Construction of plant oxygenase libraries and their functional characterization

本研究的目的是建立植物细胞色素P450单加氧酶、2-酮戊二酸依赖性双加氧酶和黄素单加氧酶的蛋白质文库，并阐明它们的生化功能。(1)植物单加氧酶数据库：从拟南芥基因组数据库中提取了3种单加氧酶（P450/FMO/2 OGD）的基因序列和推导的氨基酸序列。对蛋白质序列进行系统发育分析。此外，从MPSS和TAIR数据库中提取了不同处理下的基因表达数据。(2)P450/FMO/2 OGD的全长cDNA已从RIKEN-BRC中收集，其余克隆的全长ORF已通过RT-PCR方法扩增并分离。毕竟，大约70%的总加氧酶ORF已被分离。将FMOs和2个OGD的开放阅读框（ORF）分别导入表达载体pGEX中， ...更多信息 在E.大肠杆菌中表达，亲和层析纯化。(3)植物P450与P450-还原酶共表达系统的构建：P450的活性需要P450-还原酶的电子传递，而E.大肠杆菌没有还原酶。将pCWori-P450和pACYC-CPR共转化到细菌细胞中，构建了P450 s和CPR的共表达系统。该共表达系统使我们能够在体外和体内检测E.杆菌(4)At 3g 13610的分析：我们先前发现拟南芥根积累了大量的东莨菪素，东莨菪素是香豆素衍生物东莨菪素的β-葡萄糖苷。At 3g 13610是2 OGD基因之一，仅在根中表达，催化阿魏酰辅酶A硫酯邻位羟基化生成东莨菪内酯。因此，At 3g 13610是拟南芥中的阿魏酰辅酶A 6 '-羟化酶，并且这是一种邻位羟化酶，其是在植物中香豆素类生物合成中的关键酶。少

The aim of this study was to establish the protein libraries of plant oxygenases consisiting of cytochrome P450monooxygenases,2-oxoglutarate dependent dioxygenases, and flavin monooxygenases and to elucidate their biochemical functions.(1) Plant Monooxygenase Database: The gene sequences as well as the deduced amino acid sequences of three monooxygenases (P450/FMO/2OGD) were extracted from the genome database of Arabidopsis. The phylogenetic analyses of the protein sequences were performed. In addition, the data of the gene expression in various treatments were extracted from MPSS and TAIR database.(2) Isolation of full ORFs and expression of the recombinant proteins: The full-length cDNAs of P450/FMO/2OGD have been collected from RIKEN-BRC, and the full-ORFs of the other clones were amplified and isolated by RT-PCR approach. After all, about 70% of total oxygenase ORFs have been isolated. The ORFs of FMOs and2OGDs were introduced into the expression vector pETs, and the recombinant pr … More oteins with N-terminal His-tag were expressed in E. coli and were purified with affinity chromatography.(3) Construction of the co-expression system of plant P450s and P450-reductase; P450s require the electron-transfer from P450-reductase (CPR) for the P450 activities, but E. coli does not have the reductase. The co-expression system of P450s and CPR was constructed with the co-transformation of the two plasmids (pCWori-P450 and pACYC-CPR) into the bacterial cells. The co-expression system enabled us to detect P450 activities in in vitro and in vivo assays in E. coli.(4) Analysis of At3g13610: We previously found that Arabidopsis roots accumulate large amounts of scopolin, a β-glucoside of coumarin derivtive scopoletin. At3g13610, which is one of the 2OGD genes and exclusively expressed in roots, was found to catalyze ortho-hydroxylation of feruloyl-CoA thioester to form scopoletin. Thus, At3g13610 is Feruloyl-CoA 6'-hydroxylase in Arabidopsis, and this is an ortho-hydrozylase, which is a key enzyme in coumarins biosynthesis in hiaher plants. Less