Enzymatic synthesis of fuly stable isotope-labeled terpenoids.

稳定同位素标记的萜类化合物生物合成中间体将成为研究天然萜类化合物的有力探针。已有报道在大肠杆菌中表达了5种负责甲戊酸(MVA)向法尼基二磷酸(FDP)代谢的重组蛋白，并用酵母发酵法制备了[U-^&lt；13&gt；C_&lt；15&gt；][U-^&lt；13&gt；C_6]FDP。为了从醋酸酯合成MVA，根据脉孢霉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辅酶A合成酶(HMGS)和HMG辅酶A还原酶(HMGR)四种酶的基因。这些基因在大肠杆菌中得到了表达，重组蛋白在大肠杆菌中以可溶性形式表达。含有四种纯化蛋白：ATP、辅酶A、镁离子、NADPH和底物[U-^&lt；13&gt；C2]醋酸酯(99.9%)的酶鸡尾酒产生[U-^&lt；13&gt；C6]MVA。GC-MS分析表明，[U-^&lt；13&gt；C6]MVA的全扫描质谱图没有自然丰度峰的稀释。在合适的反应条件下，将该酶与其他从MVA产生FDP和GGDP的鸡尾酒结合，合成了[U-^&lt；13&gt；C_&lt；15&gt；]FDP和[U-^&lt；13&gt；C_&lt；20&gt；][U-^&lt；13&gt；C2；]GGDP。当测量约1 mg的样品时，我们可以得到[U-^&lt；13&gt；C_&lt；15&gt；]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.