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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Biosynthesis of Camptothecin. In Silico and in Vivo Tracer Study from [1-¹³C]Glucose¹

Graduate School of Pharmaceutical Sciences, Chiba University, 1–33 Yayoi-cho, Inage-ku, Chiba 263–8522, Japan (Y.Y., M.K., H.T., H.S., M.Y., N.A., K.S.); and Computational Biology Research Center, National Institute of Advanced Industrial Science and Technology and Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, 2–41–6 Aomi, Koto-ku, Tokyo 135–0064, Japan (M.A.)

Camptothecin derivatives are clinically used antitumor alkaloids that belong to monoterpenoid indole alkaloids. In this study, we investigated the biosynthetic pathway of camptothecin from [1-¹³C]glucose (Glc) by in silico and in vivo studies. The in silico study measured the incorporation of Glc into alkaloids using the Atomic Reconstruction of Metabolism software and predicted the labeling patterns of successive metabolites from [1-¹³C]Glc. The in vivo study followed incorporation of [1-¹³C]Glc into camptothecin with hairy roots of Ophiorrhiza pumila by ¹³C nuclear magnetic resonance spectroscopy. The ¹³C-labeling pattern of camptothecin isolated from the hairy roots clearly showed that the monoterpene-secologanin moiety was synthesized via the 2C-methyl-D-erythritol 4-phosphate pathway, not via the mevalonate pathway. This conclusion was supported by differential inhibition of camptothecin accumulation by the pathway-specific inhibitors (fosmidomycin and lovastatin). The quinoline moiety from tryptophan was also labeled as predicted by the Atomic Reconstruction of Metabolism program via the shikimate pathway. These results indicate that camptothecin is formed by the combination of the 2C-methyl-D-erythritol 4-phosphate pathway and the shikimate pathway. This study provides the innovative example for how a computer-aided comprehensive metabolic analysis will refine the experimental design to obtain more precise biological information.

¹ This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology (Japan; Grants-in-Aid for Scientific Research), by Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation (JST), and by Research for the Future Program (grant no. 00L01605; "Molecular Mechanisms on Regulation of Morphogenesis and Metabolism Leading to Increased Plant Productivity").

² Present address: Phenomenome Discoveries Inc., 204–407 Downey Road, Saskatoon, Saskatchewan, Canada S7N 4L8.

³ Present address: Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, 5–1–5, Kashiwanoha, Kashiwa-shi, Chiba 277–8562, Japan.

^* Corresponding author; e-mail ksaito{at}p.chiba-u.ac.jp; fax 81–43–290–2905.

Received July 1, 2003; returned for revision August 27, 2003; accepted September 21, 2003.

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