Crystalline Sponge Method Enabled the Investigation of a

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

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Crystalline Sponge Method Enabled the Investigation of a Prenyltransferase-terpene Synthase Chimeric Enzyme, Whose Product Exhibits Broadened NMR Signals Takaaki Mitsuhashi,† Takashi Kikuchi,§ Shotaro Hoshino,† Masahiro Ozeki,† Takayoshi Awakawa,†,⊥ She-Po Shi,∥ Makoto Fujita,*,# and Ikuro Abe*,†,⊥ †

Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan § Rigaku Corporation, 3-9-12 Matsubara-cho, Akishima-shi, Tokyo 196-8666, Japan ∥ Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China # Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

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ABSTRACT: By the genome-mining approach, a chimeric enzyme of prenyltransferase-diterpene synthase was discovered from Penicillium chrysogenum MT-12. Since its product exhibited broadened NMR signals, the structural determination by only the NMR analysis was difficult, but the crystalline sponge method successfully revealed the structure with a 6−5−5−5 fused ring system. This demonstrated that the collaboration between the genome-mining and crystalline sponge method has the potential to facilitate rapid inquiries into the unexplored chemical space of small molecules.

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Additionally, the NMR analysis may not determine the absolute configuration. Thus, even if the planar structure and relative configuration could be determined by the NMR, additional effort is required to establish the absolute configuration of the molecule. The crystalline sponge method is a recently developed X-ray technique for the structural determination of small molecules.2 When a crystal of the porous complex is incubated with small molecules, the molecules enter the crystal as guests and are trapped in it. Since the trapped guests are aligned neatly in the host crystal, the X-ray analysis of the guest-absorbed crystal enables the structural determination of the guest compounds. Therefore, the crystalline sponge method allows even noncrystalline compounds to be subjected to an X-ray analysis. Moreover, this method requires only micro- to nanogram quantities of compounds and allows the determination of the absolute configuration because the host crystal contains heavy atoms. In this work, we selected a chimeric enzyme of prenyltransferase-terpene synthase (PT-TS) as the target of a genome-mining study.3 This is a bifunctional enzyme composed of two domains, a prenyltransferase domain and a terpene synthase domain,

n the postgenomic era, the genome-mining approach is one of the most effective ways to search for novel natural products.1 This approach first involves a search for genes responsible for the production of secondary metabolites, and the targeted genes are then investigated by genetic engineering techniques. For example, when the genes are introduced into a heterologous expression host, the transformed host organism produces the compounds formed by the enzymes encoded by the DNA. Rapid developments of genome sequencing techniques have generated a wealth of DNA sequence data. Therefore, today’s challenges are methodology; for instance, (1) how to mine biosynthetic genes, (2) how to construct heterologous expression systems optimal for target genes, and (3) how to efficiently determine the structures of enzyme reaction products. To solve the structural elucidation problem, nuclear magnetic resonance (NMR) is a powerful and routinely applied technique. However, NMR analysis is not a universal method. For example, a compound that possesses more than one conformation sometimes exhibits broadened NMR signals, which is not suitable for the structural determination. Generally, if conformers are interconverting significantly slow, sharp signals derived from each conformer could be observed. When conformers are interconverting fast, signals from each conformer are averaged and sharp signals are also observed. However, if conformers are interconverting not as slow, but not fast enough for making signals sharp, the NMR signals become broadened. © XXXX American Chemical Society

Received: July 20, 2018

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DOI: 10.1021/acs.orglett.8b02284 Org. Lett. XXXX, XXX, XXX−XXX

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spectra were recorded at 50 °C, the broadened signals became sharper than the signals recorded at 25 °C (Figures S27−S31). This suggested that the high temperature accelerates the conformational changes. However, the structural determination by only the NMR analysis still remained difficult. To achieve the structural determination, we then tried the crystalline sponge method. Considering the fact that 1 exists as an oily liquid, the crystallization of this compound is not straightforward. Thus, the crystalline sponge method, which enables the crystallization-free X-ray analysis, was employed. To introduce 1 into the crystalline sponge, 5 μg of 1 was incubated with the crystalline sponge [(ZnCl2)3(tpt)2·x(solvent)]n [tpt = tris(4-pyridyl)-1,3,5-triazine] for 1 day at 50 °C,9 and then the guest-absorbed crystal was subjected to the X-ray data collection. As a result, the crystal structure of the 1/crystalline sponge complex was successfully elucidated. Two molecules of 1 per asymmetric unit were clearly observed (Figure 1 and Figures S3−S4). Consequently, 1 proved to be a precursor of the cyclopianetype diterpenes with a 6−5−5−5 fused ring system (Figure 1E). The cyclopiane-type diterpenes are known to be isolated exclusively from Penicillium genus.10 This is consistent with

which catalyze tandem reactions. The prenyltransferase domain catalyzes a chain elongation reaction to produce linear isoprenoid precursors. The terpene synthase domain then cyclizes the linear isoprenoid precursors to form the basic terpenoid carbon skeletons. This class of terpene synthase is involved in the biosynthesis of di- and sesterterpenoids in fungi. Since the first identification in 2007,4 over 200 putative PTTS chimeras have been found in the public database.3b However, despite vigorous investigations, only fewer than 20 enzymes have been characterized so far.2b,4,5 In the near future, the number of this class of enzymes should increase, as more fungi will be subjected to genome sequencing analyses. Actually, one of the bottlenecks in the investigations of these kinds of enzymes is the structural determination of their products. The products commonly exist as hydrocarbons or hydrocarbon alcohols and often possess complex fused ring systems with many stereocenters. Thus, the NMR spectra of these compounds are quite complicated and highly overlapped, especially on the high magnetic field. Moreover, the determination of the absolute configuration is also an obstacle. There is no general approach to determine the absolute configuration, and many trials and errors, such as chemical derivatization, are required. Considering the fact that the previously characterized PT-TS chimeric enzymes produce compounds with diverse and complex structures, including several novel carbon skeletons,2b,4,5 they are still fascinating targets of the genome mining. Herein, we discovered a gene of a PT-TS chimera from genetic data of Penicillium chrysogenum MT-12.6 However, the NMR signals of its product are not only highly overlapped and complicated, but also broadened. Thus, it was extremely difficult to determine the structure of the enzyme product by only NMR analysis. To solve this problem, we applied the crystalline sponge method and successfully elucidated the structure as a diterpene alcohol with a 6−5−5−5 fused ring system. Moreover, this compound has not been previously isolated from a natural source. When PT-TS chimeric enzyme candidates in the public database were examined, we noticed that PT-TS chimeras, sharing at least ∼80% similarity with each other, are conserved in many Penicillium species (Table S1). This fact encouraged us to investigate P. chrysogenum MT-12, the endophytic fungi that we previously isolated from Chinese club moss Huperzia serrata.6 As expected, even though there is no report that this strain produces di- or sesterterpenoids, one putative PT-TS chimera was found in the genome. Further sequence analysis confirmed that the typical motifs of this class of enzyme are conserved (two DDXXD and NSE/DTE motifs) (Figure S1).7 Thus, this enzyme seemed to be catalytically active. Next, to investigate the catalytic function of the PT-TS chimera, the gene was heterologously expressed in Aspergillus oryzae NSAR1,8 as this strain has been successfully utilized as a host organism for the heterologous expression of other genes of PT-TS chimeric enzymes.2b,5a−g The mycelial extract of the A. oryzae transformant was then analyzed by GC−MS, which revealed that the transformant produced the new metabolite 1 with m/z 290 [M]+ (Figure S2). Thus, 1 was expected to be a diterpene alcohol. To elucidate its structure, 1 was isolated and subjected to the NMR analysis. However, even though various NMR solvents (benzene-d6, chloroform-d, and methanol-d4) were tried, in all cases, the NMR spectra of 1 were broadened at 25 °C (Figures S6−S23). We thought that the signal broadening might be caused by conformational change of 1. Actually, when the NMR

Figure 1. (A) Crystal structure of the 1/crystalline sponge complex. Two molecules of 1 (a and b) are present in the asymmetric unit. Solvent molecules are omitted for clarity. (B) Crystal structure of 1 (guest b). (C) Crystal structure of 1 (guest b). Hydrogen atoms are omitted for clarity. (D) ORTEP drawing with 50% probability for guest b. (E) Structure of 1. B

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diterpenoids, from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) (Figure 2A). Then, GGPP is converted into 1 by a cyclization reaction catalyzed by the TS domain of PcCS (Figure 2B). The cyclization reaction proceeds via the formation of cationic intermediates (GGPP → U → V → W → X → Z → 1). Actually, the cationic intermediate X is also proposed to be formed by the previously characterized PT-TS chimeric enzymes, EvVS and PaPS.5c,i However, in the cases of EvVS and PaPS, X is not converted into 1 via Z. EvVS abstracts a proton from X to produce variediene (2), whereas PaPS converts X to phomopsene (3) via Y.11 This means that X is a bifurcation, which leads to three diterpenoids with different carbon skeletons. We believe an investigation into how these three enzymes control the fate of X is a fascinating research topic for future studies. We also performed tentative assignment of the NMR data of 1, based on the structure deduced by the crystalline sponge method (Figure 1). Consequently, it was revealed that some 13C NMR signals corresponding to the A, B, and C rings of 1 are significantly broadened at 25 °C (Figure 3, Table S2, and Figure

the fact that the homologues of the target gene are well conserved in Penicillium species. Thus, we speculate that the other homologues (Table S1) are also responsible for the production of the cyclopiane-type diterpenes, even though further investigation is required to confirm this hypothesis. Thus far, over 10 cyclopiane-type diterpene derivatives have been isolated (Figure S5). 10 Some of them exhibit potent bioactivities, such as conidiation-induction, cytotoxicity, or antibacterial activity. However, the isolation of their precursor 1 from nature has not been reported, although quite recently, another PT-TS chimera, which also seems to produce 1, was reported in a review article as an unpublished result.3b Thus, the PT-TS chimeric enzyme we found was designated as Penicillium chrysogenum cyclopiane-type diterpene synthase (PcCS) (DDBJ/EMBL/GenBank accession no. LC411963). Notably, the crystalline sponge method also revealed the absolute configuration of 1 as 1R, 4S, 5S, 6S, 9R, 11R, 15R, based on the Flack parameter [0.072(6)]. From these results, the catalytic mechanism of PcCS could be estimated (Figure 2). The PT domain of PcCS first generates geranylgeranyl diphosphate (GGPP), a linear precursor of

Figure 3. (A) Carbon atoms that exhibited broadened NMR signals at 25 °C are shown by red circles. (B) COSY correlations and key HMBC correlations.

S11). However, computational study showed the existence of conformers, which differ mainly in the conformation of the A ring moiety (Figure S32). Thus, the computational study revealed that the skeleton of 1 is not so rigid, and the conformational changes on the A ring of 1 would be a reasonable explanation for the signal broadening. Notably, the A ring of all known cyclopiane-type diterpenes is modified (Figure S5). We speculate that this is the reason why their structures could be determined by NMR analyses in the previous studies.10 The modifications on the A ring, such as the formation of CC and CO double bonds and the insertion of a hydroxyl-group and an epoxide, may contribute to fixing their conformations. In conclusion, we identified PcCS, a novel PT-TS chimeric enzyme, producing 1, by the genome-mining approach. Interestingly, the PT-TS chimeric enzymes, which exhibit high similarity to PcCS, exist in many Penicillium species. Moreover, even though the broadened NMR signals of 1 made the structural determination difficult, the crystalline sponge method achieved the clear elucidation of its complex structure with the 6−5−5−5 fused ring system. Thus, this work has demonstrated that the crystalline sponge method could accelerate genomemining studies, as the structural determination is one of the bottlenecks in this research field. Considering the fact that the genome-mining approach allows us to access cryptic genes with the potential to produce novel compounds, we believe the collaboration between the genome-mining approach and the

Figure 2. (A) Reaction catalyzed by the prenyltransferase domain of PcCS. (B) Reaction catalyzed by the terpene synthase domains of PcCS (GGPP → U → V → W → X → Z → 1). C

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crystalline sponge method will facilitate rapid inquiries into the unexplored chemical space of small molecules.

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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.8b02284. Experimental details and additional figures and tables (PDF) Accession Codes

CCDC 1855333 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_ [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Authors

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

She-Po Shi: 0000-0003-3252-3108 Makoto Fujita: 0000-0001-6105-7340 Ikuro Abe: 0000-0002-3640-888X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. K. Gomi (Tohoku University) and Prof. K. Kitamoto (The University of Tokyo) for providing the fungal heterologous expression system. This work was supported by the JST-ACCEL program in which M.F. is the principle investigator; a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI Grant Number JP16H06443 and JP17KT0095); JST/NSFC Strategic International Collaborative Research Program; and JSPS Research Fellowship for Young Scientists (to T.M.).

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REFERENCES

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DOI: 10.1021/acs.orglett.8b02284 Org. Lett. XXXX, XXX, XXX−XXX