Characterization of the FMN-Dependent Cysteine Decarboxylase from

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Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Characterization of the FMN-Dependent Cysteine Decarboxylase from Thioviridamide Biosynthesis Jingxia Lu,†,∥ Jiao Li,‡,∥ Yuan Wu,† Xianyang Fang,§ Jiapeng Zhu,*,‡ and Huan Wang*,† †

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State Key Laboratory of Coordination Chemistry, Jiangsu Key Laboratory of Advanced Organic Materials, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China ‡ School of Medicine and Life Sciences, State Key Laboratory Cultivation Base for TCM Quality and Efficacy, Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China § Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China S Supporting Information *

ABSTRACT: The biosynthesis of thioviridamide-like compounds has not been elucidated. Herein, we report that TvaF from the thioviridamide biosynthetic gene cluster is an FMNdependent cysteine decarboxylase that transforms the C-terminal cysteine of precursor peptides into a thioenol motif and exhibits high substrate flexibility. We resolved the crystal structure of TvaF bound with FMN at 2.24 Å resolution. Key residues for FMN binding and catalytic activity of TvaF have been identified and evaluated by mutagenesis studies.

motif (Figure 1C). TvaF exhibits high tolerance toward peptide substrates of various sequences, and the leader peptide of TvaA is not required for enzymatic recognition. The crystal structure of TvaF bound with FMN is resolved at 2.24 Å resolution. Key residues responsible for substrate binding and catalytic activity are identified, and their importance is evaluated by mutagenesis studies. To initiate our investigation, TvaF with an N-terminal His6 tag was expressed heterologously in E. coli BL21 (DE3) and purified by immobilized metal ion affinity chromatography (IMAC) as a yellow-colored soluble protein, indicating the presence of flavin as a cofactor. To determine the type of bound flavin, TvaF protein was heat-denatured, and the flavin was extracted by reversed-phase solid-phase extraction. Analysis by electrospray ionization mass spectrometry (ESI MS) revealed a positively charged species with a mass of 457.11 Da, which is consistent with flavin mononucleotide (FMN) (Figure S1). HPLC analysis further confirmed that the flavin cofactor has identical retention time with FMN. Thus, TvaF is an FMN-binding protein. To probe its enzymatic function, genes encoding TvaF and precursor peptide TvaA were inserted into the multiple cloning sites of the pRSFDuet-1 vector. Coexpression of these two genes in E. coli BL21 (DE3) led to the production of three TvaA derivatives, as detected by LC−MS analysis (Figure 2A). Compared with TvaA peptide, product TvaA-1a displayed a

Ribosomally synthesized and post-translationally modified peptides (RiPPs) have emerged as a major family of peptide natural products with diverse bioactivities.1 The biosynthesis of these compounds is initiated by the ribosomal synthesis of a precursor peptide, which is transformed to the mature natural product by posttranslational modification enzymes.2 Thioviridamide-like compounds are a recently characterized subgroup of RiPPs with antiproliferative and pro-apoptotic activities.3 Thioviridamide was first isolated from Streptomyces olivoviridis NA005001 (Figure 1A).4−6 Recent efforts by genome mining reveal a number of biosynthetic gene clusters (BGCs) that are closely related to that of thioviridamide, which led to the discovery of five thioviridamide-like compounds.7,8 Extensive posttranslational modifications are present in thioviridamide, including five backbone thioamides, a β-hydroxy-N1,N3dimethylhistidinium (hdmHis) residue, and a C-terminal macrocycle with an S-[(Z)-2-aminovinyl]-D-cysteine (AviCys) motif (Figure 1B and Scheme S1). C-terminal AviCys motifs are also found in other groups of RiPPs, including lanthipeptides, linaridins, and lipolanthines (Figure 1A).9−11 Oxidative decarboxylation of the C-terminal Cys in the precursor peptide by generating a (Z)-enethiol species is generally proposed to be involved in the formation of AviCys or AviMeCys in these compounds.9,12−14 However, enzymes that are responsible for AviCys formation in thioviridamide have not been characterized so far. Herein, we report that TvaF from the thioviridamide BGC is an FMN-dependent cysteine decarboxylase that catalyzes the oxidative decarboxylation of precursor peptide TvaA by generating a C-terminal thioenol © XXXX American Chemical Society

Received: May 3, 2019

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DOI: 10.1021/acs.orglett.9b01531 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

Figure 2. Modification of TvaA by TvaF through coexpression in E. coli. (A) Proposed modification of TvaA by TvaF and following transformation by nonenzymatic hydrolysis and NaBH4 reduction. (B) LC−MS analysis of TvaA peptide and its derivatives after coexpression and NaBH4 reduction. Figure 1. Chemical structure and putative biosynthetic gene cluster of thioviridamide. (A) Examples of AviCys-containing RiPPs. (B) Putative biosynthetic gene cluster of thioviridamide and the sequence of precursor peptide TvaA. Leader peptide is in black and core peptide is in red. (C) Possible AviCys formation involving the oxidative decarboxylation of C-terminal cysteine catalyzed by TvaF.

suggesting that the cyclization is mediated by one of the other enzymes in thioviridamide biosynthetic gene cluster. The identity of the enzyme(s) involved in this interesting peptide macrocyclization are currently unknown. The successful reconstitution of TvaF activity in E. coli prompted us to evaluate its substrate specificity by coexpressing TvaF and TvaA mutants (Table 1). Results showed that the C-terminal cysteine in TvaA peptide is strictly required for the oxidative decarboxylation to occur as TvaF

mass loss of 46 Da, which corresponds to oxidative decarboxylation of a cysteine residue. Products TvaA-1b and TvaA-1c had mass losses of 62 and 104 Da, respectively, relative to TvaA peptide. NaBH4 treatment of the TvaA derivatives led to reduction of TvaA-1a and TvaA-1b by generating TvaA-2a and TvaA-2b with a mass increase of 2 Da, respectively (Figure 2). In contrast, TvaA-1c was not reduced by NaBH4, suggesting the absence of an unsaturated double bond (Figure 2). Tandem MS analysis of these species indicated that TvaA-1c is the TvaA derivative with a Cterminal amide after losing the cysteine residue (Figure S2), whereas TvaA-1b and TvaA-2b contain an aldehyde and a hydroxyl group at their C-terminus, respectively (Figures S3 and S4). Thus, our data indicates that TvaA is first modified by TvaF through oxidative decarboxylation of the C-terminal cysteine to generate the unstable thioenol product TvaA-1a, which further degrades through nonenzymatic hydrolysis by generating TvaA-1b and TvaA-1c (Figure S5). Such an observation is not surprising since degradation of thioenol products generated by oxidative decarboxylation has precedent in the cases of EpiD from epidermin biosynthesis and MicD from lipolanthines biosynthesis.10,15 Thus, these results indicate that TvaF from thioviridamide biosynthesis is capable of oxidative decarboxylation of the precursor peptide TvaA. No cyclization product with AviCys ring formation was detected by LC−MS after coexpressing TvaA and TvaF in E. coli,

Table 1. Substrate Scope of TvaF toward Peptide Substrates

* The modification of TvaAS8A core peptide was carried out in vitro. The number of check marks represents the modification efficiency of TvaA peptides by TvaF.

B

DOI: 10.1021/acs.orglett.9b01531 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters was inactive toward TvaAC13A and TvaA14Ala (Figures S6 and S7). In contrast, replacement of Ser8 to Ala did not affect the modification efficiency of TvaF (Figure S8). The His12 residue of thioviridamide is heavily decorated during the maturation of thioviridamide (Figure 1A); however, whether its modification occurs before or after the formation of AviCys ring is not yet understood. To examine the importance of His12 to substrate recognition by TvaF, TvaAH12A mutant was coexpressed with TvaF in E. coli. Results showed that TvaF does not modify TvaAH12A peptide in vivo (Figure S9). In contrast, TvaAH12R peptide was modified by TvaF, however, with significantly lower efficiency compared to wild-type TvaA (Figure S10). These results indicate that although not strictly required, the presence of His12 residue adjacent to the C-terminal cysteine is important for enzyme modification by TvaF. A highly hydrophobic sequence of VMAAAA is present in the Nterminus of TvaA core peptide, where thioamidation occurs during the biosynthesis (Figure 1B). Coexpression of TvaF and TvaAAla(4−7)deletion peptide with the (Ala)4 sequence deleted resulted in the production of modified TvaAAla(4−7)deletion derivatives, indicating the (Ala)4 is not necessary for enzyme recognition by TvaF (Figure S11). In contrast, replacement of Val2 and Met3 to Ala residues notably impeded the decarboxylation, as demonstrated by low conversion of TvaAV2AM3A (Figure S12). Finally, both the full length TvaAS8A and its core peptide, TvaAS8A(2−13), can be modified by TvaF in vitro; however, the conversion of TvaAS8A(2−13) was significantly lower than its full length counterpart, indicating that the leader peptide is not strictly required but contributes to the recognition by TvaF (Figures S13 and S14). Together, our data indicates that although TvaF exhibits high tolerance toward peptide substrates of various sequences, it strictly requires the presence of a C-terminal cysteine and is sensitive to alteration at residues Val2, Met3, and His12. The relaxed substrate specificity of cysteine decarboxylases involved in AviCys formation has also been reported in the case of CypD and EpiD from the biosynthesis of cypemycin and epidermin, respectively, where only the C-terminal four residues are essential for enzymatic recognition.12,15 To provide mechanistic insight into the function of TvaF, we resolved its crystal structure at 2.24 Å resolution. In solution, TvaF forms a complex with a molecular weight ∼300 kDa estimated by size-exclusion chromatography. Consistent with this observation, crystal packing shows that TvaF assembles into a homododecamer (Figure S15). The FMN is located in a cavity at the interface of two TvaF monomers (monomer 1 and 2, Figure 3A) and interacts with primarily one monomer (monomer 1, Figure 3B). The phosphate group of FMN is hydrogen bonded with side chains of residues Ser23, Thr49, Thr99, and Thr102, as well as main chains of Gly22, Ala100, and Val131 of monomer 1. Furthermore, the main chain amide bonds of Cys111, Asp112, and Gln117 of monomer 2 provide additional binding to FMN (Figure 3B). Side-chain oxygen of Ser23 (monomer 1) forms a hydrogen bond with one carbonyl oxygen of flavin, facilitating the FMN molecule to adopt a bend conformation (Figure 3B). Superimposition of crystal structures of TvaF and EpiDH67N mutant in complex with peptide substrate showed that the overall structures of TvaF and EpiDH67N are highly similar, including the FMN binding sites (Figure 3C).16 The electron densities of residues 160− 176 of TvaF are very poor, suggesting that this region is disordered, which is similar to the crystal structure of substrate-free EpiD. In contrast, residues of the corresponding

Figure 3. Crystal structure of TvaF (PDB ID: 6JLS). (A) Close-up view of FMN bound in TvaF. (B) FMN is positioned in TvaF by a network of hydrogen bonds. (C) Superimposition of crystal structures of TvaF and EpiD bound with a peptide substrate. (D) Putative peptide binding site of TvaF lies in the cleft of two monomers.

region of peptide-bound EpiDH67N forms a defined β sheet with its peptide substrate, indicating that peptide binding would facilitate the local folding of this class of enzymes. These results suggest that TvaF might bind to its peptide substrate following a mode similar to that of EpiD, the putative peptide binding site lies in the hydrophobic cleft between monomer 1 and monomer 3 (Figure 3D), and the C-terminal cysteine might reach the FMN bound in monomer 1 for modification. A putative proton-acceptor His83 residue of TvaF monomer 2 is proposed to facilitate decarboxylation as a general base, which is highly conserved in cysteine decarboxylases such as EpiD from epidermin (Figure S16). Collectively, our results identify key residues for FMN binding and catalytic activity and suggest that the minimal catalytic unit of TvaF is composed of three adjacent monomers. To further characterize the proposed key residues important for the enzymatic activity of TvaF, mutagenesis studies were carried out by coexpressing TvaA and TvaF mutants in E. coli. When His83 was mutated into Gln, TvaFH83N exhibited no decarboxylase activity toward TvaA peptide in vivo (Figure S17). Similarly, when TvaF H83A was incubated with TvaAS8A(2−13) peptide in vitro, no modification occurred (Figure S18), supporting its role as a catalytic general base. The crystal structure of TvaF shows that residues Thr99 and Thr102 are directly involved in FMN binding. Mutation of these two residues to Ala completely abolished the activity of TvaF, indicating their importance (Figure S19). Pro97 residue of TvaF located in the loop region (residues 97−100) where Thr99 and Thr102 reside. Mutation of Pro97 to an Ala residue also abolished the activity of TvaF, indicating that the local conformation of loop97−100 is important for FMN binding (Figure S20). Based on the crystal structure of TvaF and these mutagenesis studies, we propose a plausible mechanism of oxidative decarboxylation of TvaA as shown in Figure S21.17 C

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(2) Funk, M. A.; van der Donk, W. A. Acc. Chem. Res. 2017, 50, 1577. (3) Tang, J.; Lu, J. X.; Luo, Q. F.; Wang, H. Chin. Chem. Lett. 2018, 29, 1022. (4) Hayakawa, Y.; Sasaki, K.; Adachi, H.; Furihata, K.; Nagai, K.; Shin-ya, K. J. Antibiot. 2006, 59, 1. (5) Hayakawa, Y.; Sasaki, K.; Nagai, K.; Shin-ya, K.; Furihata, K. J. Antibiot. 2006, 59, 6. (6) Izumikawa, M.; Kozone, I.; Hashimoto, J.; Kagaya, N.; Takagi, M.; Koiwai, H.; Komatsu, M.; Fujie, M.; Satoh, N.; Ikeda, H.; Shin-Ya, K. J. Antibiot. 2015, 68, 533. (7) Frattaruolo, L.; Lacret, R.; Cappello, A. R.; Truman, A. W. ACS Chem. Biol. 2017, 12, 2815. (8) Kjaerulff, L.; Sikandar, A.; Zaburannyi, N.; Adam, S.; Herrmann, J.; Koehnke, J.; Muller, R. ACS Chem. Biol. 2017, 12, 2837. (9) Sit, C. S.; Yoganathan, S.; Vederas, J. C. Acc. Chem. Res. 2011, 44, 261. (10) Wiebach, V.; Mainz, A.; Siegert, M. A. J.; Jungmann, N. A.; Lesquame, G.; Tirat, S.; Dreux-Zigha, A.; Aszodi, J.; Le Beller, D.; Sussmuth, R. D. Nat. Chem. Biol. 2018, 14, 652. (11) Izawa, M.; Kawasaki, T.; Hayakawa, Y. Appl. Environ. Microbiol. 2013, 79, 7110. (12) Ding, W.; Mo, T.; Mandalapu, D.; Zhang, Q. Synth. Syst. Biotechnol. 2018, 3, 159. (13) Ding, W.; Yuan, N.; Mandalapu, D.; Mo, T.; Dong, S.; Zhang, Q. Org. Lett. 2018, 20, 7670. (14) Ortega, M. A.; Cogan, D. P.; Mukherjee, S.; Garg, N.; Li, B.; Thibodeaux, G. N.; Maffioli, S. I.; Donadio, S.; Sosio, M.; Escano, J.; Smith, L.; Nair, S. K.; van der Donk, W. A. ACS Chem. Biol. 2017, 12, 548. (15) Kupke, T.; Kempter, C.; Jung, G.; Gotz, F. J. Biol. Chem. 1995, 270, 11282. (16) Blaesse, M.; Kupke, T.; Huber, R.; Steinbacher, S. EMBO J. 2000, 19, 6299. (17) Strauss, E.; Zhai, H.; Brand, L. A.; McLafferty, F. W.; Begley, T. P. Biochemistry 2004, 43, 15520. (18) Mo, T. L.; Yuan, H.; Wang, F. T.; Ma, S. Z.; Wang, J. X.; Li, T.; Liu, G. F.; Yu, S. N.; Tan, X. S.; Ding, W.; Zhang, Q. FEBS Lett. 2019, 593, 573.

In summary, we have reconstituted the enzymatic activity of FMN-dependent cysteine decarboxylase TvaF from the biosynthesis of thioviridamide. TvaF demonstrates flexible substrate specificity but strictly requires the presence of a Cterminal cysteine residue. The crystal structure of TvaF in complex with FMN is characterized, and key catalytic residues involved in the oxidative decarboxylation have been identified. Remarkably, TvaF shares high structural similarity with EpiD from epidermin biosynthesis and may share a similar binding mode toward peptide substrates with EpiD. A recent study suggests that all of the flavin-dependent cysteine decarboxylases involved in AviCys formation might have similar structural folds, although their primary sequences vary vastly.18 Our study provides additional insights into this class of enzymes involved in AviCys formation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b01531.

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Experimental procedures, LC−MS/MS spectra of peptides, crystal structure of TvaF protein (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Huan Wang: 0000-0003-1585-6760 Author Contributions ∥

J. Lu and J. Li contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by NSF of China (Grant 21778030 and 21861142005), the start-up fund from State Key Laboratory of Coordination Chemistry and the Fundamental Research Funds for the Central Universities (Grant 14380138 and 14380131), National key R & D Plan for Precision Medicine Research (Grant 2016YFC0905900), and Jiangsu Specially Appointed Professor Funding.

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REFERENCES

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DOI: 10.1021/acs.orglett.9b01531 Org. Lett. XXXX, XXX, XXX−XXX