Structural and functional analyses of prenyltransferase accepting aromatic prenyl acceptor as the substrate.

以芳香族异戊二烯受体为底物的异戊二烯基转移酶的结构和功能分析。

基本信息

批准号：
12680589
负责人：
YAZAKI Kazufumi
金额：
$ 1.86万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Two cDNAs encoding 4-hydroxybeazoic acid (4HB) : geranyltransferase involved in shikonin biosynthesis, which accept 4HB as the prenyl acceptor and geranylpyrophosphate as the prenyl donor, were isolated by nested RT-PCR from cultured cells of Lithospermum erythrorhizon. The identity of the putative amino acid sequences of them, designated LePGT-1 and LePGT-2, was 90 %, whereas the identities with the 4HB : hexaprenyltranseferase, a biosynthetic enzyme of ubiquinone in yeast, was only 38 %. Contrary to the yeast prenyltransferase, the mitochondrial targeting sequence is lacking in the Lithospermum proteins. Northern analyses showed that the expression pattern of both cDNA is in conformity with that of geranyltransferase activity which are regulated by addition of ammonium ion, auxin, and methyl jasmonate, as well as light irradiation, a strong inhibitor of geranyltransferase expression. The enzyme activity of both clones were measured by HPLC with the recombinant ptotein expressed in ye … More ast whose 4HB : hexaprenyltranseferase was disrupted, and a strong 4HB : geranyltransferase activity was detected in the microsomal fractions of the transformants for both cDNAs. They showed strict substrate specificity to geranyl diphosphate, while the mitochondrial 4HB : prenyltransferase showed higher preference to geranylgeranyl diphosphate as the substrate. This is the first geranyltransferase which takes an aromatic acceptor in plant secondary metabolism.In the intact plant of L. erythrorhizon, shikonin derivatives are exclusively accumulated in the root epidermis, whereas the other secondary metabolites as rosmarinic acid are also localized in cortex. This tissue specificity for shikonin accumulation was further examined by in situ hybridization to assess the contribution of LePGTs' expression using Lithospermum hairy root as a model material. The results showed that LePGT-1-specific signal was solely detected in the epidermis and root hairs. This data indicates that the expression pattern of LePGT-1 largely contribute to the tissue specificity of shikonin accumulation as a key biosynthetic enzyme. Less

通过巢式RT-PCR从紫草培养细胞中分离出编码4-羟基苯甲酸(4HB)：参与紫草素生物合成的香叶基转移酶的两个cDNA，其接受4HB作为异戊二烯基受体和香叶基焦磷酸作为异戊二烯基供体。它们的氨基酸序列被命名为LePGT-1和LePGT-2， 90%，而与酵母中泛醌生物合成酶 4HB 的同一性仅为 38%。与酵母异戊二烯转移酶相反，Northern 分析表明，紫草蛋白中缺乏线粒体靶向序列。两种cDNA都与通过添加铵离子调节的香叶基转移酶活性一致，生长素和茉莉酸甲酯，以及光照射，香叶基转移酶表达的强抑制剂。通过 HPLC 测量了两个克隆的酶活性，其中 ye … 更多 ast 中表达的重组蛋白的 4HB ：六戊二烯基转移酶被破坏，并且具有强 4HB。：在转化子的微粒体级分中检测到两种 cDNA 的香叶基转移酶活性。对香叶基二磷酸表现出严格的底物特异性，而线粒体4HB：异戊二烯基转移酶对香叶基香叶基二磷酸作为底物表现出更高的偏好性，这是植物次生代谢中第一个以芳香受体为受体的香叶基转移酶。在红草完整植物中，紫草素衍生物。仅在根表皮中积累，而其他次生代谢物如迷迭香酸也集中在皮层中。使用紫草毛状根作为模型材料，通过原位杂交进一步检查了紫草素积累的组织特异性，以评估 LePGT 表达的贡献。结果表明，仅在表皮和根毛中检测到 LePGT-1 特异性信号。该数据表明，LePGT-1 的表达模式很大程度上有助于紫草素作为关键生物合成酶积累的组织特异性。