棒状链霉菌的基因组挖掘：两种新型隐秘倍半萜合酶的表达及生化特性分析
Genome mining in Streptomyces clavuligerus: expression and biochemical characterization of two new cryptic sesquiterpene synthases.
作者信息
Hu Yunfeng, Chou Wayne K W, Hopson Russell, Cane David E
机构信息
Department of Chemistry, Brown University, Box H, Providence, RI 02912-9108, USA.
出版信息
Chem Biol. 2011 Jan 28;18(1):32-7. doi: 10.1016/j.chembiol.2010.11.008.
Two presumptive terpene synthases of unknown biochemical function encoded by the sscg_02150 and sscg_03688 genes of Streptomyces clavuligerus ATCC 27074 were individually expressed in Escherichia coli as N-terminal-His₆-tag proteins, using codon-optimized synthetic genes. Incubation of recombinant SSCG_02150 with farnesyl diphosphate (1, FPP) gave (-)-δ-cadinene (2) while recombinant SSCG_03688 converted FPP to (+)-T-muurolol (3). Individual incubations of (-)-δ-cadinene synthase with [1,1-²H₂]FPP (1a), (1S)-[1-²H]-FPP (1b), and (1R)-[1-²H]-FPP (1c) and NMR analysis of the resulting samples of deuterated (-)-δ-cadinene supported a cyclization mechanism involving the intermediacy of nerolidyl diphosphate (4) leading to a helminthogermacradienyl cation 5. Following a 1,3-hydride shift of the original H-1(si) of FPP, cyclization and deprotonation will give (-)-δ-cadinene. Similar incubations with recombinant SSCG_03688 supported an analogous mechanism for the formation of (+)-T-muurolol (3), also involving a 1,3-hydride shift of the original H-1(si) of FPP.
利用密码子优化的合成基因，将来自克拉维链霉菌ATCC 27074的sscg_02150和sscg_03688基因编码的两个生化功能未知的假定萜烯合酶分别作为N端带有His₆标签的蛋白在大肠杆菌中表达。重组的SSCG_02150与法尼基二磷酸（1，FPP）孵育产生（-）-δ-杜松烯（2），而重组的SSCG_03688将FPP转化为（+）-T-穆罗醇（3）。将（-）-δ-杜松烯合酶分别与[1,1-²H₂]FPP（1a）、（1S）-[1-²H]-FPP（1b）和（1R）-[1-²H]-FPP（1c）孵育，并对所得的氘代（-）-δ-杜松烯样品进行NMR分析，支持了一种环化机制，该机制涉及橙花叔基二磷酸（4）中间体，生成蠕孢二烯基阳离子5。FPP原来的H-1（si）发生1,3-氢迁移后，环化和去质子化将产生（-）-δ-杜松烯。对重组的SSCG_03688进行类似的孵育，支持了（+）-T-穆罗醇（3）形成的类似机制，该机制也涉及FPP原来的H-1（si）的1,3-氢迁移。
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