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。先前报道了在大肠杆菌中产生了五种负责甲羟戊酸（MVA）代谢为法呢基二磷酸（FDP）的重组蛋白。大肠杆菌（coli），并利用酵母菌发酵制备的[U-^C_6] MVA大规模生产[U-^C_6] FDP。<13><15><13>为了从[U-^C_2]乙酸酯合成[U-^C_6] MVA和[U-^C_6] GGDP，根据粗糙脉孢菌基因组数据库中的信息，克隆了乙酰辅酶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。<13><13><20><13>这些基因在E.大肠杆菌中表达，重组蛋白在大肠杆菌中以可溶性形式表达。杆菌酶混合物含有四种纯化的蛋白质，ATP，辅酶A，Mg ^&lt;2 +&gt;，NADPH和底物[U-^C_2]乙酸盐（99.9%的^C-标记），产生[U-^C_6] MVA。<13><13><13>[U-^C_6] MVA的GC-MS分析显示全扫描质谱没有稀释天然丰度峰。<13>将该酶混合物与其它由MVA生成FDP和GGDP的酶混合物在适宜的反应条件下，由[U-^C_2]乙酸酯合成[U-^C_2] FDP和[U-^C_2] GGDP，这些标记的异戊烯基二磷酸是分析萜类化合物生物合成基因产物功能的有力探针。<13><15><13><20><13>当[U-14C_] FDP的分子量约为100 μ g/mol时，可得到[U-14C_] FDP的二维~（13）C NMR谱。<13><15>测量1 mg样品。

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.