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。

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680589/
• 关键词: Lithospermum erythororhizon; prenyltransferase; 4-hydroxybenzoic acid; geranyl diphosphate; shikonin; secondary metabolism; dark-induction; yeast; 4-hydroxybenzoic acid; 小胞体局在

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

利用巢式RT-PCR技术从紫草细胞中分离到2个编码4-羟基苯甲酸（4-hydroxybeazoic acid，4 HB）的cDNA，这2个基因分别接受4-羟基苯甲酸（4 HB）作为异戊烯基受体和香叶基焦磷酸（geranylpyrophosphate）作为异戊烯基供体。LePGT-1和LePGT-2的氨基酸序列同源性为90%，而与酵母中泛醌生物合成酶4 HB：hexaprenyltransferase的同源性仅为38%。与酵母异戊烯基转移酶相反，紫草蛋白中缺乏线粒体靶向序列。北方分析表明，这两个cDNA的表达模式是一致的香叶基转移酶的活性，这是通过添加铵离子，生长素，茉莉酸甲酯，以及光照，香叶基转移酶表达的强抑制剂调节。用高效液相色谱法测定两个克隆的酶活性， ...更多信息 4HB：六异戊二烯基转移酶被破坏，并且在两种cDNA的转化体的微粒体部分中检测到强的4 HB：香叶基转移酶活性。它们对香叶基二磷酸表现出严格的底物特异性，而线粒体4 HB：异戊二烯基转移酶对香叶基香叶基二磷酸表现出更高的偏好作为底物。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。紫草素、紫草素衍生物仅在根表皮中积累，而其它次级代谢产物如迷迭香酸也定位于皮层中。以紫草毛状根为模型材料，通过原位杂交进一步研究紫草素积累的组织特异性，以评估LePGTs表达的贡献。结果表明，LePGT-1特异性信号仅在表皮和根毛中检测到。这些数据表明，LePGT-1的表达模式在很大程度上有助于紫草素积累的组织特异性作为一个关键的生物合成酶。少

项目成果

K.Yazaki: "A novel Coptis japonica multidrug resistant protein preferentially expressed in the alkaloid-accumulating rhizome"J. Exp. Botany. 52. 877-879 (2001)

K.Yazaki：“一种新型黄连多重耐药蛋白，优先在生物碱积累的根茎中表达”J.

H.Hayashi: "Molecular cloning and characterization of isomultiflorenol synthase, a new triterpene synthase from Luffa cylindrica involved in bryonolic acid"Eur. J. Biochem.. 268. 6311-6317 (2001)

H.Hayashi：“异多花酚合酶的分子克隆和表征，一种来自丝瓜的新型三萜合酶，涉及苔藓酸”Eur。

H.Yamamoto: "Enhancement of secondary metabolite productions in various plant cell cultures by co-cultivation with cork"Biosci. Biotech. Biochem.. 65・4. 853-860 (2001)

H. Yamamoto：“通过与软木共培养增强各种植物细胞培养物中的次级代谢产物”Biosci.Biochem.. 853-860（2001）。

H. Yamamoto: "Enhancement of secondary metabolite productions in various plant cell cultures by co-cultivation with cork"Biosci. Biotech. Biochem.. 65 (4). 853-860 (2001)

H. Yamamoto：“通过与软木塞共培养增强各种植物细胞培养物中次生代谢产物的产生”Biosci。

K.-B.Choi: "Molecular cloning and characterization of coclaurine N-methyltransferase from cultured cells of Coptis japonica"J. Biol. Chem.. 277・1. 830-835 (2002)

K.-B.Choi：“黄连培养细胞中的乌药碱 N-甲基转移酶的分子克隆和表征”J. Biol. 277・1 (2002)。