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

利用巢式RT-PCR技术，从紫草培养细胞中分离到两个编码紫草素生物合成相关的4HB：香叶酰基转移酶的基因，它们接受4HB为前烯受体，香叶基焦磷酸为前烯供体。它们的氨基酸序列同源性分别为LePGT-1和LePGT-2，而与酵母中辅酶Q生物合成酶4HB：六烯基转移酶的同源性仅为38%。与酵母异丙烯基转移酶相反，紫草蛋白缺乏线粒体靶向序列。Northern分析表明，这两个基因的表达模式与香叶酰转移酶活性的表达模式一致，后者受氨离子、生长素和茉莉酸甲酯以及光照射的调节。用Ye…表达的重组蛋白对两个克隆的酶活性进行了测定。其中4HB：六氢叶基转移酶被破坏的较多，在两个转化子的微体组分中都检测到较强的4HB：香叶素转移酶活性。它们对香叶基二磷酸表现出严格的底物专一性，而线粒体4HB：异丙烯基转移酶对香叶基二磷酸表现出更高的选择性。这是第一个在植物次生代谢中作为芳香受体的香叶素转移酶。在完整的红花草属植物中，紫草素的衍生物只在根的表皮中积累，而其他次生代谢产物迷迭香酸也定位在皮层中。以紫草毛状根为模型材料，通过原位杂交进一步验证了紫草素积累的组织特异性，以评估LePGTS的表达对紫草素积累的贡献。结果表明，LePGT-1特异性信号仅在表皮和根毛中检测到。这一数据表明，LePGT-1的表达模式在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。较少

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