Journal of the Korean Applied Science and Technology (한국응용과학기술학회지)

The Korean Society of Applied Science and Technology (한국응용과학기술학회)
권호 표지
DOI QR코드

DOI QR Code

Studies of vindoline metabolism in Catharanthus roseus cell cultures using deuterium-labeled tabersonine

Catharanthus roseus 세포 배양액에 deuterium이 치환된 tabersonine을 사용한 vindoline 생합성 경로 연구

  • Lee, Soo (Department of Chemical Engineering, Changwon National University) ;
  • Lee, Hyang-Yeol (Department of Biotechnology, Korea National University of Transportation)
  • 이수 (창원대학교 화공시스템공학과) ;
  • 이향렬 (한국교통대학교 생명공학과)
  • Received : 2012.02.01
  • Accepted : 2012.03.12
  • Published : 2012.03.30
Download PDF
⟨ Previous Next ⟩

Abstract

Vinca alkaloids produced from Catharanthus roseus are one of the most important natural product drugs in treatments of human cancers. These anticancer drugs are derived from coupling of the two monomeric indole alkaloids, catharanthine and vindoline. In order to investigate vindoline biosynthesis, tabersonine-$CD_3$ 1a is synthesized to use as a deuterium labeled precursor, which is distinguished clearly from the natural counterpart. We show that these deuterium labeled tabersonine 1a are successfully incorporated into the vindoline biosynthetic pathway to yield three deuterated vindoline intermediates. 16-Hydroxytabersonine-$CD_3$ (m/z 356) 2a, 16-Methoxytabersonine-$CD_3$ (m/z 370) 3a, 16-Methoxy-2,3-dihydro-3-hydroxytabersonine-$CD_3$ (m/z 388) 4a are produced from the cell suspension culture measured by UPLC/MS at 5 and 13 days after feeding tabersonine. The conversion rates from 1a to 2a and 2a to 3a are fast, whereas that from 3a to 4a is much slower. This indicates that the rate determining step among the first three vindoline biosynthesis is the last step. As a result of the slow conversion rate from 3a to 4a, the accumulation level of 16-Methoxytabersonine-$CD_3$ 3a is significantly increased up to 13 days. The accumulation ratio among 2a, 3a and 4a is 1, 2 and 0.1 at 5 days. However, the peaks of desacetoxyvindoline-$CD_3$ 5a, deacetylvindoline-$CD_3$ 6a and vindoline-$CD_3$ 7a are not found from the cell extracts even after 13 days of incubation which may indicate no presence of their corresponding enzymes.

Catharanthus roseus로부터 생산되는 빈카알칼로이드는 암을 치료하는 데에 사용되는 가장 중요한 천연물 중의 하나이다. 이러한 항암제는 두 단량체 인돌 알칼로이드인 catharanthine과 vindoline의 결합으로 합성될 수 있다. 이 중 vindoline의 생합성에 관계하는 경로를 조사하기 위해서 tabersonine의 메틸기에 중수소를 치환한 전구체인 tabersonine-$CD_3$ 1a를 합성하였다. 이는 중수소의 질량 증가로 인해 자연에서 발생하는 tabersonine과 뚜렷이 구별될 수 있도록 해 준다. 우리는 이 중소소가 치환된 tabersonine 1a가 성공적으로 vindoline 생합성경로에 편입되어 중수소가 치환된 3개의 새로운 vindoline 중간체(2a, 3a와 4a)를 생성함을 보였다. 세포현탁배양액에 tabersonine-$CD_3$ 1a를 주입한 5일과 13일째에 생성된 대사물인 16-Hydroxytabersonine-$CD_3$ (m/z 356) 2a, 16-Methoxytabersonine-$CD_3$ (m/z 370) 3a와 16-Methoxy-2,3-dihydro-3- hydroxytabersonine-$CD_3$ (m/z 388) 4a의 생성량을 UPLC/MS를 사용하여 측정하였다. 출발물 1a에서 생성물 2a, 그리고 2a에서 3a로의 전환은 빨랐던 반면 3a에서 4a로의 전환은 상대적으로 훨씬 느렸다. 따라서 각 대사물들의 상대적 전환속도를 서로 비교해 보았을 때 vindoline 생합성 과정의 첫 세 단계 중에서 가장 느린 마지막 단계가 속도결정단계임을 암시한다. 즉 대사물 3a에서 4a로의 느린 전환속도의 결과, 배양 후 13일까지도 3a의 축적률이 현저히 증가됨을 보였다. 배양 5일째의 대사물 2a, 3a와 4a의 축적비는 각각 1, 2와 0.1이었다. 그러나 desacetoxyvindoline-$CD_3$ 5a, deacetylvindoline-$CD_3$ 6a와 vindoline-$CD_3$ 7a의 피크는 배양 13일 후에도 발견되지 않아 세포현탁 배양액에 각 생합성단계와 관련된 효소들이 존재하지 않음을 알 수 있었다.

Keywords

References

  1. J. Roepke, V. Salim, M. Wu, A. M. K. Thamm, J. Murata, K. Ploss, W. Boland, and V. De Luca, Vinca drug components accumulate exclusively in leaf exudates of Madagascar periwinkle. PNAS, 107, 15287 (2010).
  2. R. van der Heijden, D. I. Jacobs, W. Snoeijer, D. Hallard and R. Verpoorte, The Catharanthus Alkaloids: Pharmacolognosy and Biotechnology. Current Medicinal Chemistry, 11, 607 (2004).
  3. H-Y. Lee, N. Yerkes, and S.E. O'Connor, Aza-Tryptamine Substrates in Monoterpene Indole Alkaloid Biosynthesis. Chemistry & Biology, 16, 1225 (2009).
  4. S. Mahroug, V. Courdavault, M. Thiersault, B. St-Pierre, V. Burlat, Epidermis is a pivotal site of at least four secondary metabolic pathways in Catharanthus roseus aerial organs. Planta, 223, 1191 (2006).
  5. J. Murata, J. Roepke, H. Gordon, and V. De Luca, The Leaf Epidermome of Catharanthus roseus Reveals It's Biochemical Specialization. The Plant Cell, 20, 524 (2008).
  6. R. Bhadra, S. Vani, and J. V. Shanks, Production of indole alkaloids by selected hairy root lines of Catharanthus roseus. Biotechnology and Bioengineering, 41, 581 (1993).
  7. J. A. Morgan, J. V. Shanks, Inhibitor studies of tabersonine metabolism in C. roseus hairy roots. Phytochemistry, 51, 61 (1999).
  8. S. Auriola, T. Naaranlahti and S. P. Lapinjoki, Synthesis of [methyl-2H]-labelled ajmalicine, yohimbine, tabersonine and catharanthine. Journal of Labelled Compounds and Radiopharmaceuticals, XXIX, 117(1990).
  9. A. Verma, K. Hartonen, and M-L. Riekkola, Optimisation of Supercritical Fluid Extraction of Indole Alkaloids from Catharanthus roseus using Experimental Design Methodology-Comparison with other Extraction Techniques. Phytochemical Analysis, 19, 52 (2008).
  10. B. St-Pierre and V. De Luca, A Cytochrome P-450 Monooxygenase Catalyzes the First Step in the Conversion of Tabersonine to Vindoline in Catharanthus roseus. Plant Physiol., 109, 131 (1995).
  11. E. De Carolis and V. De Luca, Purification, Characterization, and Kinetic Analysis of a 2-Oxoglutarate-dependent Dioxygenase Involved in Vindoline Biosynthesis from Catharantus roseus. The Journal of Biological Chemistry, 268, 5504 (1993).
  12. S. Hisiger and M. Jolicoeur, Analysis of Catharanthus roseus alkaloids by HPLC. Phytochem Rev., 6, 207 (2007).
  13. S. Rodriguez, V. Compagnon, N. P. Crouch, B. St-Pierre and V. De Luca, Jasmonate-induced epoxidation of tabersonine by a cytochrome P-450 in hairy root cultures of Catharanthus roseus. Phytochemistry, 64, 401 (2003).
  14. E. Hernandez-Doninguez, F. Campos- Tamayo, M. Carrillo-Pech, and F. Vazquez- Flota, In Catharanthus roseus Shoot Cultures for the Production of Monoterpenoid Indole Alkaloids; Methods in Molecular Biology: Plant Cell Culture Protocols; Humana Press Inc.: Totowa, NJ, 349 (2005).
  15. D. K. Liscombe, A. R. Usera, and S. E. O'Connor, Homolog of tocopherol C methyltransferases catalyzes N methylation in anticancer alkaloid biosynthesis. PNAS, vol. 107, no. 44, 18793 (2010).

Cited by

  1. Analytical Characterization of Aza-Indole Alkaloids in the Biosynthesis of Catharanthus Roseus vol.29, pp.2, 2012, https://doi.org/10.12925/jkocs.2012.29.2.10