Studies on isoprenoid biosynthetic genes isolated from prokaryotes

我们从Streptomyces sp.菌株KO-3988中克隆了一个含有4个orf的DNA片段，并通过异源表达实验证实该片段参与维维epinol {3- hydroxyypimara -9(11)，15-diene}的生物合成。在这4个orf中，ORF2和ORF4通过重组酶的实验证实分别编码一个对-共聚二磷酸（CDP）合成酶和一个香叶基香叶基二磷酸（GGDP）合成酶。ORF3与其他已知蛋白没有相似之处，也在大肠杆菌中表达并用于功能分析。纯化后的ORF3产物明显地将- cdp转化为吡玛拉-9(11)，15-二烯。由于ORF2和ORF3是最早从原核生物中发现具有这些生物合成功能的酶，因此对这两种酶的酶学性质进行了研究。ORF2可能是二聚体，其活性需要二价阳离子，如Mg^<2+>和Zn^<2+>。最适pH和温度分别为5.5和35℃。GGDP的Km值为13.7±1.0μM， kcat值为3.3 × 10^<-2>/sec。ORF3很可能是一个单体，也需要一个二价阳离子。最佳pH和温度分别为7.0和30℃。估计ent-CDP的Km值为2.6±0.2μM， kcat值为1.4 × 10^<-3>/sec。

We have cloned a DNA fragment that contained four ORFs and was confirmed to participate in viguiepinol {3-hydroxypimara-9(11),15-diene} biosynthesis by a heterologous expression experiment, from Streptomyces sp.strain KO-3988. Of the four ORFs, ORF2 and ORF4 were confirmed to encode an ent-copalyl diphosphate(CDP) synthase and a geranylgeranyl diphosphate(GGDP) synthase, respectively, by experiments using recombinant enzymes. ORF3,that did not show similarities with any other known proteins was also expressed in E.coli and used for functional analysis. The purified ORF3 product clearly converted ent-CDP into pimara-9(11),15-diene. Since ORF2 and ORF3 are the first examples of enzymes with these biosynthetic functions from prokaryotes, enzymatic properties of both enzymes were investigated. ORF2 is likely to be a dimer and requires a divalent cation such as Mg^<2+> and Zn^<2+> for its activity. The optimum pH and temperature were 5.5 and 35℃. The Km value was calculated to be 13.7±1.0μM for GGDP and the kcat value was 3.3 x 10^<-2>/sec. ORF3 is likely to be a monomer and also requires a divalent cation. The optimum pH and temperature were 7.0 and 30℃. The Km value for ent-CDP was estimated to be 2.6±0.2μM and the kcat value was 1.4 x 10^<-3>/sec.