Cloning and characterization of a norbelladine 4'-O-methyltransferase involved in the biosynthesis of the Alzheimer's drug galanthamine in Narcissus sp. aff. pseudonarcissus.
Cloning and characterization of a norbelladine 4'-O-methyltransferase involved in the biosynthesis of the Alzheimer's drug galanthamine in Narcissus sp. aff. pseudonarcissus.
复制标题
DOI：
10.1371/journal.pone.0103223
复制
发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Kutchan TM
中科院分区：
文献类型：
--
作者：
Kilgore MB;Augustin MM;Starks CM;O'Neil-Johnson M;May GD;Crow JA;Kutchan TM
加兰他敏是一种用于治疗阿尔茨海默病症状的amarylliaceae生物碱。该化合物主要从水仙花（Narcissus spp.）、雪花莲（Galanthus spp.）和夏季雪花（Leucojum aestivum）中分离得到。尽管它作为一种重要的药物，没有基因参与加兰他明的生物合成途径已确定。缺乏生物合成途径的遗传信息是许多重要植物药物合成生物学平台发展的限制因素。信息的缺乏很大程度上是由于在非模式生物中寻找生化途径酶和基因的传统方法的局限性。一种新的生物信息学方法使用了最近的一些技术改进来搜索加兰他明生物合成途径中的基因，首先针对甲基转移酶，因为这些蛋白中有很强的特征氨基酸序列。利用Illumina测序技术，构建了水仙花转录组的从头组装。BLAST用于鉴定该转录组中含有植物o -甲基转移酶特征的序列。然后使用HAYSTACK程序识别符合加兰他明在叶片、球茎和花序组织中生物合成模型的甲基转移酶。在加兰他明生物合成途径中，鉴定了一个将去甲贝拉定甲基化为4′- o -甲基去甲贝拉定的候选基因。该甲基转移酶cDNA在大肠杆菌中表达，并通过亲和层析纯化。结果发现该蛋白是加兰他明生物合成途径的去甲颠定4′- o -甲基转移酶（NpN4OMT）。
Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer’s disease. This compound is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and summer snowflake (Leucojum aestivum). Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4′-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4′-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway.
登录
查看更多内容
影响因子：
5.1
作者：
GABRIELSEN, B;MONATH, TP;PHELAN, MJ
通讯作者：
PHELAN, MJ
DOI：
10.1039/jr9630004545
发表时间：
1963-01-01
期刊：
JOURNAL OF THE CHEMICAL SOCIETY
影响因子：
--
作者：
BARTON, DHR;TAYLOR, JB;THOMAS, GM
通讯作者：
THOMAS, GM
影响因子：
--
作者：
Choi-Rhee, EJ;Cronan, JE
通讯作者：
Cronan, JE
影响因子：
3.8
作者：
GERARDY, R;ZENK, MH
通讯作者：
ZENK, MH
影响因子：
3.8
作者：
Eichhorn, J;Takada, T;Zenk, MH
通讯作者：
Zenk, MH