Studies on isoprenoid biosynthetic genes isolated from prokaryotes

我们从链霉菌KO-3988菌株中克隆了一个含有4个ORF的DNA片段，并通过异源表达实验证实其参与了viguiepinol {3-hydroxypimara-9（11），15-diene}的生物合成.在这4个ORF中，通过使用重组酶的实验证实ORF 2和ORF 4分别编码对映-柯巴基二磷酸（CDP）合酶和香叶基香叶基二磷酸（GGDP）合酶。与任何其他已知蛋白没有相似性的ORF 3也在大肠杆菌中表达并用于功能分析。纯化的ORF 3产物清楚地将ent-CDP转化为庚二烯-9（11），15-二烯。由于ORF 2和ORF 3是来自原核生物的具有这些生物合成功能的酶的第一个例子，因此研究了这两种酶的酶性质。ORF 2很可能是二聚体，并且需要二价阳离子如Mg^2+和Zn^2+来实现其活性。最适pH为5.5，最适温度为35℃。计算得出GGDP的Km值为13.7±1.0μM，kcat值为3.3 x 10^<-2>/sec。ORF 3可能是单体，并且还需要二价阳离子。最适pH为7.0，最适温度为30℃。ent-CDP的Km值估计为2.6±0.2μM，kcat值为1.4 x 10^<-3>/秒。

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.