Enzymatic synthesis of fuly stable isotope-labeled terpenoids.

萜类化合物生物合成中的稳定同位素标记中间体将成为研究天然萜类化合物的有力探针。据报道，在大肠杆菌中产生了5种负责甲羟戊酸（MVA）向法尼酯二磷酸（FDP）代谢的重组蛋白，并将酵母发酵制备的[U-^<13>C_6] MVA大规模生产[U-^<13>C_<15>] FDP。本文报道了以[U-^<13>C_2]醋酸酯为原料，酶促合成[U-^<13>C_6] MVA和[U-^<13>C_<20>] GGDP。为了从乙酸酯合成MVA，基于粗神经孢子虫基因组数据库的信息，克隆了编码乙酰辅酶a合成酶（ACS）、乙酰乙酰辅酶a合成酶（AACS）、HMG-CoA合成酶（HMGS）和HMG-CoA还原酶（HMGR） 4种酶的cdna。这些基因在大肠杆菌中表达，并在大肠杆菌中以可溶性形式产生重组蛋白。由ATP、辅酶A、Mg^<2+>、NADPH和底物[U-^<13>C_2]醋酸酯（99.9% ^<13> c标记）组成的四种纯化蛋白混合酶产生[U-^<13>C_6] MVA。GC-MS分析显示[U-^<13>C_6] MVA的全扫描质谱没有稀释天然丰度峰。将该鸡尾酒酶与其他鸡尾酒酶结合，由MVA合成FDP和GGDP，在适宜的反应条件下，由[U-^<13>C_2]醋酸酯合成[U-^<13>C_<15>] FDP和[U-^<13>C_<20>] GGDP。这些完全标记的二磷酸戊烯基是萜类生物合成基因产物功能分析的有力探针。当测量样品约1 mg时，可以得到[U-^<13>C_<15>] FDP的二维C-13核磁共振谱。

Stable isotope-labeled intermediates in terpenoid biosynthesis will be powerful probes for studies on natural terpenoids. It was previously reported that five recombinant proteins responsible for metabolism from mevalonate (MVA) to farnesyl diphosphates (FDP) were produced in E. coli, and that large scale production of [U-^<13>C_<15>] FDP from [U-^<13>C_6] MVA prepared by fermentation of an yeast was carried out. Here, enzymatic synthesis of [U-^<13>C_6] MVA and [U-^<13>C_<20>] GGDP from [U-^<13>C_2] acetate was reported.To synthesize MVA from acetate, cDNAs encoding four enzymes, acetyl-CoA synthetase (ACS), acetoacetyl-CoA synthase (AACS), HMG-CoA synthase (HMGS) and HMG-CoA reductase (HMGR) were cloned based on the information of genome database of Neurospora crassa. These genes were expressed in E. coli and the recombinant proteins were produced as soluble form in E. coli. Enzyme cocktails containing four purified proteins, ATP, Coenzyme A, Mg^<2+>, NADPH and the substrate [U-^<13>C_2] acetate (99.9% ^<13>C-labeled) produced [U-^<13>C_6] MVA. GC-MS analysis of [U-^<13>C_6] MVA showed the full-scan mass spectrum without dilution of natural abundance peaks. This enzyme cocktail was combined with the other cocktails, which produce FDP and GGDP from MVA, and the combined cocktail synthesized [U-^<13>C_<15>] FDP and [U-^<13>C_<20>] GGDP from [U-^<13>C_2] acetate under the suitable reaction conditions.These fully-labeled prenyl diphosphates is powerful probes for functional analysis of gene products of terpenoid biosynthesis. We could obtain the two-dimensional C-13 NMR spectra of [U-^<13>C_<15>] FDP when ca. 1 mg of the sample was measured.