Letter pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Insights into Pipecolic Acid Biosynthesis in Huperzia serrata Baofu Xu,†,‡ Zhen Fan,†,‡ Yongxing Lei,†,‡ Yu Ping,†,‡ Amit Jaisi,† and Youli Xiao*,†,‡,§ †
CAS Key Laboratory of Synthetic Biology, CAS Centre for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China ‡ University of Chinese Academy of Sciences, Beijing, 100039, China § CAS-JIC Centre of Excellence in Plant and Microbial Sciences, Shanghai, 200032, China S Supporting Information *
ABSTRACT: For the biosynthesis of Pip in Huperzia serrata, the mechanistic studies were evaluated. Through a series of biochemical analyses, Pip is biosynthesized through a two-step cascade reaction. Three intermediates possibly exist simultaneously as an equilibrium matter in the first-step reaction catalyzed by HsAld1, while HsSard4 performs as a ketimine reductase and chemoselectively and stereoselectively takes 1,2-dehydropipecolic acid as the preferred substrate in vitro.
P
Scheme 1. Proposed Biosynthesis Pathways of Pip
iperidine alkaloids containing a saturated heterocyclic sixmember piperidine ring have been pharmacologically used to treat cancer, mannosidosis, hepatitis C, and Gaucher disease.1 These types of alkaloids are mainly isolated from the plant kingdom,2 especially in the lycopodiaceae plant family, such as Huperzia serrata (H. serrata)3 and are also isolated from some microorganisms (see Figure 1).4,5 Pipecolic
from L-Lys in a single step (Route 1).4b,7d Although genome mining in higher plants for ornithine cyclodeaminase like (OCD-like) genes fitting into Route 1 has been reported, no biochemical activities were observed.8 The mechanistic details of the two-step enzymatic biosynthesis of Pip through 1,6dehydropipecolic acid (1,6-DP, 4) (Route 2) or 1,2dehydropipecolic acid (1,2-DP, 5) (Route 3) as intermediates, have also been investigated (see Scheme 1).6,7 Two genes AtAld1 and AtSard4are involved in Pip biosynthesis in Arabidopsis thaliana (A. thaliana), and they were proposed to fit into Route 3.6 In brief, AtAld1 transfers the α-amino group of LLys to pyruvate or 2-oxoglutarate, following the Schiff base that is spontaneously formed between the residual amino group and the newly produced carbonyl to generate 5, which isomerizes to an enaminic isomer 2,3-DP (6) (see Scheme 1).6c,d Therefore, 6 was proposed as a key intermediate to be directly reduced by AtSard4 to form Pip (Scheme 1, Route 3).6d Noteworthy, Pip was also detected in H. serrata,10a which is a fir moss plant that produces huperzine A (HupA), which is used for the treatment of Alzheimer’s Disease.3a Multiple lycopodium alkaloids that have been isolated from H. serrata are known to contain the Pip core scaffold“the piperidine
Figure 1. Representative medicinal natural products containing a piperidine ring scaffold.
acid (Pip), which is a nonprotein amino acid harboring a piperidine ring, is believed to be the biosynthetic precursor of piperidine-type alkaloids, such as rapamycin from streptomycete,4 swainsonine from fungi,5 and castanospermine from plants3 (see Figure 1). Recently, it has been reported that Pip also participates in plant immunity by acting as a regulator of systemic acquired resistance (SAR).6 Generally, three major biosynthetic routes were proposed for converting L-lysine (LLys) to Pip in organisms (see Scheme 1).7,3c,5b,6d Gatto et al. reported the mechanistic details of RapL, a cyclodeaminase from Streptomyces hygroscopicus, catalyzing the formation of Pip © XXXX American Chemical Society
Received: February 12, 2018
A
DOI: 10.1021/acs.orglett.8b00523 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters ring”. Therefore, Pip can be a promising precursor for the biosynthesis of alkaloids of this family (Figure 1). Given that H. serrata belongs to fir moss, which is much lower than A. thaliana, different strategies for biosynthesis of Pip may be used in H. serrata. To gain a better understanding of the piperidine ring formation in H. serrata, two genes homologous with AtAld1 and AtSard4 were mined from the transcriptome data.10b HsAld1 (Ald1 from H. serrata) and HsSard4 (Sard4 from H. serrata) show 74% and 43% identities with those from A. thaliana, respectively (see Table S1 in the Supporting Information). Moreover, the HsSard4 may also belong to an OCD-like family with a relative high identity (49%) (recall Table S1). Accordingly, Pip possibly is biosynthesized in a similar way, compared to A. thaliana (Route 3), or in a way fitting into Route 1. Moreover, insights into how 6 is formed (regardless of whether it remains in the majority, compared with 5), how Sard4 reduces it, and the absolute stereochemistry have not been well studied previously. To gain insights into piperidine ring biosynthesis in H. serrata, a series of biochemical analyses were performed with the two selected genes. HsAld1 and HsSard4 were cloned, expressed, and purified with an N-terminal His-tag in a heterologous E. coli BL21(DE3) expression system (for details, see Figure S1 in the Supporting Information (SI)). Because of the low expression and poor solubility issues, HsAld1 was further optimized with 45 amino acids truncated in N-terminal, named as Tr45HsAld1. The biochemical activity assays for Tr45HsAld1 and HsSard4 were analyzed by using a derivatization reagent, fluoro-2,4-dinitrobenzene (FDNB), which could convert any primary and secondary amines to the corresponding DNB-derivatized products (see Figure 2A, as well as Scheme S1 in the SI). Since HsSard4 belongs to an OCD-like family (Table S1), the OCD-like reaction was first evaluated to fit into Route 1. However, no signal corresponding to DNB-Pip standard (std) was observed in an OCD-like reaction catalyzed by HsSard4 alone (Figure S2 in the SI) indicating a lack of cyclodeaminase activity and the Route 1 model was excluded. Thereafter, assays fitting into Route 2 or 3 were performed. Compared to the control, no new peak was detected except for the byproduct, DNB-Ala in Tr45HsAld1 assay (trace (2) in Figure 2B). However, if the reaction was carried out with Tr45HsAld1, followed by adding HsSard4/ NADH or NaBH4, a new signal corresponding to DNB-Pip can be detected (see traces (3) and (4) in Figure 2B vs trace (5) in Figure 2B), which indicates the presumed double bond in dehydropipecolic acid isomers (4, 5, or 6), which can be reduced by HsSard4/NADH or NaBH4. Note that 6 was not detected, which is different from that previously reported,6d which may imply that the ε-amino group of L-Lys was eliminated by Tr45HsAld1 (Scheme 1, Routes 2 and 3). Taken together, it can be predicted that Pip may be synthesized through Route 2 by Tr45HsAld1 and HsSard4. To validate the “Route 2” prediction above, the amino group of L-Lys, which was transformed to pyruvate by HsAld1, was investigated. A common chemical reducing reagent, NaBH4, was used in the enzymatic reaction mixtures which can readily reduce molecule harboring in the Schiff base to produce either the corresponding linear amino alcohol11a or ring closure compound.11b Herein, only the ring closure compound was detectable (Figure 2), probably as a result from the pH of the solution being adjusted to ∼10 when chemical reduction was performed. Based on that, we took advantage of NaBD4 to introduce the deuterium atom into the potential dehydropipe-
Figure 2. Analyses of activities of Tr45HsAld1 and HsSard4. (A) The reaction scheme and derivative method. (B) Ion-extracted LC-MS chromatograms of Tr45HsAld1-NaBH4/HsSard4 assays (legend: (1) control (no enzyme), (2) Tr45HsAld1 + L-Lys + pyruvate + PLP, (3) Tr45HsAld1 + L-Lys + pyruvate + PLP + NaBH4, (4) Tr45HsAld1 + HsSard4 + L-Lys + pyruvate + PLP + NADH, and (5) DNB-Ala, DNBPip, and DNB-Lys stds). The [M−H]− m/z values of DNB-Ala, DNBPip, DNB-2,3-DP, and DNB-Lys are calculated as 254.0419, 294.0732, 292.0575, and 477.1012, respectively.
colic acid key intermediates (4 or 5) produced from Tr45HsAld1 reaction. The location of the introduced deuterium atom could resolve the amino transfer question raised above (see Scheme S2 in the SI). Based on the mass spectrometry and NMR analyses, we deduced the number and position of the deuterium atom(s) introduced. The deuterium labeled compound, designated 11a, generated from the Tr45HsAld1−NaBD4 reaction, with a similar molecular polarity of DNB-Pip (11) was purified by silica gel column chromatography. In the 1H NMR spectrum of the purified 11a, the signal at 4.17 ppm was missing, corresponding to the hydrogen of tertiary C-2 position (see traces (1) and (2) in Figure 3A). In addition, the related deuterium signal was detected through 2H NMR analysis (see trace (3) in Figure 3A). High-resolution mass spectrum (HRMS) analysis showed that the [M−H]− m/z value of the 11a was 295.07880, with an increase of 1.00586, compared to the DNB-Pip standard (Figure 3B), indicating that only one deuterium atom was introduced. In addition, the stereochemistry of Pip from HsAld1-NaBD4 assay was also evaluated using the chiral derivatization reagent, N-α-(2,4-dinitro-5-fluorophenyl)-L-valinamide (FDNP-L-Val-NH2, 12) (for details, see the SI).9 The Pip-derivative, DNP-L-Val-NH2-D/L-Pip (13) generated from HsAld1−NaBD4 assay was racemic, compared to D-13 and L13 (see Figure 3C, as well as Figure S3 in the SI) which was consistent with results in Figure 3A (see Route 3′ in Scheme S2). Taken together, 11a was unambiguously DNB-[2-2H]-DLPip (see Figure 3A, as well as Scheme S2). Thus, 5 instead of 4 was directly converted to [2-2H]-DL-Pip through chemical reduction and was derivatized to DNB-[2-2H]-DL-Pip. According to the location of the deuterium atom, which is B
DOI: 10.1021/acs.orglett.8b00523 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
Figure 4. HsSard4 specifically catalyzed 5 as a substrate. (A) Hypotheses of HsSard4-catalyzed reduction. (B) ESI-HRMS of 11 produced in Tr45HsAld1−HsSard4 coupling assay with L-Lys(d9) or (C) L-Lys. Calculated [M−H]− m/z values of deuterated 11c and nondeuterium labeled 11b are 302.1250 and 294.0745, respectively.
select enaminic isomer (6) as the preferred substrate, two hydrogens will be introduced into the pipecolic acid ring in nonexchangeable form (Figure 4A). Through HRMS analysis of product derivative produced by Tr45HsAld1 coupled with HsSard4, we determined that the [M−H]− m/z values of DNBPip derivative 11b or 11c from L-Lys or L-Lys(d9) were 294.0745 or 302.1250, respectively (see Figures 4B and 4C). The mass was increased by 8.0505, which indicated that only one deuterium atom of L-Lys(d9) was lost after it was catalyzed by Tr45HsAld1 and HsSard4. Based on the results, it can be deduced that HsSard4 can reduce ketimine 5 directly to Pip instead of reducing the CC double bond of its enamine isomer (6). Similar assays were done with L-Lys in 90% D2O and no deuterated Pip was detected (see Figure S4D in the SI), which was consistent with the results described above. However, when the assay was done stepwise with NaBH4 as the reducing reagent, H/D exchange of H-3 of Pip (Scheme 1, Route 3) was observed (Figure S4B). In addition, the H/D exchange observed here proceeds in an enzyme-independent and spontaneous manner, based on the results of incubating 5 in D2O (for details, see Figures S5 and S6 in the SI), indicating the dynamic interconversion of intermediates (see Figure S7 in the Supporting Information). Until now, the chirality of Ala and Pip produced from the Ald1-Sard4 coupling assay was still unclear. Thus, we performed analyses on HPLC with chiral derivatization reagent, FDNP-L-Val-NH2.9 It was shown that Tr45HsAld1 can transform pyruvate to L-Ala (see traces (2) and (3) in Figure 5) and HsSard4 can reduce 5 to L-Pip (see trace (3) in Figure 5). However, racemic Pip can be produced through chemical reduction by NaBH4 after Tr45HsAld1 catalysis (see Figure 3C, as well as Figure S3 in the SI). Taken together, the results presented above reveal that Tr45HsAld1 could truly transform the α-amino group of L-Lys to pyruvate to produce L-Ala and 3, and the latter spontaneously forms 5. And possibly, 5 can further isomerize into 6 spontaneously. HsSard4 as a ketimine reductase can then reduce 5, instead of its enamine isomer 6, to produce a single chirality compound: L-Pip (10) (see Scheme 2). Moreover, based on the H/D exchange assays (Figures S4−S7 in the SI), it can be deduced that 3, 5, and 6 possibly exist simultaneously
Figure 3. α-Amino group of L-Lys was transferred into pyruvate by Tr45HsAld1 (see hypothesis in Scheme S2). (A) NMR analyses of DNB-Pip derivatives (legend: (1) 1H NMR of synthesized 11, (2) 1H NMR, and (3) 2H NMR of 11a generated from Tr45HsAld1−NaBD4 coupled assay). (B) ESI-HRMS analyses of DNB-Pip derivatives (legend: (1) ESI-HRMS of synthesized 13, and (2) 11a produced from Tr45HsAld1−NaBD4 coupled assay). (C) Ion-extracted LC-MS chromatograms of DNP-Pip derivatives (legend: (1) 13, obtained by Tr45HsAld1−NaBH4 coupled assay; (2) D-13, and (3) L-13). Calculated [M−H]− m/z value of 13 is 408.1525.
located at C-2 in DNB-[2-2H]-DL-Pip, we deduced that the Schiff base is located at the carbon adjoining carboxyl group (C-2 of Pip, as shown in Scheme 1). Consequently, the αamino group of L-Lys was transferred by Tr45HsAld1 to pyruvate, which was contrary to predictions made based on the nondetection of 6 in common activity analysis (see trace (2) in Figure 2B). Predictably, after the α-amino group of L-Lys was eliminated by Tr45HsAld1, ε-amino-α-ketocaproic acid (KAC, 3) was formed, followed by spontaneously transformation to 5 in a reversible way, according the Schiff base theory. Moreover, 5 was proposed to isomerize into 6 via the action of the AtAld1 protein.6d None of the three intermediates were detected directly in our study through derivatization with FDNB, probably because of the Schiff base molecular form (5), harboring a tertiary amine, remaining in the majority, compared to 3 and 6 in the reaction solution. However, which of them, especially 5 or 6, was selectively catalyzed by HsSard4? To further investigate the selectivity issue, the following HsSard4 enzymatic assay in the presence of a deuterated substrate L-(2,3,3,4,4,5,5,6,6-d9)-lysine (L-Lys(d9)) was carried out. As proposed previously, AtSard4 could reduce 6 instead of 5 to Pip (see Scheme 1, Route 3).6d The deuterium atom of the α-carbon of L-Lys(d9) should be lost after deamination (Figure 4A), and it would continue to lose another one at the C-3 position if it isomerizes into enaminic form. Theoretically, if Sard4 selectively reduces the Schiff base ketimine molecular form (5), only one hydrogen will be introduced into pipecolic acid ring in nonexchangeable form. In contrast, if Sard4 could C
DOI: 10.1021/acs.orglett.8b00523 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
15JC1400402). We thank Dr. Yining Liu, Dr. Yuanhong Shan, and Mr. Shizheng Bu in the Core Facility Centre of SIPPE for mass spectrometry and NMR assistance.
■
Figure 5. Chirality analyses of Ala and Pip produced by Tr45HsAld1 and HsSard4 with LC-MS. Legend: (1) control (no enzymes); (2) Tr45HsAld1 + L-Lys; (3) Tr45HsAld + HsSard4 + L-Lys; (4) DNP-LVal-NH2-L-Pip std; (5) DNP-L-Val-NH2-D-Pip std; (6) DNP-L-ValNH2-L-Ala std; and (7) DNP-L-Val-NH2-D-Ala std. The [M−H]− m/z values of DNP-L-Val-NH2-Ala and DNP-L-Val-NH2−Pip are calculated as 368.1212 and 408.1525, respectively.
Scheme 2. Proposed Mechanism of Piperidine Ring Formation Catalyzed by Tr45HsAld1 and HsSard4
through transformation to each other in equilibrium (see Scheme 2). And probably, 5 is the major molecular form in aqueous solution under physiological condition. In addition, more details about how Tr45HsAld1 transfers amino group into pyruvate and how HsSard4 stereoselectively reduces imine are not well-established. Further detailed relevant mechanistic investigation is currently underway in our laboratory.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b00523. General information, experimental procedures, Figures S1−S8, and Schemes S1 and S2, data tables, NMR spectra, and DNA sequences (PDF)
■
REFERENCES
(1) (a) Seto, B. Clin. Transl. Med. 2012, 1, 29. (b) Dorling, P. R.; Huxtable, C. R.; Colegate, S. M. Biochem. J. 1980, 191, 649. (c) Allan, G.; Ouadid-Ahidouch, H.; Sanchez-Fernandez, E. M.; Risquez-Cuadro, R.; Fernandez, J. M. G.; Ortiz-Mellet, C.; Ahidouch, A. PLoS One 2013, 8, e76411. (d) Sánchez-Fernández, E. M.; Rísquez-Cuadro, R.; Chasseraud, M.; Ahidouch, A.; Mellet, C. O.; Ouadid-Ahidouch, H.; Fernández, J. M. G. Chem. Commun. 2010, 46, 5328. (e) Li, Z.; Li, T.; Dai, S.; Xie, X.; Ma, X.; Zhao, W.; Zhang, W.; Li, J.; Wang, P. G. ChemBioChem 2013, 14, 1239. (2) (a) Pinder, A. R. Nat. Prod. Rep. 1992, 9, 17. (b) Plunkett, A. O. Nat. Prod. Rep. 1994, 11, 581. (c) O’Hagan, D. Nat. Prod. Rep. 1997, 14, 637. (d) O’Hagan, D. Nat. Prod. Rep. 2000, 17, 435. (3) (a) Ma, X.; Gang, D. R. Nat. Prod. Rep. 2004, 21, 752. (b) Liu, Y. C.; Zhang, Z. J.; Su, J.; Peng, L. Y.; Pan, L. T.; Wu, X. D.; Zhao, Q. S. Nat. Prod. Bioprospect. 2017, 7, 405. (c) Bunsupa, S.; Yamazaki, M.; Saito, K. Mini-Rev. Med. Chem. 2017, 17, 1002. (4) (a) Vezina, C.; Kudelski, A.; Sehgal, S. N. J. Antibiot. 1975, 28, 721. (b) Gatto, G. J.; Boyne, M. T.; Kelleher, N. L.; Walsh, C. T. J. Am. Chem. Soc. 2006, 128, 3838. (5) (a) Dewick, P. M. Medicinal Natural Products: A Biosynthetic Approach;John Wiley & Sons: Chichester, West Sussex, U.K., 2002, Vol. 2, pp 291−403. (b) Ren, Z.; Song, R.; Wang, S.; Quan, H.; Yang, L.; Sun, L.; Zhao, B.; Lu, H. J. Microbiol. Biotechnol. 2017, 27, 1897. (6) (a) Návarová, H.; Bernsdorff, F.; Döring, A. C.; Zeier, J. Plant Cell 2012, 24, 5123. (b) Bernsdorff, F.; Döring, A. C.; Gruner, K.; Schuck, S.; Bräutigam, A.; Zeier, J. Plant Cell 2016, 28, 102−129. (c) Ding, P.; Rekhter, D.; Ding, Y.; Feussner, K.; Busta, L.; Haroth, S.; Xu, S.; Li, X.; Jetter, R.; Feussner, I.; Zhang, Y. Plant Cell 2016, 28, 2603. (d) Hartmann, M.; Kim, D.; Bernsdorff, F.; Ajami-Rashidi, Z.; Scholten, N.; Schreiber, S.; Zeier, T.; Schuck, S.; Reichel-Deland, V.; Zeier, J. Plant Physiol. 2017, 174, 124. (e) Sun, T.; Busta, L.; Zhang, Q.; Ding, P.; Jetter, R.; Zhang, Y. New Phytol. 2018, 217, 344. (7) (a) Schütte, H. R.; Seelig, G. Z. Naturforsch. 1967, 22b, 824−826. (b) Gupta, R. N.; Spenser, I. D. J. Biol. Chem. 1969, 244, 88−94. (c) Broquist, H. P. Annu. Rev. Nutr. 1991, 11, 435. (d) Kuo, M. S.; Yurek, D. A.; Mizsak, S. A.; Cialdella, J. I.; Baczynskyj, L.; Marshall, V. P. J. Am. Chem. Soc. 1999, 121, 1763. (e) Walsh, C. T.; O’Brien, R. V.; Khosla, C. Angew. Chem., Int. Ed. 2013, 52, 7098. (8) Sharma, S.; Shinde, S.; Verslues, P. E. BMC Plant Biol. 2013, 13, 182. (9) Bhushan, R.; Kumar, R. Anal. Bioanal. Chem. 2009, 394, 1697. (10) (a) Wu, S.; Fan, Z.; Xiao, Y. Synth. Syst. Biotechnol. 2018, 3, 44. (b) Yang, M.; You, W.; Wu, S.; Fan, Z.; Xu, B.; Zhu, M.; Li, X.; Xiao, Y. BMC Genomics 2017, 18, 245. (11) (a) Dey, R.; Banerjee, P. Org. Lett. 2017, 19, 304. (b) Xu, B.; Lei, L.; Zhu, X.; Zhou, Y.; Xiao, Y. Phytochemistry 2017, 136, 23.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Youli Xiao: 0000-0002-4803-3333 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by CAS (Grant No. XDPB0402 and 153D31KYSB20170121) and SCST (Grant No. D
DOI: 10.1021/acs.orglett.8b00523 Org. Lett. XXXX, XXX, XXX−XXX
