Studies on isoprenoid biosynthetic genes isolated from prokaryotes

我们从链霉菌KO-3988中克隆了一个含有4个ORF的DNA片段，通过异源表达实验证实该片段参与了viguiepinol{3-羟基-9(11)，15-二烯}的生物合成。通过重组酶实验，在4个ORF中，ORF2和ORF4分别编码了一个内酯二磷酸(CDP)合酶和一个香叶基二磷酸(GGDP)合酶。ORF3，与任何其他已知的蛋白没有相似之处，也在大肠杆菌中表达并用于功能分析。纯化的ORF3产物明显地将ent-CDP转化为pimara-9(11)，15-二烯。由于ORF2和ORF3是从原核生物中首次发现具有这些生物合成功能的酶，因此我们对这两种酶的酶学性质进行了研究。ORF2很可能是二聚体，需要二价阳离子，如镁离子和锌离子才能发挥活性。最适pH为5.5，最适温度为35℃。计算出GGDPKm值为13.7±1.0kCAT M，μ值为3.3x10~(-2)/秒。ORF3很可能是单体，也需要二价阳离子。最适pH为7.0，最适温度为30℃。ENT-CDP的Km值为2.6±0.2μM，kcat值为1.4×10~(-3)/s。

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.