木本曼陀罗中一条新的TRI基因克隆与酶活功能鉴定  

Cloning and enzymatic function characterization of a novel tropinone reductase I(DaTRI 2) in Datura arborea

作　　者：强玮 夏科[2] 赵许朋[3] 付维 满建民[1] 张明生[1] QIANG Wei;XIA Ke;ZHAO Xu-peng;FU Wei;MAN Jian-min;ZHANG Ming-sheng(School of Life Sciences/Resources Conservation and Germplasm Innovation in Mountainous Region Ministry of Education,Guizhou University,Guiyang 550025,China;Guangxi Institute of Botany,Chinese Academy of Sciences,Guilin 541006,China;Guiyang University,Guiyang 550005,China)

机构地区：[1]贵州大学生命科学学院/山地植物资源保护与种质创新教育部重点实验室,贵州贵阳550025 [2]中国科学院广西植物研究所,广西桂林541006 [3]贵阳学院,贵州贵阳550005

出　　处：《药学学报》2019年第3期574-581,共8页Acta Pharmaceutica Sinica

基　　金：国家重点研发计划课题(2016YFC0502604);贵州省科技计划重大专项课题(黔科合平台人才[2017]5411-06);贵州省科技创新人才团队建设专项资金(黔科合平台人才[2016]5624);贵州省教育厅创新群体重大研究项目(黔教合KY字[2016]023);贵州大学国家级重点培育项目(黔科合平台人才[2017]5788);贵州大学引进人才科研项目(贵大人基合字[2017]58);贵州省中药材现代产业技术体系建设项目(GZCYTX-02)

摘　　要：托品酮还原酶I(tropinone reductase I, TRI)是托品烷生物碱(tropane alkaloids, TAs)合成途径中游分支点处的关键酶,可引导托品酮代谢流进入TAs合成,因此是TAs代谢工程重要的靶标基因。本研究从木本曼陀罗(Datura arborea)中克隆到了一条新的TRI基因,命名为DaTRI2 (GenBank登录号为MH705164)。DaTRI2基因cDNA全长1 135 bp,与DaTRI序列一致性为96.8%,预测编码272个氨基酸。DaTRI2蛋白具备茄科TRI保守的结合NADPH的TGXXXGXG基序、结合底物托品酮的11个保守氨基酸残基以及发挥催化活性的N-S-Y-K四联体基序。进化关系上, DaTRI2和茄科的TRI成员聚为一支,与Datura属的TRI亲缘关系最近。对DaTRI2进行原核表达,纯化的重组蛋白能催化托品酮还原反应和托品氧化反应,最适pH值分别为8.0和9.6。DaTRI2在pH=6.4时,对托品酮的Km和Vmax分别为210.05μmol·L^(-1)和69.6 nkat·mg^(-1) protein,在pH=9.6时,对托品的K_m和V_(max)分别为188.03μmol·L^(-1)和114 nkat·mg^(-1) protein。qPCR检测表明DaTRI2在幼叶中表达量最高,其次是须根。DaTRI2基因的克隆和酶活力分析为深入研究木本植物中TAs的生物合成分子机制奠定了基础,同时为TAs代谢工程提供了一个更高效的候选靶基因。Tropinone reductase I(TRI) is a key branch point enzyme in the midstream of tropane alkaloids(TAs) biosynthesis pathway and represents an important target for TAs metabolic engineering, which can lead to metabolic flux of substrate tropinone to TAs. A novel TRI gene was isolated from Datura arborea, a woody resource plant, and designated as DaTRI2(GenBank accession number is MH705164). The full-length cDNA of DaTRI2 with 1 135 bp exhibits a high sequence homology(96.8%) with DaTRI, and is predicted to encode a protein of 347 amino acids. Deduced DaTRI2 protein contain a conserved TGXXXGXG motif involved in NADPH binding, the catalytic N-S-Y-K tetrad motif and eleven amino acid residues important for binding to its substrate tropinone. The phylogenetic analysis revealed that DaTRI2 and other TRIs from Solanaceous plants belong to the same cluster and DaTRI2 exhibited closest phylogenetic proximity to TRIs from Datura. DaTRI2 was expressed in E. coli and the purified recombinant protein can catalyze both tropinone reduction and tropine oxidation with an optimum pH value of 8.0 and 9.6, respectively. When tropinone was used as the substrate, the Km and Vmaxvalues of DaTRI2 at pH 6.4 were 210.05 μmol · L^-1 and 69.6 nkat · mg^-1 protein respectively, while the Km and Vmax values for tropine as the substrate were 188.03 μmol · L^-1 and 114 nkat · mg^-1 protein respectively, at pH 9.6.DaTRI2 transcript was most abundant in the young leaf, followed by the root. Cloning of DaTRI2 gene and biochemical analysis of recombinant DaTRI2 facilitate further research on the molecular mechanism on TAs biosynthesis in woody plants and provide a more potent candidate for TAs metabolic engineering.

关 键 词：托品酮还原酶 木本曼陀罗 托品烷生物碱 酶反应动力学 

分 类 号：R931[医药卫生—生药学]