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Unprecedented cyclization catalyzed by a cytochrome P450 in benzastatin biosynthesis Hayama Tsutsumi, Yohei Katsuyama, Miho Izumikawa, Motoki Takagi, Manabu Fujie, Noriyuki Satoh, Kazuo Shin-ya, and Yasuo Ohnishi J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b02769 • Publication Date (Web): 02 May 2018 Downloaded from http://pubs.acs.org on May 2, 2018

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Journal of the American Chemical Society

Unprecedented cyclization catalyzed by a cytochrome P450 in benzastatin biosynthesis Hayama Tsutsumi1, Yohei Katsuyama1,2,*, Miho Izumikawa3,+, Motoki Takagi3, Manabu Fujie4, Noriyuki Satoh4, Kazuo Shin-ya5 and Yasuo Ohnishi1,2,* 1

Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 2 Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 3

Japan Biological Informatics Consortium (JBIC), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan Okinawa Institute of Science and Technology Graduate University, 1919-1 Tancha, Onna-son, Kunigami-gun, Okinawa 904-0495, Japan 5 National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan + Deceased on 23 December 2015 Supporting Information Placeholder 4

ABSTRACT: Benzastatins have unique structures probably derived from geranylated p-aminobenzoic acids. The indoline and tetrahydroquinoline scaffolds are presumably formed by cyclization of the geranyl moiety, but the cyclization mechanism was unknown. We studied the benzastatin biosynthetic gene cluster of Streptomyces sp. RI18; functions of the six enzymes encoded by it were analyzed by gene disruption in a heterologous host and in vitro enzyme assays. We propose the biosynthetic pathway for benzastatins in which a cytochrome P450 (BezE) is responsible for the cyclization of geranylated pacetoxyaminobenzoic acids; BezE catalyzes elimination of acetic acid to form an iron nitrenoid, nitrene transfer to form an aziridine ring, and nucleophilic addition of hydroxide ion to C-10 and chloride ion to C-9 to generate the indoline and tetrahydroquinoline scaffolds, respectively. Discovery of this enzyme, which should be termed cytochrome P450 nitrene transferase, provides an important insight into the functional diversity of cytochrome P450.

Introduction Cyclization is one of the most important reactions in the biosynthesis of natural products; it produces characteristic molecular scaffolds, which are often important for the bioactivity of natural products as represented by the βlactam ring. Various cyclization reactions, such as the Diels–Alder reaction, oxidative cyclization, and nucleophilic addition, are used for natural product biosynthesis.1,2 However, some natural products seem to be synthesized by still unknown cyclization reactions. Cytochrome P450s are useful biocatalyst catalyzing various oxidation reactions including hydroxylation,3–6 epoxidation,7,8 and biaryl coupling9–11 (Scheme 1a).12 Most cytochrome P450s require redox partners (e.g., ferredoxin and ferredoxin reductase) to reduce the ferric heme iron during their catalytic cycle. Some can use peroxides to bypass the reduction of the heme iron. In the biosynthesis of natural products, cytochrome P450s play important roles to generate structural diversity13–15 and some are responsible for oxidative cyclization (e.g., in the biosynthesis of virid-

icatumtoxin, griseofulvin, and fumitremorgin C).2 Recently, several researchers reported that cytochrome P450 mutants are capable of catalyzing carbene and nitrene transfer reactions as artificial reactions (Scheme 1b).16 For instance, engineered P450BM3 was used for the olefin cyclopropanation via the carbene transfer reaction without any oxidative step.17 In this reaction, engineered P450BM3 forms an ironcarbenoid species for the carbene transfer reaction.17 Thus, cytochrome P450s are potentially capable of catalyzing these transfer reactions, but such cytochrome P450s have so far not been found in nature. Benzastatin derivatives are natural products isolated from Streptomyces species (Figure 1).18–25 For example, JBIR-67 (5a), 7-hydroxyl benzastatin D (6′d), virantmycin (6d), and JBIR-73 (6′e) were isolated from Streptomyces sp. RI18. Benzastatin derivatives show interesting bioactivities, such as neuronal cell protection19 and antiviral activities,22 and have diverse structures, such as indoline and tetrahydroquinoline scaffolds, that are probably derived from geranylated p-aminobenzoic acid (PABA) or PABA

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Scheme 1. Reactions catalyzed by cytochrome P450 and proposed cyclization reactions catalyzed by BezE. (a) Oxidative reactions catalyzed by cytochrome P450. (b) Mechanisms of aziridine ring formation catalyzed by engineered cytochrome P450. (c) Putative mechanism for the cyclization of geranyl moiety to generate indoline and tetrahydroquinoline scaffolds, which is based on the previously predicted cyclization reaction via epoxidation and epoxide-opening for benzastatins D and E.16 (d) Possible cyclization reactions catalyzed by BezE. Red and blue arrows indicate the reactions to synthesize indoline and tetrahydroquinoline scaffolds, respectively. Oxygen derived from H2O is shown in green. derivatives, such as p-aminobenzamide. The biosynthesis of ben-zastatins D and E was proposed to be initiated from geranylation of p-aminobenzamide followed by epoxidation and cyclization in Streptomyces nitrosporeus 3064 (Scheme 1c).26 However, neither the enzymes involved in these reactions nor any gene clusters for the biosynthesis of benzastatin derivatives were known for a long time. Wehad determined a draft sequence of the Streptomyces sp. RI18 genome, which led the identification of the biosynthetic gene cluster (bez gene cluster).27

duced by Streptomyces sp. RI18 (Figure 2). Three (bezD, bezH, and bezI) of the 10 genes are probably involved in the supply of the precursors, isoprenyl pyrophosphate and PABA. BezF, a putative UbiA-type polyprenyltransferase,28 appears to be responsible for the geranylation of PABA.

Here we analyzed the bez gene cluster by a heterologous gene expression system and in vitro assays of recombinant enzymes. We propose a probable benzastatin biosynthesis pathway involving an unprecedented cyclization reaction catalyzed by a cytochrome P450 (Scheme 1d). Results In silico analysis and heterologous expression of bez gene cluster Biosynthetic gene clusters similar to the bez cluster were discovered in Streptomyces olivaceus, Streptomyces niveus, Streptomyces ipomoeae, Streptomyces acidiscabies, and Saccharothrix espanaensis (Figure 2). Sequence comparison between the bez cluster and these gene clusters indicated that 10 genes (bezA-J) are responsible for the biosynthesis of the benzastatin derivatives pro-

Figure 1. Structures of benzastatin derivatives. *, †, ‡, and § indicate benzastatin derivatives isolated from Streptomyces sp. RI18, Streptomyces nitroporeus 3064, S. nitroporeus AM-2722, and Streptomyces sp. SANK 60101, respectively.


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Figure 2. Benzastatin biosynthetic gene cluster from Streptomyces sp. RI18 and homologous clusters from other actinomycetes. bezD encodes a putative trans-isoprenyl pyrophosphate synthase.31 bezH and bezI encode a PabA-PabB fusion protein and PabC homologues, respectively. PabA, PabB, and PabC are responsible for the biosynthesis of PABA.32 Thus, BezD and BezH-BezI are probably the enzymes for the supply of the precursors, isoprenyl pyrophosphate and PABA, respectively, to enhance the production of benzastatins. BezA and BezB are putative methyltransferases, showing homologies to a γ-tocopherol methyltransferases, which catalyzes benzene ring methylation,29 and RebM, which catalyzes the O-methylation of a sugar,30 respectively. Two cytochrome P450s BezC and BezE were predicted to be responsible for incorporation of a hydroxy group or epoxidation, which presumably occurs prior to the cyclization. BezG is a putative N-acetyltransferase and BezJ is a protein homologous to the N-oxygenase AurF, which catalyzes oxidation of the arylamine of PABA to synthesize pnitrobenzoate.33–35 The bez cluster was analyzed by heterologous gene expression and several putative benzastatin biosynthesis pathways were predicted.27 However, the cyclization mechanism of benzastatin remained elusive because the productivity of some benzastatins was unstable. To express the bez cluster more stably than what was achieved in the previous work,27 a chromosomeintegrative expression plasmid, pTYM-bezA-J, harboring bezA, bezB, bezC, and the bezD-J operon, was constructed. Each gene or gene set was placed under the control of the tipA promoter (Supplementary Figure 1). Streptomyces lividans was transformed with this plasmid and metabolites produced by the recombinant strain were analyzed by liquid chromatography-tandem mass spectrometry (LCMS/MS). As a result, production of the following benzastatin derivatives was reproducibly observed; 7-hydroxyl benzastatin J (2a), 7-hydroxyl benzastatin B (2b), 7hydroxyl O-demethylbenzastatin A (2c), JBIR-67 (5a), 7hydroxyl benzastatin F (5b), 7-hydroxyl Odemethylbenzastatin D (6′c), O-demethylvirantmycin (6c), 7-hydroxyl benzastatin D (6′d), virantmycin (6d), benzastatin K (7a), and methylbenzastatin K (7b) (Figure 3b, characterization of these compounds is described in Supplementary Information, with reference to Supplementary

Tables 2-7 and Supplementary Figures 2, 3 and 18-54; see Scheme 2 for their structures). JBIR-73 (6′e) was not produced in this system. Compounds 7a and 7b are a novel class of benzastatin derivatives, which possess a benz[b]azepine scaffold. This result demonstrates that bezA-J are responsible for benzastatin biosynthesis. However, the profile of benzastatin derivatives produced by the recombinant S. lividans strain was different from that produced by Streptomyces sp. RI18 (Figure 3a and b). The recombinant strain produced much more 2a, 5a, and 7a and much less 2c, 6′d, and 6d than Streptomyces sp. RI18. Moreover, only the recombinant strain produced 6′c and 6c. These differences are probably caused by differences in gene expression; bezA, bezB, bezC, and bezD-J are under the control of different native promoters in Streptomyces sp. RI18, while all of them were forcibly expressed using the tipA promoter in the heterologous expression system. In vivo analysis of the function of bez genes To analyze the function of each biosynthesis enzyme gene, a series of heterologous expression plasmids, each of which lacks one of the biosynthesis enzyme genes (bezA, bezB, bezC, bezE, bezG, and bezJ), was constructed and transferred into S. lividans (Supplementary Table 1). Deletion of bezG or bezJ abolished the ability to produce compounds that possess bicyclic scaffolds (5a, 5b, 6c, 6d, 6′c, 6′d, 7a, and 7b); the ∆bezG and ∆bezJ strains produced only “linear” benzastatins (2a, 2b, and 2c) (Figure 3g and h). The production of benzastatin derivatives with bicyclic scaffolds was restored by introducing the deleted gene (bezG or bezJ) into each mutant using another chromosome integration plasmid (pTYM2k) (Supplementary Figure 4). These results indicate that bezG and bezJ are essential for the second ring formation. Inactivation of



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bezE abolished the production of three of the four benzastatins with a tetrahydroquinoline scaffold (6c, 6′c, and 6d), and decreased the yield of the two benzastatins with an indoline scaffold (5a and 5b) and one of the four benzastatins with a tetrahydroquinoline scaffold (6′d, 6′d was only detectable in MS analysis) (Figure 3f). These defects of the ∆bezE strain were restored by introducing bezE into the mutant using pTYM2k (Supplementary Figure 4). This result suggests that BezE is not necessarily required for the formation of indoline and tetrahydroquinoline scaffolds, but has an important role in this step. Taken together with the results of bezE, bezG, and bezJ deletion, we speculated that biosynthetic intermediates that are amenable to the second ring cyclization should be produced by BezG and BezJ, and that BezE should facilitate and control the cyclization of these intermediates. Because formation of the benz[b]azepine scaffold (7a and 7b) was enhanced in the ∆bezE strain (Figure 3f), the cyclization generating the benz[b]azepine scaffold seems to occur non-enzymatically. Formation of the indoline and tetrahydroquinoline scaffolds also seems to occur non-enzymatically with a much lower efficiency compared with the benz[b]azepine scaffold formation. Meanwhile, BezE appears to be essential for the introduction of a chloro group to produce 6c and 6d. Therefore, we speculated that the reaction catalyzed by BezE should involve the introduction of a chloro group. The ∆bezC strain could not produce the linear benzastatin 2c and any of the four benzastatins having a tetrahydroquinoline scaffold (6c, 6′c, 6d, and 6′d) (Figure 3e). This suggests that BezC catalyzes the hydroxylation of 2b to produce 2c, and that the hydroxyl group at C-17 is essential for the formation of a tetrahydroquinoline scaffold. The ∆bezA strain produced only 2a, 5a, and 7a (Figure 3c). This result clearly shows that BezA catalyzes methylation at C-13, and indicates that the methyl group at C-13 is essential for the hydroxylation at C-17 by BezC. The ∆bezB strain lost the ability to produce 6d and 6′d, and accumulated 6c and 6′c (Figure 3d), suggesting that the O-methylation reaction catalyzed by BezB is the final step of 6d and 6′d biosynthesis, and that chlorination occurs prior to this reaction (Scheme 2). Non-enzymatic degradation of virantmycin (6d) Because the chloro group of virantmycin (6d) is a good leaving group and the thiol group of ergothioneine has a high nucleophilicity,36 we assumed that JBIR-73 (6′e) is non-enzymatically synthesized from 6d by the nucleophilic attack of the thioketone of ergothioneine (Scheme 2 and Supplementary Figure 5b). To prove this assumption, a physiological amount of ergothioneine was incubated with 6d in HEPES buffer (pH 7.4) for 1 h. As expected, formation of 6′e was observed (Supplementary Figure 5a). This result suggests that 6′e is synthesized nonenzymatically from 6d and ergothioneine. In addition, conversion of 6d into 6′d and its isomer 6′d* was also observed, suggesting that 6′d and 6′d* are also synthesized by the non-enzymatic hydrolysis of 6d.

Figure 3. Heterologous expression of the benzastatin biosynthetic gene cluster. LC-MS/MS analysis of benzastatins extracted from (a) Streptomyces sp. RI18, S. lividans strains harboring (b) pTYM-bezA-J, (c) pTYMbezA-J∆bezA, (d) pTYM-bezA-J∆bezB, (e) pTYM-bezAJ∆bezC, (f) pTYM-bezA-J∆bezE, (g) pTYM-bezA-J∆bezG, (h) pTYM-bezA-J∆bezJ, and (i) pTYM19gt (negative control). Compound 6′d* was predicted to be an isomer of 6′d by comparing its tandem mass spectrum with that of 6′d (Supplementary Figure 6). The compounds observed only by MS analysis are depicted in gray. In vitro analysis of the enzymes for the modification of the geranyl moiety To obtain further insights into the biosynthetic pathway for benzastatin derivatives, we first analyzed modification of the geranyl moiety by BezA and BezC in vitro. Recombinant BezA and BezC were prepared as Nterminally His6-tagged proteins (Supplementary Figure 7). The recombinant BezC showed a Soret band shift from 420 to 450 nm as a typical cytochrome P450, when CO was bubbled into the BezC solution, indicating that BezC was produced as an active form (Supplementary Figure 8d). As described above, BezA and BezC were indicated to catalyze the methylation at C-13 and the following hydroxylation at C-17, respectively. However, against our expectation, 2a and 2b were not served as substrates of BezA and BezC, respectively (Supplementary Figure 8a and b). Therefore, we hypothesized that the modification of the geranyl moiety should occur at an earlier step, namely on geranyl pyrophosphate (GPP, 1a). When BezA was incu-


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bated with 1a and S-adenosylmethionine (SAM), Sadenosyl-L-homocysteine was formed, suggesting that BezA catalyzes methylation of 1a (Supplementary Figure 8c). To identify the product of BezA reaction, we aimed to transfer it onto PABA using recombinant BezF. Because BezF appears to be a membrane-bound prenyltransferase, we prepared crude BezF protein solubilized from the membrane fraction of the recombinant S. lividans expressing bezF. We confirmed that the crude BezF solution possessed a distinct geranylation activity; 2a was produced when PABA and 1a were incubated with the crude BezF solution (Figure 4d and Supplementary Figure 9). BezA was removed from the BezA reaction mixture with ultrafiltration and the reaction product was transferred onto PABA by the crude BezF solution. As a result, we observed the production of 2b (Figure 4b). This result clearly shows that BezA catalyzes C-methylation of 1a to synthesize methylgeranyl pyrophosphate (MGPP, 1b). Next, we analyzed the function of BezC using a BezA-BezC one-pot reaction. BezA and BezC were incubated with 1a, SAM, NADH, ferredoxin (CamA), and ferredoxin reductase (CamB), and the product was transferred onto PABA as described above. As a result, we observed the formation of 2c (Figure 4a). When BezA was removed from the reaction mixture, only 2a was detected (Figure 4c), indicating that hydroxylation of GPP could not occur prior to methylation also in vitro. Taken together, we concluded that BezA and BezC catalyze sequential methylation and hydroxylation of 1a to synthesize hydroxymethylgeranyl pyrophosphate (HMGPP, 1c). In addition, BezF was shown to have a relaxed donor substrate specificity; BezF can catalyze the geranylation of PABA using any of 1a, 1b, and 1c. In vitro analysis of BezG and BezJ To investigate the functions of BezG and BezJ, we prepared recombinant enzymes (Supplementary Figure 7). Regardless of our extensive attempts to observe BezJ activity using various possible substrates and electron providers, we could not detect any oxidation activity of BezJ. Therefore, we decided to deduce the function of BezJ with a feeding experiment. We synthesized phydroxyaminobenzoic acid (PHABA) and fed it to the ∆bezJ strain. As a result, the ∆bezJ strain restored the production of benzastatin derivatives with indoline and tetrahydroquinoline scaffolds (Supplementary Figure 10). Therefore, BezJ was suggested to catalyze the oxidation of PABA to synthesize PHABA. According to this result, PHABA was predicted to be a substrate of BezG. When PHABA was incubated with BezG and acetyl-CoA, we observed the formation of a compound whose m/z corresponded to acetylated PHABA ([M+H]+ = m/z 196) (Supplementary Figure 11a). The product was highly unstable and could not be isolated, but it showed a characteristic fragment due to the loss of acetate ([M]+ = m/z 136) in MS/MS analysis (Supplementary Figure 11b). BezG did not show any acetylation activity against PABA (Supplementary Figure 11c), indicating that the acetylation occurs on the hydroxyl group. Therefore, we concluded that the

product of BezG reaction is p-acetoxyaminobenzoic acid (PAcABA). It should be noted that in this experiment we used BezG of Streptomyces niveus, which shows 87% amino acid sequence identity with BezG of Streptomyces sp. RI18, because we could not produce the Streptomyces sp. RI18 BezG in an active form in Escherichia coli.

Figure 4. In vitro analysis of BezA and BezC. BezA, BezC, CamA, and CamB were incubated with 1a, SAM, and NADH. After the reaction, products were transferred onto PABA by BezF. (a) In the presence of all the enzymes, 2c was detected. (b) When BezC, CamA, CamB, and NADH were removed from the reaction mixture, 2b was detected. (c) When BezA was removed from the reaction mixture, 2a was detected. (d) When BezF was incubated with 1a and PABA, 2a was detected. In vitro analysis of BezE According to the obtained results, we predicted that BezE, annotated as a cytochrome P450, should catalyze the cyclization of geranylated PAcABA derivatives (4a, 4b, and 4c) by inducing the acetate elimination coupled with the cyclization reaction. To confirm this prediction, recombinant BezE was prepared; it showed a Soret band shifting from 420 to 450 nm, when CO was bubbled into the BezE solution (Supplementary Figures 7 and 12a). Because the possible substrates (4a, 4b, and 4c) of BezE are highly unstable, we tried one-pot reaction of BezE, BezF, and BezG using 1a and chemically synthesized PHABA as substrates. When these three enzymes were incubated with 1a and PHABA, 5a was produced as expected (Figure 5a). When BezE was removed from the reaction mixture, 7a was produced (Figure 5b), which was consistent with the in vivo experiment (Figure 3f). In addition, a small amount of 5a was observed in the absence of BezE, probably due to the nonenzymatic cyclization of 4a.



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Figure 5. In vitro analysis of BezE. (a) When BezE, BezF, and BezG were incubated with PHABA, 1a, and acetyl-CoA, formation of 5a was observed. (b) When BezE was removed from the reaction mixture of (a), 7a and a small amount of 5a were observed. (c) First, 1b was synthesized from GPP (1a) by BezA. After this reaction, BezE, BezF, BezG, and PHABA were added into the reaction mixture. As a result, formation of 5b was observed. (d) When BezE was removed from the second reaction of (c), formation of 7b was observed. (e) First, 1c was synthesized from 1a by BezA and BezC. After this reaction, BezE, BezF, BezG, and PHABA were added into the reaction mixture, resulting in the formation of 6c and 6′c. (f) When BezE was removed from the second reaction of (e), 6c and 6′c were not formed. (g-h) BezEFG reaction using stable isotopelabeled compounds. Mass spectra of 5a produced by the BezEFG reaction are shown: (g) normal reaction; (h) in the presence of 50% H218O; (i) in the presence of 18O2. In an independent time-course experiment, a small amount of 7a, as well as a large amount of 5a, was detected in the BezEFG reaction, while a large amount of 7a and a trace amount of 5a were detected in the BezFG reaction at 120min incubation (5a was only detectable in MS analysis) (Supplementary Figure 13a). These results indicate that BezE enormously facilitate 5a formation. Interestingly, the addition of a redox partner did not affect the rate of 5a formation in the BezEFG reaction (Supplementary Figure 13b). To obtain further insights into the cyclization mechanism, we conducted in vitro analysis of BezE using H218O and 18O2. As a result, in the presence of 50% H218O, approximately 50% of 5a was labeled with 18O in the BezEFG reaction (Figure 5g and h). In contrast, in the presence of 18O2, 18O was not incorporated into 5a (Figure 5i). These results strongly indicate that cyclization never occurs via epoxidation and that BezE is not a monooxygenase. In addition, we showed that hemin (an iron-containing porphyrin) could not catalyze the formation of 5a, indicating that the protein scaffold of BezE is required for the cyclization reaction (Supplementary Figure 13c). BezE also catalyzed the formation of 5b when 1b, instead of 1a, was used as a substrate (Figure 5c). Note that 1a should be included in the reaction mixture because we prepared 1b with the enzymatic conversion of 1a by BezA. This is why 5a was also produced in this experi-

ment. When BezE was removed from the reaction mixture, 7b was formed (Figure 5d), which was also consistent with the in vivo experiment (Figure 3f). Next, we analyzed the role of BezE in tetrahydroquinoline synthesis. Compound 1c, which had been synthesized from 1a by BezA and BezC in vitro, was added to the reaction mixture containing BezE, BezF, BezG, and PHABA. As a result, we observed the formation of 6c and 6′c (Figure 5e). Compounds 5a and 5b were also detected because 1a and 1b should be included in the reaction mixture. When BezE was removed from the reaction mixture, no formation of these compounds was observed (Figure 5f). From these results, we concluded that BezE is responsible for the second ring cyclization of benzastatins and the introduction of a Cl group at C-9. Because 6d was easily hydrolyzed to 6′d in the reaction buffer (Supplementary Figure 5), we think that 6′c observed in the reaction is a shunt product synthesized by the hydrolysis of 6c. Next, we analyzed the substrate-binding spectrum of BezE. Because 4a, 4b, and 4c (actual substrates of BezE) were unstable, 2a, 2b, and 2c were used for the analysis as the substrate analogs. The addition of these compounds to BezE resulted in type II shift, which is observed when nitrogen binds to the heme iron (Supplementary Figure 12b). The dissociation constants were determined to be 3.45 ± 0.86 µM (2a), 2.01 ± 0.29 µM (2b), and


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1.56 ± 0.60 µM (2c) (Supplementary Figure 12c). This result indicates that the heme of BezE binds with the amino group of 4a, 4b, and 4c during the catalysis. Discussion In this study, we propose the following biosynthetic pathways for benzastatin derivatives (Scheme 2). BezA and BezC are involved in the modification of 1a. BezA methylates 1a to produce 1b, and BezC hydroxylates 1b to produce 1c. Meanwhile, BezJ and BezG catalyze the sequential hydroxylation and O-acetylation of PABA to produce PAcABA. Then, BezF assembles these building blocks to produce three geranylated PAcABA derivatives (4a, 4b, and 4c). These derivatives are cyclized by BezE to produce benzastatins with indoline and tetrahydroquinoline structures. O-Methylation of 6c catalyzed by BezB results in 6d. Compound 6d is further converted to 6′e by the nonenzymatic nucleophilic attack of ergothioneine. Compounds 6′c and 6′d are probably synthesized mainly from 6c and 6d, respectively, by non-enzymatic hydrolysis. Compound 6′c also might be produced from non-enzymatic cyclization of 4c because a trace amount of 6′d was detected in the ∆bezE strain. Feeding of the “linear” benzastatin 2a to a recombinant S. lividans strain expressing bezE, bezG, and bezJ did not lead to the formation of 5a (Supplementary Figure 14a). Feeding of the “linear” benzastatins 2b and 2c to the ∆bezA and ∆bezC mutants, respectively, also did not lead to the formation of cyclized benzastatins (Supplementary Figure 14b and c). These results strongly indicate that the “linear” benzastatins (2a, 2b, and 2c) are shunt products synthesized by geranylation of PABA. Potentially, geranylated PHABA derivatives can also be synthesized by BezF. However, these compounds were not detected in the ∆bezG strain, and therefore they seem to be unstable. This notion was also supported by the observation that the yield of “linear” benzastatins (2a, 2b, and 2c) produced by the ∆bezG strain was significantly lower than that produced by the ∆bezJ strain (Figure 3g and h). Similarly, the N-acetoxy intermediates (4a, 4b, and 4c) seem to be unstable because they were not detected in the bezE strain and in vitro experiments using BezG and BezF. When we analyzed current genome database, we found that many actinobacteria possess putative benzastatin biosynthetic gene clusters, suggesting that this system is widely distributed among actinobacteria (Supplementary Figure 15). The formation of indoline and tetrahydroquinoline structures is the most interesting reaction in benzastatin biosynthesis. We initially expected that these compounds were synthesized by the cyclization of “linear” benzastatins (2a, 2b, and 2c) as shown in Scheme 1c. This reaction is most likely to proceed via epoxidation of the double bond followed by nucleophilic attack of the aromatic amine to the epoxide, in an analogous manner to polyether biosynthesis as proposed by Yoo et al.26,37 However, this mechanism could not explain why two putative oxygenases (BezJ and BezE) and a putative N-acetyltransferase (BezG) are required for the correct cyclization, why 18O of H218O was

introduced into 5a, and how a benz[b]azepine scaffold is synthesized. Instead of the oxidation of a double bond, our studies indicate that this cyclization should be initiated by activation of aromatic amine by the successive N-oxidation and O-acetylation as depicted in Schemes 1d and 2. Then, the activated aromatic amine is subjected to the cyclization reaction. The cytochrome P450 BezE promotes and controls the cyclization reaction to form indoline and tetrahydroquinoline scaffolds. This reaction probably proceeds via (i) elimination of the acetoxy group resulting in iron nitrenoid formation, (ii) aziridine ring formation by the nitrene transfer to the nearest double bond of the geranyl moiety, and (iii) aziridine ring opening by the nucleophilic attack of HO− to C-10 and Cl− to C-9 to generate the indoline and tetrahydroquinoline scaffolds, respectively (Scheme 1d). Importantly, BezE does not catalyze any oxidative reaction in this step. This is consistent with the observation that BezE catalyzed indoline scaffold formation in the absence of any redox partners (ferredoxin, ferredoxin reductase, and NAD(P)H), which are usually required for the reaction catalyzed by cytochrome P450s. The feeding experiment using 18O also supports this hypothesis; 18O of H218O, not 18O of 18O2, was incorporated into 5a. The BezE-catalyzing reaction resembles the nitrene transfer reactions catalyzed by some cytochrome P450 mutants.38 In this reaction, engineered cytochrome P450s eliminate N2 from an azide substrate and form a putative iron nitrenoid reactive intermediate. Iron nitrenoid is used for the nitrene transfer reaction to produce an aziridine derivative. Although an azide is commonly used as a source of nitrene in these artificial reactions, it is reported that an N-acetoxy group also can be the source of nitrene.39 Therefore, the above-proposed reaction mechanism of BezE is convincing. Furthermore, type II substrate binding spectra of BezE with 2a, 2b, and 2c strongly support the proposed reaction mechanism. Type II shift is usually observed when an amine of an inhibitor binds to the heme to inhibit the oxidative reaction. Because 2a, 2b, and 2c are analogs of 4a, 4b, and 4c, respectively, the observed shifts strongly indicate that the amines of 4a, 4b, and 4c also should coordinate with the heme iron during catalysis. This binding state is suitable for iron nitrenoid formation. Furthermore, hemin did not catalyze 5a formation, suggesting that protein scaffold of BezE is critical for this reaction. Taken together, our results demonstrate that the nitrene transfer reaction used by artificial cytochrome P450s is also used in nature to biosynthesize natural products. Phylogenetic analysis showed that BezE and BezE homologues encoded by the putative benzastatin biosynthetic gene clusters are evolutionally related to AmphL,40 NysL,41 PimD,42 PenM,43 and PntM,43 all of which catalyze monooxygenation with reducing partners (Supplementary Figure 16b). Thus, BezE seems to have evolved from a P450 catalyzing monooxygenation to develop its unique nitrene transfer activity. Among BezE and BezE homologues, the heme binding region, including the Cys residue that coordinates with heme iron, is well conserved (Supplementary Figure 16a).


Journal of the American Chemical Society NH2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

NH2

BezF

PPO

HO

R1 13

HO O PABA

GPP (1a) BezA

BezJ H N

PPO

O

GPPs

Page 8 of 10

17

R2

R1=R2=H: 7-hydroxyl benzastatin J (2a) R1=Me, R2=H: 7-hydroxyl benzastatin B (2b) R1=Me, R2=OH: 7-hydroxyl O-demethylbenzastatin A (2c)

OH

HO

H N

MGPP (1b)

O PHABA

BezC

O

BezG O O NH

PPO OH

If R2=H NE

O NH

BezF

HMGPP (1c) O PAcABA

R1

O

GPPs

R2 R1=R2=H: (4a) R1=Me, R2=H: (4b) R1=Me, R2=OH: (4c)

PABA modification

If R2= OH NE

H N

HO

R1

O R1=H: JBIR-67 (5a) R1=Me: 7-hydroxyl benzastain F (5b)

cyclization

BezB

O

NE

10 9

HO

H N

O

H N NE

OH O 7-hydroxyl benzastatin D (6'd)

HO

Cl O O-demethylvirantmycin (6c)

BezB

HO

9

OH 10

OH

H N

NE

OH O 7-hydroxyl O-demethylbenzastatin D (6'c)

H H N

If R2=H BezE or NE (weak)

If R2= OH BezE

OH

HO

H N

O R1=H: benzastain K (7a) R1=Me: methylbenzastain K (7b)

HO

HO

GPP modification

R1

HO

Cl

HO

O

O

N+

N

O virantmycin (6d)

H N

S

JBIR-73 (6'e)

HO

O

Scheme 2. Proposed biosynthetic pathway for benzastatin derivatives. NE, nonenzymatic; PABA, p-aminobenzoic acid; GPP, geranyl pyrophosphate; MGPP, methylgeranyl pyrophosphate; HMGPP, hydroxymethylgeranyl pyrophosphate. Meaning of compound number and suffix: 1, GPP derivatives; 2, “linear” benzastatins derived from PABA; 4, biosynthesis intermediates derived from GPP derivatives and PAcABA; 5, benzastatins with an indoline scaffold; 6, benzastatins with a tetrahydroquinoline scaffold; 6′, degradation products of 6; 7, benzastatins with a benz[b]azepine scaffold; a, no modification on the geranyl moiety; b, a methyl group on the geranyl moiety; c, methyl and hydroxy groups on the geranyl moiety; d, methyl and methoxy groups on the geranyl moiety; e, substitution of the chloro group of 6d with ergothioneine. The acetoxy group formed on the aromatic amine is unstable and subjected to hydrolysis or photolysis to generate an active phenylnitrenium ion.44 Takeuchi et al. reported that phenylnitrenium ion can react with a double bond to synthesize an aziridinium ion and that the aziridinium ion can be hydrolyzed easily.45 Thus, even in the absence of BezE, non-enzymatic cyclization can proceed to synthesize not only 5a and 5b, but also 6′c. However, in this non-enzymatic cyclization, 7a and 7b are synthesized in higher amounts. We speculate that the phenylnitrenium ion should directly react with the methyl group (C-17) to generate a seven-membered ring because the allylic C−H bond is relatively weak. Because 7a and 7b are synthesized in larger amounts in the absence of BezE than in the presence of BezE, we consider that these compounds are shunt products. Therefore, we propose that BezE is required for the proper second-ring cyclization of benzastatin derivatives.

Intriguingly, the hydroxyl group of the geranyl moiety seems to determine the cyclization specificity. When the geranyl moiety has been hydroxylated at C-17, the cyclization occurs to generate a tetrahydroquinoline scaffold, while an indoline scaffold is generated in the absence of this hydroxyl group. According to the synthetic study on benzastatin E, the absolute stereochemistry of 5a and 5b should be (9S,10R) (Supplementary Table 8).23,46 In contrast, the absolute stereochemistry of 6d was reported to be (9R,10R), indicating that 6c also should have the same stereochemistry, (9R,10R).47 These indoline and tetrahydroquinoline compounds can be synthesized from the similar aziridine intermediates with (9S,10S) stereochemistry by SN2 reaction (Scheme 1d and Supplementary Figure 17). We speculate that the steric hindrance caused by the presence of the hydroxyl group at C-17 inhibits the nucleophilic attack of HO− by shutting out HO− from the active site. Thus, a Cl− ion, probably captured near the active site, can efficiently react with C-9 to synthesize the tetra-


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hydroquinoline scaffold. In the absence of this hydroxyl group at C-17, HO− can enter into the cavity that would be occupied by the hydroxyl group. We speculate that this cavity is closer to C-10 than C-9, so that the nucleophilic attack of HO− can occur to C-10 to generate the indoline scaffold. Another interesting point in the reaction catalyzed by BezE is how the chloro group is introduced. Most chloro groups of secondary metabolites are introduced by oxidases, such as flavin-dependent halogenases.48–50 There are also enzymes that catalyze a nucleophilic halogenation of SAM by using L-methionine as a leaving group.51,52 Halogenation in the benzastatin biosynthetic pathway uses neither of these mechanisms and probably occurs nucleophilically, coupled with aziridine ring opening. We speculate that the active site of BezE has a specific site to accommodate Cl− and a catalytic acid to enhance the aziridine ring opening, so that BezE can specifically catalyze nucleophilic addition of Cl− at C-9 when 4c was used as a substrate (Scheme 1d). However, further structural and biochemical studies on BezE are required for proposing enzymatic mechanism for halogenation. In summary, analysis of the benzastatin biosynthetic gene cluster enabled us to propose unprecedented cyclization and halogenation reactions catalyzed by BezE. To the best of our knowledge, BezE is the first native cytochrome P450 that was reported to catalyze nitrene transfer. We propose “P450 nitrene transferase” for BezE. Accession codes The bez gene cluster was deposited in the DNA Data Bank of Japan (DDBJ) under the accession number LC177364. ASSOCIATED CONTENT Supporting Information. The supporting information is available free of charge on the ACS publication website. Materials and methods, Supplementary tables, and supplementary figures about experimental details, structural elucidation (tandem mass spectra and NMR spectra), functional analysis of bez genes in vivo and in vitro, wide distribution of bez cluster homologues, and sequence alignment and phylogenetic analysis of BezE and P450s (PDF). AUTHOR INFORMATION Corresponding Authors *Y.O.; [email protected] *Y. K.; [email protected] Notes The authors declare no competing financial interests. Acknowledgements

We thank H. Onaka (pTYM19gt and pTYM2k), Y. Zhang (GZB05-dir), and A. Miyanaga and A. Arisawa (pET28bcamA and pET28b-camB) for providing experimental materials. This research was supported in part by a funding program for next generation world-leading researchers from the Bureau of Science, Technology, and Innovation Policy, Cabinet Office, Government of Japan (to Y.O.), a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) (to Y.K.), a grant from the New Energy and Industrial Technology Development Organization (NEDO) of Japan (to K.S. and N.S.), and Japan Society for the Promotion of Science (JSPS) A3 Foresight Program. REFERENCES (1) Pang, B.; Wang, M.; Liu, W. Nat. Prod. Rep. 2016, 33, 162. (2) Tang, M. C.; Zou, Y.; Watanabe, K.; Walsh, C. T.; Tang, Y. Chem. Rev. 2017, 117, 5226. (3) Xu, L. H.; Fushinobu, S.; Takamatsu, S.; Wakagi, T.; Ikeda, H.; Shoun, H. J. Biol. Chem. 2010, 285, 16844. (4) Shrestha, P.; Oh, T. J.; Liou, K.; Sohng, J. K. Appl. Microbiol. Biotechnol. 2008, 79, 555. (5) Takahashi, S.; Nagano, S.; Nogawa, T.; Kanoh, N.; Uramoto, M.; Kawatani, M.; Shimizu, T.; Miyazawa, T.; Shiro, Y.; Osada, H. J. Biol. Chem. 2014, 289, 32446. (6) Pohle, S.; Appelt, C.; Roux, M.; Fiedler, H. P.; Süssmuth, R. D. J. Am. Chem. Soc. 2011, 133, 6194. (7) Gaisser, S.; Lill, R.; Staunton, J.; Méndez, C.; Salas, J.; Leadlay, P. F. Mol. Microbiol. 2002, 44, 771. (8) Malla, S.; Thuy, T. T. T.; Oh, T. J.; Sohng, J. K. Arch. Microbiol. 2011, 193, 95. (9) Funa, N.; Funabashi, M.; Ohnishi, Y.; Horinouchi, S. J. Bacteriol. 2005, 187, 8149. (10) Zhao, B.; Lamb, D. C.; Lei, L.; Kelly, S. L.; Yuan, H.; Hachey, D. L.; Waterman, M. R. Biochemistry. 2007, 46, 8725. (11) Zhao, B.; Guengerich, F. P.; Bellamine, A.; Lamb, D. C.; Izumikawa, M.; Lei, L.; Podust, L. M.; Sundaramoorthy, M.; Kalaitzis, J. A.; Reddy, L. M.; Kelly, S. L.; Moore, B. S.; Stec, D.; Voehler, M.; Falck, J. R.; Shimada, T.; Waterman, M. R. J. Biol. Chem. 2005, 280, 11599. (12) Girvan, H. M.; Munro, A. W. Curr. Opin. Chem. Biol. 2016, 31, 136. (13) Podust, L. M.; Sherman, D. H. Nat. Prod. Rep. 2012, 29, 1251. (14) Zhang, X.; Li, S. Nat Prod Rep. 2017, 34, 1061. (15) Rudolf, J. D.; Chang, C. Y.; Ma, M.; Shen, B. Nat Prod Rep. 2017, 34, 1141. (16) Brandenberg, O. F.; Fasan, R.; Arnold, F. H. Curr. Opin. Biotechnol. 2017, 47, 102. (17) Coelho, P. S.; Brustad, E. M.; Kannan, A.; Arnold, F. H. Science. 2013, 339, 307. (18) Kim, W. G.; Kim, J. P.; Kim, C. J.; Lee, K. H.; Yoo, I. D. J. Antibiot. 1996, 49, 20. (19) Kim, W. G.; Kim, J. P.; Koshino, H.; Shin-Ya, K.; Seto, H.; Yoo, I. D. Tetrahedron. 1997, 53, 4309. (20) Kim, W. G.; Ryoo, I. J.; Park, J. S.; Yoo, I. D. J. Antibiot. 2001, 54, 513.



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