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

两个编码4-羟基唑酸（4HB）的cDNA：与舍氏蛋白生物合成有关的黄烷基转移酶，它们接受4Hb为蛋白基受体，而乙酰磷酸酯作为培养基供体的培养基细胞分离出培养基的RT-PCR从Litherthr的细胞中分离出来。指定的LEPGT-1和LEPGT-2的假定氨基酸序列的识别率为90％，而4HB：六帕烯基转移酶的鉴定是酵母中荧光素酮的生物合成酶，酵母中的鉴定仅为38％。与酵母前转移酶相反，线粒体靶向序列缺乏岩性蛋白质。北部分析表明，两种cDNA的表达模式都符合香精转移酶活性的表达模式，这些cDNA的表达模式通过添加铵离子，生长素和黄甲酸甲酯以及光照射（一种强烈的天形转移酶表达的强抑制剂）来调节。通过HPLC测量了两个克隆的酶活性，并用在ye中表达的重组ptotoin进行了……更多的AST，其4Hb：4Hb：己糖基烯基转移酶被破坏，并且在两种cDNAS的转化剂的微粒馏分中都检测到强的4Hb：Geranylysylansferase活性。他们显示出严格的底物对二磷酸乙酰磷酸盐的特异性，而线粒体4HB：前转移酶表现出更高的偏爱与乙酰磷酸乙酰磷酸的偏爱。这是第一个在植物二次代谢中采用芳香族受体的黄烷基转移酶。在完整的Erythrorhizon的完整植物中，Shikonin衍生物仅在根表皮中积累，而其他次生代谢物也是assmarinic Acid的其他二次代谢。通过原位杂交进一步研究了这种对湿康蛋白丙烯酸的组织特异性，以评估使用岩石质毛毛状根作为模型材料的LEPGTS表达的贡献。结果表明，在表皮和根毛中仅检测到LEPGT-1特异性信号。该数据表明，LEPGT-1的表达模式在很大程度上有助于作为关键的生物合成酶的Shikonin积累的组织特异性。较少的