Yamazaki Yasuyo, Kitajima Mariko, Arita Masanori, Takayama Hiromitsu, Sudo Hiroshi, Yamazaki Mami, Aimi Norio, Saito Kazuki
Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
Plant Physiol. 2004 Jan;134(1):161-70. doi: 10.1104/pp.103.029389. Epub 2003 Dec 4.
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.
喜树碱衍生物是临床上使用的抗肿瘤生物碱，属于单萜吲哚生物碱。在本研究中，我们通过计算机模拟和体内实验研究了喜树碱从[1-¹³C]葡萄糖（Glc）的生物合成途径。计算机模拟研究使用代谢原子重建软件测量了Glc掺入生物碱的情况，并预测了[1-¹³C]Glc连续代谢产物的标记模式。体内实验通过¹³C核磁共振光谱跟踪[1-¹³C]Glc掺入到蛇根草毛状根中的喜树碱情况。从毛状根中分离出的喜树碱的¹³C标记模式清楚地表明，单萜类番木鳖苷部分是通过2C-甲基-D-赤藓糖醇4-磷酸途径合成的，而不是通过甲羟戊酸途径。途径特异性抑制剂（磷霉素和洛伐他汀）对喜树碱积累的差异抑制支持了这一结论。色氨酸的喹啉部分也如代谢原子重建程序所预测的那样，通过莽草酸途径被标记。这些结果表明，喜树碱是由2C-甲基-D-赤藓糖醇4-磷酸途径和莽草酸途径结合形成的。本研究为计算机辅助综合代谢分析如何完善实验设计以获得更精确的生物学信息提供了创新范例。
