, Y.; Zhao, J.; Ji, X.; Mo, T.; Qianzhu, H.; Tu, T.; Deng, Z.; Yu, Y.; Chen, F.; Zhang, Q. Angew. Chem., Int. Ed. 2017, 56, 3857. (17) Zhang, Z.; Mahanta, N.; Hudson, G. A.; Mitchell, D. A.; van der Donk, W. A. J. Am. Chem. Soc. 2017, 139, 18623. (18) Jin, W. B.; Wu, S.; Jian, X. H.; Yuan, H.; Tang, G. L. Nat. Commun. 2018, 9, 2771.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Yinlong Guo: 0000-0003-2493-2876 Wen Liu: 0000-0003-2729-1787 D
DOI: 10.1021/acs.orglett.9b00293 Org. Lett. XXXX, XXX, XXX−XXX
