NRPS Protein MarQ Catalyzes Flexible Adenylation and Specific S

Letters Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX

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NRPS Protein MarQ Catalyzes Flexible Adenylation and Specific S‑Methylation Tingting Huang, Yingyi Duan, Yi Zou, Zixin Deng, and Shuangjun Lin* State Key Laboratory of Microbial Metabolism, Joint International Laboratory on Metabolic & Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China

ACS Chem. Biol. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 08/31/18. For personal use only.

S Supporting Information *

ABSTRACT: Maremycins are a group of structurally diverse 2,5-diketopiperazine natural products featuring a rare amino acid building block, S-methyl-L-cysteine (Me-Cys). Three freestanding nonribosomal peptide synthetase (NRPS) proteins from the maremycins biosynthetic pathway were proposed for the formation of the 2,5-diketopiperazine scaffold: MarQ, MarM, and MarJ. MarQ displays flexible adenylation activity toward Cys, Me-Cys, Ser, and (S)-2,3diaminopropanoic acid (DAP) and transfers these substrates to MarJ, which is the discrete peptidyl carrier protein (PCP). MarQ could also activate several other amino acids. The embedded methyltransferase (MT) domain in MarQ specifically catalyzes the thiol methylation of MarJ-tethered Cys. The in vitro reconstitution of MarQ and MarJ further provides clear evidence for the reaction sequence of methylation step on Cys. Our study on MarJ/Q tridomain cassette gains valuable insights into maremycins structure diversity and will be exploited to incorporate Me-Cys into natural products by combinatorial biosynthesis.

S

thioactin and thioxamycin,5 and nonribosomal peptides (NRPs), including echinomycin,6 thiocoraline,7 and the maremycin family.8,9 The enzymes and mechanisms for Smethylation of RiPPs remain enigmatic. Recently, from a cryptic RiPP pathway, a SAM (S-adenosyl-L-methionine)dependent methyltransferase PtyS is confirmed to modify the thiol groups of Cys residues in the core peptide in vitro, yet the corresponding natural product proteusin-like peptide has not been isolated.10 Echinomycin and thiocoraline are important members of the chromodepsipeptide family that function as DNA bisintercalators. In echinomycin cluster, a SAM methyltransferase Ecm18 first catalyzes the methylation of one of the S atoms of the disulfide bond of triostin A, then a rearrangement occurs to form the thioacetal bridge of echinomycin.11 In thiocoraline biosynthetic pathway, the bifunctional TioN was capable of catalyzing L-Cys adenylation and S-methylation.12 While TioN catalyzed the methylation of both PCP-Cys and AMP-Cys, the order of methylation and loading is still unknown in vivo. Diketopiperazines, containing peculiar heterocyclic system, have been found in many alkaloid natural products.13 Maremycins are a group of structurally diverse 2,5diketopiperazine derivatives that are composed of a (2S,3S)β-methyl tryptophan (Me-Trp) and a Me-Cys or L-serine (Ser) with additional modifications. To the best of our knowledge, maremycins and closely related FR900452 are the only group

ulfur is a functionally important element present in primary metabolites, such as thiamin, molybdopterin, and biotin, and their biosynthetic pathways diverge completely.1 Sulfur is also found in many microbial natural product peptides, while most of them are produced by ribosome or nonribosomal peptide synthetases (NRPSs) using cysteine (Cys) or methionine (Met) for sulfur incorporation.2 Diverse sulfur-containing moieties in these compounds have a major impact on the biological activities, and recently, various enzymatic mechanisms for C−S bond formation have been investigated.3,4 S-methyl-L-cysteine (Me-Cys) is a rare nonproteinogenic residue in natural product scaffolds (see Figure 1). For example, Me-Cys has been observed in ribosomally synthesized post-translationally modified peptides (RiPPs), such as

Received: April 19, 2018 Accepted: August 27, 2018

Figure 1. Representative S-methyl-L-cysteine containing natural products. The Me-Cys moieties are highlighted in light gray. © XXXX American Chemical Society

A

DOI: 10.1021/acschembio.8b00364 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology

light on the relevance of the two standalone NRPS proteins to the diverse maremycins structures. Based on previous bioinformatics analyses, it has been suggested that MarM uses aromatic amino acid phenylalanine (Phe) and MarQ activates Cys, according to the specificityconferring code of the A domain.19 However, the prediction does not correlate well with the amino acids that are incorporated into the maremycins backbone. To investigate the real substrates of the two A domains in vitro, we cloned, expressed, and purified MarM (146.9 kDa) and MarQ (89.9 kDa) as N-terminal His6-tagged proteins. Purified proteins were subjected to ATP-PPi release assay. Various amino acids have been tested, including Cys, Ser, Me-Cys, and tryptophan (Trp) derived (2S,3S)-β-Me-Trp, (2S,3R)-β-Me-Trp and (2S,3S)-2-amino-3-(2-oxindole) butyric acid (AIBA). The activity was measured at 360 nm by a coupling spectrophotometric analysis. These assays showed that MarM activated Trp-derived amino acids but not Phe or Cys, supporting the belief that MarM uses Trp-derived Me-Trp as a building block (see Figure S2 in the Supporting Information). The 2-oxindole motif in AIBA was catalyzed by MarE, which is a homologue of FeII/heme-dependent Trp 2,3-dioxygenase.20 Thus, MarG/H/ I catalyzes the specific formation of (2S,3S)-β-Me-Trp from Trp, and then MarE catalyzes the unique mono-oxygenation of the substrate to form AIBA. MarM was envisioned to activate AIBA for the maremycins assembly. MarQ exhibited the highest activation activity for Cys, which is consistent with the specificity code of MarQ A domain. MarQ also showed comparative adenylation activities for MeCys and Ser (Figure S2). In order to compare the catalytic efficiency of MarQ toward these three substrates, the kinetic analysis has been performed. The kinetic data of MarQ for Cys are approximately the same as those for Me-Cys, but ∼4-fold greater than those for Ser (see Table 1 and Figure S3 in the

of diketopiperazines bearing Me-Cys as a crucial building block. We have previously identified the maremycins gene cluster from Streptomyces sp. B9173, and characterized two methyltransferases MarI and MarF (see Figure 2A): (i) MarI is

Figure 2. Methyltransferases involved in maremycins biosynthesis. (A) Maremycins gene cluster. Methyltransferases MarF and MarI are highlighted in light gray, while NRPS related enzyme MarM, Q, and J are shown in black. (B) Domain organization of three NRPS proteins. Legend: A, NRPS adenylation domain; T, peptidyl carrier protein; C, condensation domain; MT, methyltransferase domain; UN, a domain with unknown function. MarQ-MT domain integrated between a2 and a3 motif of MarQ A domain; (C) Phylogenetic analysis of various MT domains. The S-MT, N-MT, and O-MT separate in the different clade.

involved in the formation of (2S,3S)-β-Me-Trp. The in vitro work confirmed that MarG functions as an L-Trp aminotransferase to give indolepyruvate from L-Trp, which is then converted to (R)-β-methylindolepyruvate by the methyltransferase MarI. Epimerization by MarH and second transamination by MarG yield (2S,3S)-β-Me-Trp.14 (ii) MarF was identified in vivo as an indole N-methyltransferase after isolation of the N-demethylated maremycins analogues from the marF knockout mutant.15 Therefore, among 17 predicted open reading frames of maremycins gene cluster, no reasonable stand-alone methyltransferase has been found to catalyze the Smethylation of L-Cys. Biosynthetic studies on maremycins revealed the NPRS origin of the diketopiperazine core. A typical NRPS module contains a set of core domains: a condensation (C) domain, an adenylation (A) domain, and a peptidyl carrier protein (PCP) domain.16,17 In addition, many auxiliary functional domains that catalyze, e.g., epimerization, methylation, cyclization, introduce a variety of chemical modifications to the NRP backbones to further enlarge their structural diversity.18 In maremycins gene cluster, marM encodes an NRPS enzyme that contains a C domain, an A domain, a PCP, and an unknown (UN) domain sharing very low similarity to other known NRPS domains. The second NRPS gene marJ codes for a stand-alone PCP, containing the conserved core GGHSLK for 4′-phosphopantetheine (Ppant) binding (Ser is the active site for PCP to accept Ppant from CoA). MarQ, the third NRPS enzyme, contains an A domain interrupted by an MT domain (see Figure 2B and Figure S1 in the Supporting Information). Inactivation of both marM and marQ abolished the production of all the maremycins components confirmed that these NRPS genes were responsible for maremycins biosynthesis.15 Upon the domain assignment of the three NRPS proteins, it was difficult to predict the overall arrangement of maremycins NRPS assembly line. Here, we report that MarQ collaborates with PCP domain MarJ for L-Cys activation and subsequent Smethylation to incorporate Me-Cys into maremycins peptide backbone. Our study explores the substrates specificity and incorporation efficiency by MarQ and MarJ, and further sheds

Table 1. KM and kcat for Adenylation Activity of MarQ-A Domaina substrate

KM (mM)

kcat (min−1)

kcat/KM (mM−1 min−1)

Cys Me-Cys Ser

0.44 ± 0.13 0.58 ± 0.17 1.92 ± 0.57

5.85 ± 0.80 6.64 ± 0.53 5.89 ± 1.10

13.31 11.32 3.06

a

By plotting the initial velocity of product formation against varying concentrations of substrates, the KM and kcat were calculated by direct fitting to the Michaelis−Menten equation.

Supporting Information). These in vitro findings are consistent with the different components of maremycins in vivo. Maremycin D originated from Ser is the minor component. Other maremycins all contain Me-Cys, indicating that Cys must be methylated prior to the condensing with the Trp derivative. According to the kinetic data, the methylation of Cys can occur either before activation by MarQ or after loading of Cys onto the PCP. MarQ consists of NRPS A and MT domains. The MT domain inserts between a2 and a3, the two conserved signature motifs of A domain, and the NRPS didomain protein TioN in thiocoraline pathway represents the same domain insertion pattern. It is entirely different from canonical N-MT or O-MT domains from NRPS megasynthetases, which normally exist between the a8 and a9 motifs (see Figure S1).21,22 A multiplesequence alignment indicated that MT domain in MarQ B

DOI: 10.1021/acschembio.8b00364 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Letters

ACS Chemical Biology showed 33%−43% identities with conventional NRPS MT domains and shared several conserved motifs for SAM binding, e.g., the glycine-rich motif I (GxGxG), and Motif II/Y (Figure S4 in the Supporting Information). The highly conserved key residue tyrosine (Tyr) has been reported to form an adduct with SAM in all types of MT domains.23 Despite the special integrated location between the a2 and a3 motifs of the MarQ A domain, the sequence similarity and conserved motifs suggest that MarQ-MT is functional and is able to process the Cys methylation step. To decipher methylation activity of MT domain in MarQ, we performed the enzymatic reaction in vitro. A reaction containing MarQ, Cys, or Me-Cys was performed for 40 min, and the reaction solution was treated with 1-fluoro-2,4dinitrobenzene (FDNB) as a derivatization reagent for Cys and Me-Cys detection. HPLC-MS analysis showed that MarQMT was unable to catalyze the methylation of the free amino acid Cys in the presence of SAM (see Figure S5 in the Supporting Information). During NRP biosynthesis, the A domain reaction involves (i) the initial amino acid recognition and binding occurring in the corresponding A domain to form the aminoacyl adenylate and (ii) the adenylated substrate undergoing a nucleophilic attack by the thiol of the Ppant arm of the PCP domain, forming a thioester bound aminoacyl-S-PCP. To test if methylation of L-Cys by the MarQ-MT occurs on amino acid-AMP, we first performed the MarQ reaction with Me-Cys or Cys in the presence of ATP, and both Cys-AMP and MeCys-AMP intermediate can be detected by using LC-MS analyses. However, when MarQ was incubated with ATP, Cys, and SAM, we could not detect Me-Cys-AMP, despite multiple attempts (see Figure S6 in the Supporting Information). Thus, MarQ is distinct from its ortholog TioN from thiocoraline pathway with the in vitro methylating activity on Cys-AMP. Taken together, MarQ-MT was inactive against free Cys or Cys-AMP, indicating that the methylation activity might be restricted to Cys loading onto the Ppant group of holo PCP. In maremycins gene cluster, marJ encodes the only freestanding PCP. We then hypothesized that MarQ activated and tethered Cys to the HS-pantetheinyl group of holo MarJ to form the substrate of the MT domain in MarQ. The holo MarJ was generated in vivo by coexpression with Sfp24 (Bacillus subtilis PPTase expressed from plasmid pSV20). Expression of MarJ without Sfp was used as a control. After protein expression and purification, the identity of holo MarJ was confirmed by high-performance liquid chromatography (HPLC) and electrospray ionization quadrupole-time-of-flight mass spectroscopy (ESI-Q-TOF MS) analyses, giving 13427.49 Da (calcd: 13427.05 for holo-MarJ). The mass for holo-MarJ increased by ∼340 Da, which validated that the Ppant arm was covalently attached to the Ser residue of apoMarJ (13086.65 Da, calcd: 13086.55 for apo-MarJ) (see Figures 3A and 3B). After confirmation of the modification of MarJ with Ppant, we further examined whether amino acid could be loaded onto holo-MarJ. The covalent loading assay containing MarQ, holoMarJ, ATP, TCEP (tris(2-carboxyethyl)phosphine), and amino acids was incubated at 28 °C for 40 min and quenched by being frozen at −80 °C freezer. By HPLC and Q-TOF MS analyses, we observed the formation of MarJ-tethered amino acids products (see Figure 3C, traces b−d): (a) MarJ-Cys (m/ z: 13529.48 Da; calcd: 13529.20 Da); (b) MarJ-Ser (m/z: 13513.59 Da; calcd: 13513.90 Da); (c) MarJ-Me-Cys (m/z:

Figure 3. Characterization of adenylation and methylation activity of MarQ in the presence of MarJ: (A) HPLC analyses of apo MarJ and holo MarJ; (B) Q-TOF MS analyses of apo-MarJ, holo-MarJ, MarJCys, and MarJ-Me-Cys generated from methylation reaction of MarJCys catalyzed by MarQ; (C) HPLC analysis of in vitro assays of MarJ and MarQ reconstitution system. The data (e.g., 13427.49 Da) are the mass of MarJ at different states. Each assay was replicated three times. HPLC profile was monitored at UV 210 nm.

13543.70 Da; calcd: 13543.90 Da). High-resolution MS analysis clearly demonstrated that Ser, Cys, and Me-Cys can be activated by MarQ and tethered onto MarJ, respectively (see Figures 3B and 3C, traces b−d). However, we found a large amount of holo-MarJ (13427.49 Da) remained in the Ser loading reaction, suggesting that MarQ has relatively lower activity toward Ser, consistent with the previous kinetic data (Figure 3C, trace b). Among these isolated maremycins, only maremycin D contains the diketopiperazine core derived from L-Ser and other maremycins with higher titer were sulfurcontaining diketopiperazines derived from L-Cys. In vitro tethering efficiency of NRPS MarQ and MarJ can be traced back to the production of maremycins. MarQ A domain revealed activity for Ser, Cys, and Me-Cys, and covalently tethered to holo-MarJ. We next tested the methylation activity of MT domain in MarQ with MarJC

DOI: 10.1021/acschembio.8b00364 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology

Another S-MT domain is found in TioN from thiocoraline biosynthetic pathway.11 Phylogenetic analysis showed that MarQ-MT and TioN-MT (share 43% sequence identity) formed a separate clade (Figure 2C), which represented a unique group of stand-alone NRPS A-MT didomain that were involved in thiol methylation on PCP tethered Cys. A domain in TioN displayed restricted substrate specificity toward Cys, while MT domain catalyzed specific S-methylation of both Cys-PCP and Cys-AMP in vitro.25,26 The special MT domain from MarQ and TioN provides a synthetic biology tool to increase the NRPs diversity.27 Here, in maremycins biosynthetic pathway, the discrete MarQ collaborates with its partner MarJ to form a simple tridomain cassette activates both Cys and Ser. Maremycin A/B, G, and FR900452 are all contain Me-Cys derived from L-Cys, and the exocyclic methylene group of maremycin D is derived from Ser. The substrate flexibility MarQ-A domain enables for the diverse maremycins as it opens the door to accept various substrates as NRPS building blocks, while the NRPS embedded MarQ-MT domain that is amenable to further specific S-methylation. The combinatorial potential of stand-alone MarQ/MarJ tridomain cassette might be exploited to incorporate the rare Me-Cys into peptide natural products or specifically introduce a methyl group in β position by NRPS engineering. Given the lack of structural information on complete MarQ and MarJ, the interactions of proteins involved in the maremycins NRPS system are still difficult to predict. It can be expected that structural data on the MarJ/Q tridomain system can generate reasonable models. Our study confirmed the variation of maremycins components and provided a potential synthetic biology tool for C−S bond formation in the biosynthesis of complex natural products.

tethered amino acids. When MarJ-Cys was incubated with SAM and MarQ, we observed the 0.1 min peak shift that should be the methylation product by comparison with the Me-Cys loading reaction (Figure 3C, trace e). Q-TOF MS analysis gave a molecular weight of m/z 13543.68 Da, an increase of 14 Da, confirming the transfer of a methyl group from SAM to MarJ-Cys (Figure 3B). Since Me-Cys is a nonproteinogenic amino acid, Cys is the natural substrate of MarQ and MT domain catalyzes S-methylation of Cys after being tethered to MarJ. SAM-dependent O-, N-, C-methyltransferases are frequently associated with natural product biosynthetic pathways, and the methyl group typically is transferred from SAM through an SN2 mechanism to the nucleophilic group, such as −OH and −NH2.23,4 When MarJ-Ser was incubated with SAM and MarQ, however, no methylated MarJ-Ser can be detected, suggesting that MarQ-MT cannot catalyze methylation of hydroxyl group (Figure 3C, trace f). The finding implicated that MT in MarQ is a sulfur-specific methyltransferase. To test this hypothesis, we investigated (S)-2,3-diaminopropanoic acid (DAP) offering another nucleophilic group −NH2 as a surrogate. DAP was activated and loaded onto MarJ by MarQ (Figure 3C, trace h) but methylation of MarJ-DAP was not observed in the presence of SAM. (Figure 3C, trace g). All the results clearly demonstrate that MarQ-MT is a truly specific methyltransferase toward the thiol group of MarJtethered Cys (see Scheme 1). Scheme 1. Pathway for the Formation of MarJ-Me-Cys by MarQa

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METHODS

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ASSOCIATED CONTENT

General Experimental Procedures. A detailed description of general procedures, methods, strains, cloning, protein expression, and enzymatic activity assays, kinetics analyses of MarQ, in vitro reconstitution of MarJ and MarQ tridomain system is given in the Supporting Information.

a

The mechanism of SAM-dependent methylation of thiol group is surrounded by a dashed line.

S Supporting Information *

Here, our biochemical characterization confirmed that the natural amino acids L-Cys or L-Ser can be activated as their aminoacyl-AMPs by MarQ-A domain followed by tethering to PCP MarJ. The SAM-dependent MarQ-MT domain specifically catalyzes the methylation of thiol group on MarJ-tethered L-Cys to generate MarJ-Me-Cys. Herein, there may be some functional differences between MarQ-MT and other NRPS MT domains. Sequence alignments indicated that MarQ-MT and its NRPS MT domain homologues harbor similar conserved motifs (see Figure S4). Typically, the MT domains for N-methylation or O-methylation are usually located at the C-terminal of the associated A domains, whereas the MarQMT domain is inserted between motifs a2 and a3 (a critical conserved motif for adenylation). The different position of MT here should lead to the special fold of the A-MT didomain complex, which transfers the methyl group to the β-SH group of the amino acid substrate. Since MarQ-MT is specific for −SH, it can be postulated that hydrogen bonds may form between the key amino acid residues of MarQ-MT and the −NH2 or −OH group of PCP tethered substrates, thereby preventing the methylation.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.8b00364. Methods, MarQ and MarQ A domain analysis, multiple sequence alignments, figures, tables for strains, plasmids, and primers (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Shuangjun Lin: 0000-0001-9406-9233 Present Address †

College of Pharmaceutical Science and Chinese Medicine, Southwest University, Chongqing, PRC. Author Contributions

T.H. and S.L. designed the experiments. T.H., Y.D., and Y.Z. performed the experiments. T.H., Z.D., and S.L. analyzed the data and assembled the manuscript. D

DOI: 10.1021/acschembio.8b00364 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology Notes

(17) Koglin, A., and Walsh, C. T. (2009) Structural insights into nonribosomal peptide enzymatic assembly lines. Nat. Prod. Rep. 26, 987−1000. (18) Walsh, C. T., Chen, H. W., Keating, T. A., Hubbard, B. K., Losey, H. C., Luo, L. S., Marshall, C. G., Miller, D. A., and Patel, H. M. (2001) Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines. Curr. Opin. Chem. Biol. 5, 525−534. (19) Rausch, C., Weber, T., Kohlbacher, O., Wohlleben, W., and Huson, D. H. (2005) Specificity prediction of adenylation domains in nonribosomal peptide synthetases (NRPS) using transductive support vector machines (TSVMs). Nucleic Acids Res. 33, 5799−5808. (20) Zhang, Y., Zou, Y., Brock, N. L., Huang, T., Lan, Y., Wang, X., Deng, Z., Tang, Y., and Lin, S. (2017) Characterization of 2-Oxindole Forming Heme Enzyme MarE, Expanding the Functional Diversity of the Tryptophan Dioxygenase Superfamily. J. Am. Chem. Soc. 139, 11887−11894. (21) Hacker, C., Glinski, M., Hornbogen, T., Doller, A., and Zocher, R. (2000) Mutational analysis of the N-methyltransferase domain of the multifunctional enzyme enniatin synthetase. J. Biol. Chem. 275, 30826−30832. (22) Ansari, M. Z., Sharma, J., Gokhale, R. S., and Mohanty, D. (2008) In silico analysis of methyltransferase domains involved in biosynthesis of secondary metabolites. BMC Bioinf. 9, 454−474. (23) Struck, A. W., Thompson, M. L., Wong, L. S., and Micklefield, J. (2012) S-adenosyl-methionine-dependent methyltransferases: highly versatile enzymes in biocatalysis, biosynthesis and other biotechnological applications. ChemBioChem 13, 2642−2655. (24) Nakano, M. M., Corbell, N., Besson, J., and Zuber, P. (1992) Isolation and characterization of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacillus subtilis. Mol. Gen. Genet. 232, 313−321. (25) Labby, K. J., Watsula, S. G., and Garneau-Tsodikova, S. (2015) Interrupted adenylation domains: unique bifunctional enzymes involved in nonribosomal peptide biosynthesis. Nat. Prod. Rep. 32, 641−653. (26) Mori, S., Pang, A. H., Lundy, T. A., Garzan, A., Tsodikov, O. V., and Garneau-Tsodikova, S. (2018) Structural basis for backbone Nmethylation by an interrupted adenylation domain. Nat. Chem. Biol. 14, 428−430. (27) Lundy, T. A., Mori, S., and Garneau-Tsodikova, S. (2018) Engineering bifunctional enzymes capable of adenylating and selectively methylating the side chain or core of amino acids. ACS Synth. Biol. 7, 399−404.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Science Foundation of China (Nos. 31425001, 21632007, and 21661140002 to S.L., and No. 31600049 to T.H.), and the Postdoctoral Science Foundation of China (No. 2016M590357). We thank W. Liu from Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences for providing pSV20 plasmid.

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