Construction of plant oxygenase libraries and their functional characterization

本研究旨在建立由细胞色素p450单加氧酶、2-氧戊二酸依赖的双加氧酶和黄素单加氧酶组成的植物加氧酶的蛋白文库，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3种单加氧酶（P450/FMO/2OGD）的基因序列及推导出的氨基酸序列。对蛋白序列进行系统发育分析。此外，从MPSS和TAIR数据库中提取不同处理下的基因表达数据。(2)完整orf的分离和重组蛋白的表达：从RIKEN-BRC中收集P450/FMO/2OGD的全长cdna，并通过RT-PCR方法扩增和分离其他克隆的完整orf。毕竟，大约70%的加氧酶orf已经被分离出来。将FMOs和2ogds的orf导入到表达载体pet中，在大肠杆菌中表达了更多带有n端His-tag的蛋白，并通过亲和层析纯化。(3)植物p450与p450还原酶共表达系统的构建；P450需要P450还原酶（CPR）的电子转移才能产生P450活性，但大肠杆菌不具有这种还原酶。将pCWori-P450和pACYC-CPR两种质粒共转化到细菌细胞中，构建p450与CPR共表达体系。该共表达系统使我们能够在体外和体内检测P450在大肠杆菌中的活性。(4) At3g13610的分析：我们之前发现拟南芥根部积累了大量的东莨菪碱，这是香豆素衍生物东莨菪碱的β-糖苷。At3g13610是2OGD基因之一，仅在根中表达，被发现催化阿魏酰辅酶a硫酯的邻羟基化形成东莨菪碱。因此，At3g13610是拟南芥中的阿魏酰辅酶a 6′-羟化酶，是高等植物中香豆素生物合成的关键酶。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