Studies on isoprenoid biosynthetic genes isolated from prokaryotes

原核生物类异戊二烯生物合成基因的研究

• 批准号: 16310153
• 负责人: DAIRI Tohru
• 金额: $ 4.8万
• 依托单位: Toyama Prefectural University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16310153/
• 关键词: isoprenoid; cyclase; Actinomycetes; ent-copaly1 diphosphate; pimara-9 (11),15-diene; furaquinocin A; furaquinocin A; copalyl diphosphate; pimara-9(11), 15-diene; KS505a; isoprenoid; cyclase; Actinobacteria; mevalonate pathway

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.

我们从链霉菌菌株 KO-3988 中克隆了包含四个 ORF 的 DNA 片段，并通过异源表达实验证实其参与 viguiepinol {3-羟基pimara-9(11​​),15-二烯}的生物合成。 ORF、ORF2 和 ORF4 被证实编码对映柯巴基二磷酸 (CDP) 合酶和香叶基香叶基通过使用重组酶进行的实验，未显示与任何其他已知蛋白质相似的二磷酸（GGDP）合酶也在大肠杆菌中表达，并用于功能分析。 -9(11​​),15-二烯 由于 ORF2 和 ORF3 是原核生物中具有这些生物合成功能的酶的第一个例子，因此对这两种酶的酶学特性进行了研究。 ORF2可能是二聚体，需要二价阳离子如Mg^2+和Zn^2+来发挥其活性。最适pH和温度为5.5和35℃。计算出的Km值为13.7。 GGDP为±1.0μM且kcat值为3.3×10^-2/秒。ORF3可能是单体并且还需要二价阳离子。最适pH和温度为7.0和30℃。估计ent-CDP的Km值为2.6±0.2μM，kcat值为1.4×10^-3/秒。

项目成果

Presence of copalyl diphosphate synthase gene in an actinomycete possessing the mevalonate pathway

• DOI: 10.7164/antibiotics.57.739
• 发表时间: 2004-11-01
• 期刊: JOURNAL OF ANTIBIOTICS
• 影响因子: 3.3
• 作者: Kawasaki, T;Kuzuyama, T;Dairi, T
• 通讯作者: Dairi, T