Thiolation Protein-Based Transfer of Indolyl to a Ribosomally

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Thiolation Protein-Based Transfer of Indolyl to a Ribosomally Synthesized Polythiazolyl Peptide Intermediate during the Biosynthesis of the Side Ring System of Nosiheptide yanping qiu, Yanan Du, Fang Zhang, Rijing Liao, shuaixiang zhou, Chao Peng, Yinlong Guo, and Wen Liu J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b11367 • Publication Date (Web): 04 Dec 2017 Downloaded from http://pubs.acs.org on December 4, 2017

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Journal of the American Chemical Society

Thiolation Protein-Based Transfer of Indolyl to a Ribosomally Synthesized Polythiazolyl Peptide Intermediate during the Biosynthesis of the Side Ring System of Nosiheptide Yanping Qiu1,#, Yanan Du1,#, Fang Zhang2, Rijing Liao3, Shuaixiang Zhou1, Chao Peng4, Yinlong Guo2, and Wen Liu1,5,6,* 1

State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China 2 State Key Laboratory of Organometallic Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China 3 Xuhui Central Hospital, Shanghai Clinical Center, Chinese Academy of Sciences, Shanghai 200031, China 4 Shanghai Science Research Center, Chinese Academy of Sciences, Shanghai 200031, China 5 State Key Laboratory of Microbial Metabolism, School of Life Science & Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China 6

Huzhou Center of Bio-Synthetic Innovation, 1366 Hongfeng Road, Huzhou 313000, China

Supporting Information Placeholder ABSTRACT: Nosiheptide, a potent bicyclic member of the family of thiopeptide antibiotics, possesses a distinctive L-Trp-derived indolyl moiety. The way in which this moiety is incorporated into a ribosomally synthesized and post-translationally modified thiopeptide remains poorly understood. Here, we report that NosK, an α/β-hydrolase fold protein, mediates the transfer of indolyl from NosJ, a discrete thiolation protein, to a linear pentathiazolyl peptide intermediate rather than its genetically-encoded untreated precursor. This intermediate results from enzymatic processing of the peptide precursor, in which five of the six L-Cys residues are transformed into thiazoles but Cys4 selectively remains unmodified for indolyl substitution via a thioester exchange. Determining the timing of indolyl incorporation, which expands the chemical space of a thiopeptide framework, facilitates mechanistic access to the unusual logic of post-translational modifications in the biosynthesis of nosiheptide-type thiopeptide members that share a similar compact side-ring system.

nos: A

B

C

D

Peptide Precursor

F

G

H

Indolyl Incorporation

I

JK

L

M N

Thiazole Formation

O

P

Others

Core Peptide SerCysThrThrCysGlucCysCysCysSerCysSerSer

Leader Peptide NH2

NosM (1)

COOH H N

O

O NH2

OH

NH2

N COOH

S

Nosiheptide (NOS)

S

N N

HN

Indolyl Moiety

N S

O

HO

S

N H

L-Trp

O

NH HN

O

O

S H N

N

Thiopeptide antibiotics are a growing class of sulfur-rich, ribosomally synthesized and post-translationally modified peptide natural products.1 These antibiotics possess a wide variety of biological properties, e.g., anti-infection, antitumor and immunosuppressive characteristics,2 and have therefore long been the targets of intensive synthetic and biosynthetic attempts to improve their biological activities and overcome associated physiochemical drawbacks for clinical use.3 Each thiopeptide arises from posttranslational modifications (PTMs) of a peptide precursor that contains an N-terminal leader peptide (LP) and a C-terminal core peptide (CP) (Figure 1). CP is rich in L-Cys and L-Ser/Thr residues and is the sequence subjected to myriad PTMs during the formation of a macrocyclic framework that features a sixmembered heterocyclic domain central to multiple azoles and dehydroamino acids.4

E

NH N

COOH S O

O O OH

N H

3-Methyl-2-Indolic Acid (MIA)

Figure 1. Biogenesis and structure of bicyclic thiopeptide NOS. The genes coding for the ribosomally synthesized precursor peptide (black), indolyl incorporation (red) and thiazole formation (yellow) are highlighted in the nos cluster. The route for forming MIA (blue) and associated indolyl moiety (in a dashed rectangle) within the side-ring system of NOS is indicated.

While the establishment of a shared thiopeptide framework requires common PTMs,5 the specialization of this framework for over 100 different thiopeptide members largely depends on a number of specific PTMs. The biosynthesis of nosiheptide (NOS), an archetypal member of the family of thiopeptide antibiotics, is no exception to this principle.6 NOS is bicyclic and possesses an

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Figure 2. Preparation of indolyl donor. (A) Route for thiolation protein-based MIA activation. (B) In vivo production profiles of S. actuosus strains for analyzing the necessity of nosJ by HPLC (UV at 330 nm). i, wild-type; ii, △nosJ by in-frame deletion; iii, in trans expression of nosJ in △nosJ mutant; and iv, site-specific mutation for changing S to A within the conserved motif of NosJ. (C) In vitro assays of NosJ in various forms by HPLC-HR-MS. i, apo-form (Calcd. 10531.1758; Found 10531.1807; Er. 0.5 ppm); ii, holo-form (Calcd. 10871.2613; Found 10871.2648; Er. 0.3 ppm); and iii; NosJ-S-MIA (acyl-form, Calcd. 11028.3142; Found 11028.3360; Er. 2.0 ppm).

indolyl moiety that is appended to the characteristic thiopeptide core through (thio)ester linkages to form a compact side-ring system (Figure 1). This unusual moiety is biologically relevant, and its selective fluorination led to an improvement in the antiinfection activity of the molecule.7a In NOS biosynthesis, the indolyl moiety is derived from an LTrp residue independent of the peptide precursor (NosM, 1).7 This residue undergoes a complex radical-mediated rearrangement to process its carbon side chain and produce 3-methyl-2-indolic acid (MIA) as a key intermediate (Figure 1). Subsequent steps in the formation of the side-ring system remains poorly understood, particularly the process by which MIA is processed and incorporated with the ribosomally synthesized peptide precursor-derived portion. MIA is essentially a non-proteinogenic amino acid. Given that non-proteinogenic amino acids can be used by non-ribosomal peptide synthetases (NRPSs) in peptidyl assembly,8 MIA could be processed similarly to monomers in NRPS catalysis, which typically involves an adenylation protein/domain for monomer activation and a thiolation protein/domain for channeling via a phosphopantetheinyl (Ppant) group coming from coenzyme A (CoA) (Figure 2A). In accordance with this hypothesis, we reanalyzed the biosynthetic gene cluster of NOS (Figure 1),9 in which the three closely clustered genes, nosI, nosJ and nosK, attracted our attention. It has been proposed that nosI and nosK encode an ATP-dependent acyl-CoA synthetase and an α/β-hydrolase fold protein, respectively. In contrast, nosJ was previously unassigned and codes for a 79-aa protein containing the thiolation protein/domain-characteristic motif DSLETV (Figure S1), in which the L-Ser residue could be phosphopantetheinylated. The inactivation of nosJ in the NOS-producing Streptomyces actuosus strain completely abolished NOS production, which was partially restored by in trans supplementation of nosJ, demonstrating the necessity of this gene for NOS biosynthesis (Figure 2B). In particular, the site-specific mutation of L-Ser to L-Ala within the conserved motif DSLETV led to a NOS-nonproducing phenotype identical that observed in the ∆nosJ mutant strain (Figure 2B),

supporting the hypothesis that the deduced protein NosJ functions as a discrete thiolation protein. To perform activity assays in vitro, we overexpressed and purified the three proteins NosI, NosJ and NosK from Escherichia coli BL21(DE3) (Figure S2). The resulting NosJ protein existed in an unmodified apo-form and was then transformed into its phosphopantetheinylated holo-form by Sfp,10 a Ppant transferase (PPTase) from Bacillus subtilis (Figures 2C and S3-S5). In the presence of NosI and MIA, the holo-form of NosJ was effectively acylated via thioesterification in an ATP-dependent manner to yield NosJ-S-MIA (Figures 2C and S6). In addition, we coexpressed NosI and NosJ in E. coli BAP1, which carries the PPTase gene sfp for the in vivo phosphopantetheinylation of apoNosJ to holo-NosJ.11 In the supplementation with exogenous MIA, this co-expression system yielded holo-NosJ and NosJ-SMIA simultaneously. These findings, in addition to similar results that were recently reported by two other research groups,12 demonstrated that NosI is an adenylation protein for the activation and transfer of MIA to NosJ. In particular, Boal/Booker et al. showed that NosK catalyzes the transfer of MIA from NosJ to a conserved L-Ser residue and produces NosK-O-MIA, an acyl-Ser enzyme intermediate.12a NosK is most likely an α/β-hydrolase fold indolyltransferase; however, the timing of its activity during PTMs of the ribosomally synthesized precursor peptide remained to be determined. To examine whether the unmodified ribosomally synthesized peptide precursor can accept the indolyl group from NosJ, we produced NosM in E. coli BL21(DE3) (Figures S2 and S7). To prepare indolyl donor, we incubated NosI and holo-NosJ (the latter of which was purified from E. coli BAP1) with an excess of MIA, yielding the thiolation protein-based intermediate NosJ-SMIA. In the presence of NosK, the transfer of MIA from NosJ to the peptide precursor NosM was not observed on careful Highperformance liquid chromatography with mass spectrometric detection (HPLC-MS). Consistently, feeding MIA to the E. coli BAP1 strain in which NosM was co-expressed with NosK and the NosJ-S-MIA-forming complex composed of NosI and NosJ failed to produce the predicted acylated NosM variant. These results excluded the possibility that the selective acylation of NosM proceeds as the first PTM in NOS biosynthesis to protect Cys4 against cyclodehydration and dehydrogenation, the process by which the other five Cys residues of the CP sequence are exclusively transformed into thiazoles. To determine whether indolylation occurs on a modified peptide intermediate in NOS biosynthesis, we co-expressed NosM and proteins necessary for thiazole formation (the cyclodehydratase NosG, the dehydrogenase NosF and the Ocin-ThiF-like protein NosH for precursor peptide engaging) in E. coli BL21(DE3).5 The combination of NosM with NosGFH produced a -100 Da product (2) compared with the untreated precursor peptide (Figure 3A), and subsequent high resolution (HR)-MS/MS analysis further supported the hypothesis that 2 is a pentathiazolyl peptide resulting from the selective transformation of five L-Cys residues during successive cyclodehydration-dehydrogenation reactions (Figure S10). NosM was subjected to Ala-scanning of all six LCys residues in the CP sequence to produce precursor peptide variants 1-C1A, 1-C2A, 1-C3A, 1-C4A, 1-C5A or 1-C6A in the thiazole-forming E. coli system, and the corresponding polythiazolyl products were purified to examine the changes in molecular weight (Table 1 and Figure S11). Only the mutation of C4A had no effect on thiazole number and led to a product (2-C4A) with a -100 Da shift due to pentathiazole formation. The mutations of C1A, C2A, C3A and C6A resulted in the products 2-C1A, 2C2A, 2-C3A and 2-C6A, respectively, which share a -80 Da shift arising from tetrathiazole formation. These findings confirmed the involvement of residues Cys1, Cys2, Cys3 and Cys6 in the


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Figure 3. Indolyl incorporation with a pentathiazolyl peptide intermediate. (A) PTM route of the precursor peptide (1) for NosGFH-mediated thiazole (yellow) formation (yielding intermediate 2) and subsequent NosK-catalyzed MIA (blue) transfer (producing 3), as well as the release of MIA in the presence of glycerol (yielding shunt product 4). (B) In vitro analysis of the transfer of MIA to intermediate 2 by HPLC (UV at 245 nm). i, in the absence of NosK; and ii, iii and iv, in the presence of NosK for 10 min, 2 h and 6 h, respectively.

effects of NosGFH treatment. In contract, the mutation of C5A led to a -62 Da product (2-C5A*), consistent with the notion that dehydrogenation following trithiazole formation (yielding 2-C5A) occurs in E. coli BL21(DE3). HR-MS/MS analysis further revealed the positions of the trithiazoles, which are derived from Cys1, Cys2 and Cys3, and particularly a disulfide linkage formed between Cys4 and Cys6 in the CP sequence of 2-C5A* (Figure S12). This disulfide linkage protects Cys6 and could allow this residue to escape from processing by the thiazole synthase complex; alternatively, Cys6-based thiazole formation is dependent of a similar Cys5-based conversion. Cys4 is clearly the only unmodified L-Cys residue after treating NosM with the thiazole synthase complex NosGFH, which selectively acts on the other five L-Cys residues of the CP sequence (Cys1, Cys2, Cys3, Cys5 and Cys6) during the formation of pentathiazolyl peptide intermediate 2 (Figure 3A). Next, we incubated 2 with the indolyl donor NosJ-S-MIA, which was prepared in situ as described above by mixing NosI, holo-NosJ and MIA. Remarkably, in the presence of NosK, the MIA group was effectively transferred from NosJ to 2 in a timedependent manner (Figures 3B, S8 and S9), yielding 3, a product confirmed to be acylated with MIA via HR-MS/MS analysis (Figures 3A and S13). In a control reaction in which 2 was replaced by its variant 2-C4A, MIA transfer failed to occur (Table 1 and Figure S16D). These results indicated that the pentathiazolyl peptide intermediate 2 is an indolyl acceptor and that processing the precursor peptide to this intermediate precedes functionalizing its Cys4 residue with MIA via a thioester exchange. Interestingly,

excess MIA in the reaction mixture can be completely consumed; under this reaction condition, acylated product 3 reached a maximum, gradually diminished and then completely disappeared over a 6-h incubation period (Figures 3B and S9). Meanwhile, a new product (4) appeared later than 3, accompanying the regeneration of 2, and became dominant in the late stage of the reaction. We scaled up this reaction and accumulated a sufficient quantity of 4 for structural elucidation. Spectral analysis revealed that this product is an ester derivative of MIA and glycerol (Figures 3A and S14), the latter of which is a protein-protective agent introduced into the reaction mixture when using enzymes stored at low temperature. Clearly, 3 appears to be unstable and tends to suffer from nucleophilic attack in the absence of further pathwayassociated PTMs. We then evaluated whether NosK activity tolerates the variation in the indolyl acceptor (Table 1). Individual replacement of pentathiazolyl peptide intermediate 2 with its tetrathiazolyl variants 2-C1A, 2-C2A, 2-C3A and 2-C6A led to effective production of the corresponding acylated products 3-C1A, 3-C2A, 3C3A and 3-C6A (Figures S15 and S16). Thus, the formation of pentathiazoles is not necessary for NosK-mediated MIA transfer. However, this reaction failed to occur with 2-C5A (Figure S16F), a trithiazolyl variant of 2 that was generated by treating 2-C5A* with tris(2-carboxyethyl)phosphine to release Cys4 and Cys6, suggesting that the transfer of indolyl to Cys4 relies on Cys5based thiazole formation. To examine whether NosK catalysis is applicable to the exchange between the thioester and ester linkages, we prepared 2-C4S, a pentathiazolyl variant of 2 in which Cys4 is substituted with an L-Ser residue, by mutating NosM to variant 1-C4S in the aforementioned thiazole-forming E. coli system (Table 1 and Figures S15D and S16E). Consequently, the acylated product 3-C4S, in which MIA conjugates the pentathiazolyl peptide via an ester linkage, was produced effectively, confirming the specificity of the Cys4 position for indolyl installation. Interestingly, this catalysis mimics the process of MIA Table 1. Calculated and found MS data of 1, 2 and 3 and their variants in this study.

Variants

Calcd. M.W. (Da)

Found M.W. (Da)

Er. (ppm)

1-C1A

7013.9712

n,d

--

1-C2A

7013.9712

n,d

--

1-C3A

7013.9712

n,d

--

1-C4A

7013.9712

n,d

--

1-C4S

7029.9661

n,d

--

1-C5A

7013.9712

n,d

--

1-C6A

7013.9712

n,d

--

2-C1A

6933.8663

6933.8881

3.1

2-C2A

6933.8663

6933.8503

2.3

2-C3A

6933.8663

6933.8627

0.5

2-C4A

6913.8401

6913.8273

1.9

2-C4S

6929.8350

6929.8178

2.5

2-C5A*

6951.8769

6951.9002

3.6

2-C6A

6933.8663

6933.8922

3.7

3-C1A

7090.9192

7090.9237

0.6

3-C2A

7090.9192

7090.9009

2.6

3-C3A

7090.9192

7090.8959

3.3

3-C4A

--

n,d

--

3-C4S

7086.8879

7086.8834

0.6

3-C5A

--

n,d

--

3-C6A

7090.9192

7090.9132

0.9

n.d: no detected



incorporation in the biosynthesis of nocathiacins (NOCs),13 a group of naturally occurring analogs of NOS, through intermediates analogous to 2-C4S and 3-C4S because NocM, the precursor peptide of NOCs, contains a CP sequence identical to that of 1C4S. Most likely, the NOC and NOS biosynthetic pathways share a similar set of PTM enzymes (e.g., NocLIJ-GFH-K and NosLIJGFH-K with 52-89% identities) during the formation of their sidering systems; in each case, the indolyl group is appended to L-Ser or L-Cys residue via an ester or a thioester linkage at the same position. The study described herein provides insights into the less understood biosynthetic process of the side-ring system of NOS, as how and when the biologically important indolyl residue is incorporated were determined. Booker et al. recently demonstrated that NosN, a Class C S-adenosylmethionine (SAM)-dependent radical protein, catalyzes the transfer of a carbon atom from SAM to the MIA moiety that is tethered to a trithiazolyl pentapeptide and then the cyclization of the pentapeptide by forming an ester linkage between its L-Glu residue and the newly introduced C1 unit.14 Intriguingly, this pentapeptide is a simplified synthetic mimic of the indolylated pentathiazolyl peptide 3, supporting that 3 is a key intermediate prior to the functionalization of the tethered MIA moiety during the formation of the side-ring system. Increasing evidence has indicated that PTMs of ribosomally synthesized precursor peptides are comparable to NRPSs in terms of the creation of structural complex molecules. Here, the characterization of indolyl incorporation and its timing in NOS biosynthesis highlights an unusual PTM paradigm that merges with NRPS-like biosynthetic machinery (i.e., for indolyl donor preparation) in the expansion of the structural diversity of thiopeptide antibiotics. In conclusion, we determined the timing of the incorporation of MIA into the bicyclic thiopeptide antibiotic NOS. MIA is first activated as monomers in NRPS catalysis, yielding a thiolation protein-based indolyl donor, NosJ-S-MIA. In the presence of the indolyltransferase NosK, this non-proteinogenic α-amino acid is then transferred from NosJ to 2, a pentathiazolyl peptide intermediate instead of its unmodified precursor 1, to produce the unstable acylated product 3. The formation of 2 relies on the activities of the enzyme complex NosGFH, which converts five of the six LCys residues in the CP sequence of 1 to thiazoles during successive cyclodehydration and dehydrogenations. Both thiazole formation and indolyl transfer appear to be highly position-selective, given that the former common PTM leaves Cys4 unmodified for indolyl installation by the latter specific PTM via thioester exchange. This reactions, which expands the chemical space of a thiopeptide framework, exemplify the unusual biosynthetic logic in the development of peptide-based structural complexity through the interdependence of common and specific PTMs.

The authors declare no competing financial interests.

ACKNOWLEDGMENT We thank Prof. Heinz G. Floss at the University of Washington for providing of the NOS-producing strain S. actuosus ATCC 25421. This work was supported in part by grants from NSFC (31430005, 21520102004, 21472231, 21605154 and 21621002), CAS (QYZDJ-SSW-SLH037 and XDB20020200), STCSM (17JC1405100 and 15JC1400400), MST (2017ZX09101003-006010), K. C. Wang Education Foundation and Chang-Jiang Scholars Program of China.

REFERENCES (1) Ortega, M. A.; van der Donk, W. A., Cell Chem. Biol. 2016, 23, 31. (2) Bagley, M. C.; Dale, J. W.; Merritt, E. A.; Xiong, A., Chem Rev. 2005, 105, 685. (3) (a) Just-Baringo, X.; Albericio, F.; Alvarez, M., Angew. Chem. Int. Ed. Engl. 2014, 53, 6602; (b) Lin, Z.; He, Q.; Liu, W., Curr. Opin. Biotechnol. 2017, 48, 210. (4) Chen, M.; Liu, J.; Duan, P.; Li, M.; Liu, W., Natl. Sci. Rev. 2017, 4, 553-575. (5) Burkhart, B. J.; Schwalen, C. J.; Mann, G.; Naismith, J. H.; Mitchell, D. A., Chem. Rev. 2017 , 117, 5389. (6) Wang, S.; Zhou, S.; Liu, W., Curr. Opin. Chem.Biol. 2013, 17, 626. (7) Zhang, Q.; Li, Y.; Chen, D.; Yu, Y.; Duan, L.; Shen, B.; Liu, W., Nat. Chem. Biol. 2011, 7, 154. (8) Marahiel, M. A., J. Pept. Sci. 2009, 15, 799. (9) Yu, Y.; Duan, L.; Zhang, Q.; Liao, R.; Ding, Y.; Pan, H.; Wendt-Pienkowski, E.; Tang, G.; Shen, B.; Liu, W., ACS Chem. Biol. 2009, 4, 855. (10) Quadri, L. E.; Weinreb, P. H.; Lei, M.; Nakano, M. M.; Zuber, P.; Walsh, C. T., Biochemistry 1998, 37, 1585. (11) Pfeifer, B. A.; Admiraal, S. J.; Gramajo, H.; Cane, D. E.; Khosla, C., Science. 2001, 291, 1790. (12) (a) Badding, E. D.; Grove, T. L.; Gadsby, L. K.; LaMattina, J. W.; Boal, A. K.; Booker, S. J., J. Am. Chem. Soc. 2017, 139, 5896; (b) 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. (13) Ding, Y.; Yu, Y.; Pan, H.; Guo, H.; Li, Y.; Liu, W., Mol. BioSyst. 2010, 6, 1180. (14) LaMattina, J. W.; Wang, B.; Badding, E. D.; Gadsby, L. K.; Grove, T. L.; Booker, S. J. J. Am. Chem. Soc. 2017, doi: 10.1021/jacs.7b08492.

ASSOCIATED CONTENT Supporting Information Supplementary Methods, Results, Figures and Tables. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *[email protected]

Author Contributions #

Y.Q. and Y.D. contributed equally to this work

Notes


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Thiolation Protein-Based Transfer of Indolyl to a Ribosomally

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