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

采用巢式RT-PCR技术，从紫草草培养细胞中分离到2个编码4-羟基beazoic acid （4HB）的cdna：参与紫草素生物合成的香叶基转移酶，以4HB为戊烯基受体，以香叶基焦磷酸为戊烯基供体。它们的推定氨基酸序列（命名为LePGT-1和LePGT-2）的一致性为90%，而与酵母中泛素的生物合成酶4HB：六烯基转移酶的一致性仅为38%。与酵母戊烯基转移酶相反，紫草蛋白缺乏线粒体靶向序列。Northern分析表明，这两种cDNA的表达模式与香叶转移酶的表达模式一致，而香叶转移酶的活性受添加铵离子、生长素和茉莉酸甲酯以及强抑制剂光照射的调控。用高效液相色谱法测定了这两个克隆的酶活性，其中4HB：六烯丙烯基转移酶被破坏，在这两个cdna转化子的微粒体部分检测到较强的4HB：香叶基转移酶活性。它们对二磷酸香叶基表现出严格的底物特异性，而线粒体4HB：戊烯基转移酶对二磷酸香叶基表现出更高的偏好。这是植物次生代谢中第一个接受芳香受体的香叶基转移酶。在完整植株中，紫草素衍生物只在根表皮积累，而迷迭香酸等次生代谢产物也定位于皮质。以紫草毛状根为模型材料，通过原位杂交进一步检测LePGTs的表达对紫草素积累的组织特异性。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作为一种关键的生物合成酶，其表达模式在很大程度上决定了紫草素积累的组织特异性。少

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