Two Cytochrome P450 Enzymes from Streptomyces sp. NRRL S-1868

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

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Two Cytochrome P450 Enzymes from Streptomyces sp. NRRL S‑1868 Catalyze Distinct Dimerization of Tryptophan-Containing Cyclodipeptides Huili Yu and Shu-Ming Li* Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Robert-Koch-Straße 4, 35037 Marburg, Germany Downloaded via UNIV PARIS-SUD on August 20, 2019 at 12:10:37 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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ABSTRACT: Heterologous expression in Streptomyces coelicolor and in vitro enzyme characterization proved that two P450 enzymes, AspB and NasB, from Streptomyces sp. NRRL S-1868 catalyze two new dimerization patterns of tryptophancontaining cyclodipeptides. Structure elucidation of the metabolites revealed an N1−C7′ dimer of two cWP molecules as the predominant product of AspB and C3−C7′ connected cWP with cWA as that of NasB.

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DKP modification have been identified,3,15,16 only two biosynthetic pathways implying their dimerization were reported: the formation of (−)-ditryptophenaline, a C3−C3′linked cWF derivative from a NRPS-dependent pathway (Scheme 1i),17 and naseseazine C (3c) from a CDPSdependent pathway (Scheme 1ii).18 Cytochrome P450 enzymes catalyze the dimerization in both cases.

ndole alkaloids derived from cyclodipeptides (CDPs) containing a 2,5-diketopiperazine (DKP) scaffold constitute an important class of secondary metabolites that are biosynthesized predominately by microorganisms.1−4 Among them, the dimeric DKP alkaloids with fascinating biological activities ranging from anticancer and antimicrobial to immunosuppressive have attracted increasing attention in recent years.2,4,5 A number of tryptophan-based dimeric DKP alkaloids derived from cyclo-L-Trp-L-Pro (cWP, 1) and cyclo-LTrp-L-Ala (cWA, 2) have been isolated from microbes. Aspergilazine A (3a),6,7 naseseazine B (3b)/naseseazine A (4a),8,9 and naseseazine C (3c)7,10 are N1−C7′, C3− C7′(2R,3S), and C3−C6′(2S,3R) linked examples, respectively (Figure 1).

Scheme 1. Known Biosynthetic Pathways of Dimeric DKP Alkaloids

Figure 1. Examples of dimeric DKP alkaloids isolated from marinederived fungus and Streptomyces.

The CDPS pathway-associated P450 enzymes have been recently reported to catalyze diverse intriguing chemical transformations including inter- and intramolecular C−C bond formation, aromatization of the DKP ring, and guaninyl-transfer reactions.16,19 Encouraged by these findings, we intended to identify more P450 enzymes for DKP modification, especially those for metabolism of tryptophancontaining CDPs.

The connection positions and stereochemistry of the dimeric DKP frameworks affording diverse bioactivities, especially at the quaternary stereocenter of C3, require enormous efforts during their organic synthesis.11,12 In nature, the DKP moieties can be biosynthesized by two different enzyme groups, i.e., the nonribosomal peptide synthetases (NRPSs) using free amino acids as substrates and the aminoacyl tRNA-dependent cyclodipeptide synthases (CDPSs).3,13,14 Although a number of tailoring enzymes for © XXXX American Chemical Society

Received: July 29, 2019

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DOI: 10.1021/acs.orglett.9b02666 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters In a previous study, we identified nine CDPSs from different Streptomyces strains by heterologous expression in E. coli, which catalyze the formation of tryptophan-containing CDPs.20 Inspection of their genomic surroundings showed that most of these CDPS genes are associated with putative cytochrome P450 genes. Three of such P450 enzymes function as guaninyl transferases.19,21 Two of the identified CDPSs in that study are from Streptomyces sp. NRRL S-1868. WP_078873129 (CWPS1NS1868, renamed to AspA in this study) catalyzes the formation of cWP (1), while WP_078872750 (CWXS1NS1868, NasA) assembles both cWP (1) and cWA (2). Both CDPS genes are directly adjacent to a P450 gene, i.e., aspB and nasB, followed by a putative transporter or regulator (Figure 2).

Figure 2. Schematic comparison of asp and nas clusters from Streptomyces sp. NRRL S-1868 with nasc cluster from Streptomyces sp. CMB-MQ030.

The CDPSs and P450s of the two mentioned clusters resemble each other (Table S1). They also show high homology with the members of the recently identified nasc cluster18 (Figure 2). Multiple sequence alignments of the putative P450s AspB and NasB with structure-defined P450s from bacteria revealed the conserved P450 featuring motifs (Figure S1): the highly conserved A/G241G242XXT245 motif in the long I helix22 (referring the number of EryF23), E280XXR283 motif in the K helix,24 and G347XXXC351 motif in the hemebinding loop.22 Phylogenetic analysis of the two P450s with the functionally characterized bacterial P450s (Figure S2) indicated that they distribute in the same subclade with NascB, which catalyzes the C3−C6′ linkage of two cWP molecules to form naseseazine C (3c, Scheme 1) with a 2S,3R stereochemistry.18 It could be therefore speculated that the asp and nas clusters are also responsible for the formation of dimeric DKP alkaloids with AspB and NasB as dimerization enzymes. For functional proof of the two clusters, we used Streptomyces coelicolor M114625 as the expression host. The coding sequence of AspA was first cloned into the replicative vector pPWW50A26 and transformed into M1146 (Tables S2 and S3). The aspA transformant was cultivated in liquid YMG media at 28 °C for 7 days. The ethyl acetate extracts of the bacterial cultures were subjected to LC−MS analysis by using M1146 carrying pPWW50A as a control strain. In comparison to that of the negative control (Figure 3i), the extract of an aspA transformant showed the sole product peak with the expected [M + H]+ ion at m/z 284.1394 for cWP (1) (Figure 3ii), which was consistent with the result obtained from E. coli transformant.20 Subsequently, aspA and aspB were coexpressed via pPWW50A in M1146. The asp(AB) transformant was cultivated and treated as described above. Compared to that of the aspA transformant (Figure 3ii), three additional

Figure 3. HPLC analysis of the S. coelicolor M1146 transformants with and without precursor feeding. [M + H]+ ions with a tolerance range of ±0.005 were detected at m/z 284.139 (1), 258.124 (2), 565.256 (3a−3d), 539.240 (4a/4b), and 513.225 (5a/5b).

products with 3a as the predominant and 3c and 3d as minor peaks were detected in the asp(AB) transformant (Figure 3iii). Notably, the CDPS product 1 was consumed almost completely. The [M + H]+ ions of the products at m/z 565.256 ± 0.005 correspond well to those of cWP dimers. In analogy, nasA was cloned alone and together with nasB into pPWW50A and expressed in M1146. LC−MS analysis of the transformant harboring nasA revealed both cWP (1) and cWA (2) as the main products (Figure 3iv) with a slightly different ratio (1:1) compared to that in the E. coli transformant (1:1.5),20 which was probably due to the different hosts. LC−MS analysis of the extract of a nas(AB) transformant revealed the presence of six new product peaks as three pairs, 3a/3b, 4a/4b, and 5a/5b, regarding their [M + H]+ ions (Figure 3v). Among them, 4a was detected as the B

DOI: 10.1021/acs.orglett.9b02666 Org. Lett. XXXX, XXX, XXX−XXX

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Scheme 2. Biosynthesis of Dimeric DKPs in Streptomyces sp. NRRL S-1868

predominant product. The [M + H]+ ions of 3a/3b, 4a/4b, and 5a/5b at m/z 565.256 ± 0.005, 539.240 ± 0.005, and 513.225 ± 0.005 indicate dimerization of 1, connection of 1 with 2, and dimerization of 2, respectively. To confirm the metabolism of 1 by AspB as well as 1 and 2 by NasB for dimerization, their coding sequences were cloned alone into pPWW50A and expressed in M1146. As shown in Figure 3vii,x, no product was detected in either P450 transformant. Two-day-old cultures of M1146 transformants containing pPWW50A as control and aspB expression construct pHY121 were then fed with 100 μM 1 and cultivated further for 7 days. Inspection of the LC−MS results showed that 1 was completely metabolized in the AspB biotransformation culture and 3a, 3c, and 3d were accumulated with similar ratios as that in the asp(AB) transformant (Figure 3iii,viii). As expected, no conversion of 1 was observed in the control transformant harboring pPWW50A (Figure 3vi). Similarly, 1 and 2 were metabolized by NasB to the six peaks as detected in the nas(AB) transformant (Figures 3v,ix,xi). 4a was detected as major product and 3b and 5a were more accumulated than that in the nas(AB) transformant. This could be caused by different availabilities of endogenous and fed substrates. These results demonstrated that the DKPs produced by CDPSs were taken as natural substrates by P450s AspB and NasB for dimerization. For structure elucidation, 3a, 3c, and 3d were isolated from a large-scale fermentation broth of a transformant harboring asp(AB) (4 L) and 3a, 3b, 4a, 4b, 5a, and 5b from that with nas(AB) (12 L). Detailed interpretation of the NMR spectra of the analytically pure products (Tables S4−S9, Figures S3− S25) and comparison with literature data confirmed that 3a samples isolated from both asp(AB) and nas(AB) transformants have the same structure as aspergilazine A.6 In analogy, 3c was identified as naseseazine C.10 The NMR data of 3d (Table S6, Figures S10−S12) corresponded well with that of the chemically synthesized iso-naseseazine B.9 Products 3b, 4a, and 5b were identified to be naseseazine B, naseseazine A, and NAS-1, respectively.8,9,18 Compound 5a (termed naseseazine D) was predicted to be a homodimer of 2 according to its [M + H]+ ion at m/z 513.2266. Its NMR data (Table S8, Figures S19−S24) strongly resembled that of naseseazine A,8,9 with the exception for the replacement of pyrrolidine signals in 4a by a methyl group. Key correlations of H8′−C3 and H12a−C7′ were observed in the HMBC spectrum of 5a (Figure S23). Its stereochemistry was confirmed by NOE correlations of H2−H11, H2−H8′, H12a-H11, and H12a-H6′ (Figure S24). Due to the low product yield, an insufficient amount of 4b was obtained for NMR analysis. From the HR-EIMS data, 4b should be a coupling product of 1 and 2. Among the seven identified dimers, 3a is connected via an N1−C7′ bond, while the other products are different C3-aryl pyrroloindoline derivatives: C3−C6′ coupling in 3c, 3d, and 5b or C3−C7′ coupling in 3b, 4a, and 5a. In the proposed structures, 3b, 3d, 4a, and 5a have a 2R,3S chirality, while 3c and 5b carry a 2S,3R chirality, which was confirmed by CD analysis (Figure S26). The total product yields of the dimeric DKPs isolated from asp(AB) and nas(AB) transformants were calculated to be 260 and 46 mg/L, respectively, with aspergilazine A (3a) as the predominant product for the asp cluster (98%) and naseseazine A (4a) for the nas cluster (84%) (Scheme 2). These data imply that AspB catalyzes mainly the N1−

C7′dimerization of 1 and NasB the C3−C7′ coupling between 1 and 2. Both reactions differ clearly from those of DtpC and NascB (Scheme 1). In a recent publication,18 NascB is proposed to catalyze the formation of naseseazine C via a radical-mediated intermolecular addition. In analogy, NasB could form an N1• radical by abstracting a hydrogen from N1 of cWA (2) followed by radical migration leading to the formation of the pyrroloindoline C3 radical. Attacking of this radical by C7′ of cWP (1) from behind side and elimination of one hydrogen will result in the major product naseseazine A (4a). For AspB, the coupling of the N1• radical of cWP (1) with C7′ of the second cWP (1) and loss of one hydrogen will generate the predominant product aspergilazine A (3a). LC−MS analysis of the fermentation broth of Streptomyces sp. NRRL-S1868 revealed the presence of 1, 2, 3a, 3b, 4a, 4b, 5a, and 5b, almost the same metabolite profile of nas(AB) transformant, with 4a and 3b (both via a C3−C7′ coupling) as main products (Figures 3v and S27). Compound 3a was only detected as a minor product. It is plausible that the genes of the asp cluster are not expressed under the tested conditions. To verify the P450 function in vitro, we cloned aspB and nasB into pET28a (+) vector and overexpressed them in E. coli Rosetta (DE3) (Tables S2 and S3). Induction with 0.5 mM IPTG at 18 °C for 20 h and purification with Ni-NTA agarose C

DOI: 10.1021/acs.orglett.9b02666 Org. Lett. XXXX, XXX, XXX−XXX

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with 3a as the predominant product (Figures 3iii and 4ii). A conversion of 20% was achieved with 1 mM 1 and 10 μM AspB after incubation at 30 °C for 12 h. Negative control with heat-inactivated AspB showed no conversion of 1 (Figure 4i). In comparison to AspB, NasB showed a lower enzyme activity. In the incubation mixture of NasB with 1 and 2, the product profile differs slightly from that of heterologous expression. This could be caused by different availabilities of both substrates in vivo and in vitro. However, 4a was still detected as major product, followed by 3b (Figure 4iv). When 1 or 2 alone was incubated with NasB, 3b or 5a was detected as the predominant product (Figure 4v,vi). Compounds 3b, 4a, and 5a share the same coupling pattern (C3−C7′) and the same stereochemistry (2R,3S), proving again NasB as a C3α arylation enzyme. The reactions catalyzed by AspB and NasB also follow the Michaelis−Menten kinetics. KM values from 50 to 93 μM and turnover numbers (kcat) from 0.01 to 0.023 s−1 were determined for the enzymes (Table S10 and Figures S31−S33). In conclusion, we identified in this study two similar cdpsassociated two-gene clusters for the formation of dimeric DKP alkaloids. The CDPS AspA assembles cWP (1), which was converted by the cytochrome P450 AspB to aspergilazine A (3a), an N1−C7′ linked homodimer. NasA produces both cWP (1) and cWA (2) for dimerization by NasB to naseseazine A (4a), a C3−C7′ connected heterodimer as the main product. Homodimers with the same linkage pattern and stereochemistry, i.e., naseseazine B (3b) and neseseazine D (5a), were detected as minor products. All of these substances were also detected as enzyme products of the recombinant AspB and NasB. The biosynthetic pathways of aspergilazine A (3a), naseseazine A (4a), and naseseazine B (3b) have not been reported prior to this study. The dimerization reactions catalyzed by AspB and NasB differ clearly from those of the two known cytochrome P450 enzymes illustrated in Scheme 1 in their connection positions and stereochemistry.

afforded recombinant proteins with high purity (Figure S28). UV−Vis spectroscopic analysis of the purified recombinant P450s revealed the typical absorption maximum of the sodium dithionite reduced FeII−CO complex at approximately 450 nm (Figures S29 and S30). The enzyme activity of AspB was investigated by using 1 as substrate and the commercially available spinach ferredoxin and ferredoxin-NADP+ reductase for electron transport. In the case of NasB, 1, 2, or 1 and 2 were used as substrates. The enzyme products were identified on LC−MS (Figure 4) by comparing their retention times, UV spectra, and [M + H]+ ions with those of the products isolated from S. coelicolor transformants. Incubation of AspB resulted in the same product profile as that of heterologous expression,

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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.9b02666.

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Experimental details, spectroscopic data (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: shuming.li@staff.uni-marburg.de. Tel/Fax: + 496421-28-22461/25365. ORCID

Shu-Ming Li: 0000-0003-4583-2655 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the ARS Culture Collection (NRRL) for providing Streptomyces strains and R. Kraut and S. Newel (University of Marburg) for LC−MS and NMR analyses. The Bruker micrOTOF QIII mass spectrometer was financially supported in part by a grant from the Deutsche Forschungsgemeinschaft

Figure 4. LC−MS analysis of the in vitro assays with AspB and NasB. D

DOI: 10.1021/acs.orglett.9b02666 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters (INST 160/620-1 to S.-M.L.). H.Y. is a scholarship recipient of the China Scholarship Council (201306220024).

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REFERENCES

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DOI: 10.1021/acs.orglett.9b02666 Org. Lett. XXXX, XXX, XXX−XXX