天蓝色链霉菌中的基因组挖掘：一种新型倍半萜合酶的分子克隆与表征
Genome mining in Streptomyces coelicolor: molecular cloning and characterization of a new sesquiterpene synthase.
作者信息
Lin Xin, Hopson Russell, Cane David E
机构信息
Department of Chemistry, Brown University, Box H, Providence, Rhode Island 02912-9108, USA.
出版信息
J Am Chem Soc. 2006 May 10;128(18):6022-3. doi: 10.1021/ja061292s.
Abstract
The terpene synthase encoded by the SCO5222 (SC7E4.19) gene of Streptomyces coelicolor was cloned by PCR and expressed in Escherichia coli as an N-terminal-His6-tag protein. Incubation of the recombinant protein, SCO5222p, with farnesyl diphosphate (1, FPP) in the presence of Mg(II) gave a new sesquiterpene, (+)-epi-isozizaene (2), whose structure and stereochemistry were determined by a combination of 1H, 13C, COSY, HMQC, HMBC, and NOESY NMR. The steady-state kinetic parameters were kcat 0.049 +/- 0.001 s-1 and a Km (FPP) of 147 +/- 14 nM. Individual incubations of recombinant epi-isozizaene synthase with [1,1-2H2]FPP (1a), (1R)-[1-2H]-FPP (1b), and (1S)-[1-2H]-FPP (1c) and NMR analysis of the resulting deuterated epi-isozizaenes supported an isomerization-cyclization-rearrangement mechanism involving the intermediacy of (3R)-nerolidyl diphosphate (3).
摘要
通过PCR克隆了天蓝色链霉菌SCO5222（SC7E4.19）基因编码的萜烯合酶，并在大肠杆菌中作为N端His6标签蛋白进行表达。在Mg(II)存在下，将重组蛋白SCO5222p与法呢基二磷酸（1，FPP）一起孵育，得到了一种新的倍半萜（+）-表异紫穗槐烯（2），其结构和立体化学通过1H、13C、COSY、HMQC、HMBC和NOESY NMR相结合的方法确定。稳态动力学参数为kcat 0.049±0.001 s-1，Km（FPP）为147±14 nM。将重组表异紫穗槐烯合酶分别与[1,1-2H2]FPP（1a）、（1R）-[1-2H]-FPP（1b）和（1S）-[1-2H]-FPP（1c）一起孵育，并对所得氘代表异紫穗槐烯进行NMR分析，支持了一种涉及（3R）-橙花叔基二磷酸（3）中间体的异构化-环化-重排机制。
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