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Molecular discection and structural biology of aromatic substrate prenyltransferase

芳香族底物异戊二烯基转移酶的分子解析和结构生物学

基本信息

批准号：
14380286
负责人：
YAZAKI Kazufumi
金额：
$ 7.1万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

Prenyltrransferases accepting aromatic substrates occur in a wide range from bacteria to human, which play important roles in biosyntheses of quinone compounds mediating electron transfer, and also in those of many plant secondary metabolites. However, the discovery of genes encoding these enzymes and molecular analyses of them were hardly reported., because most of them are membrane-bound proteins. In this study, we used p-hydroxybenzoic acid : prenyltransferase as a model, e.g. LePGT involved in naphthoquinone biosynthesis and Coq2 for ubiquinone biosynthesis, and made attempts to elucidate their biochemical and enzymatic reaction mechanism.First we made an attempt to make a chimeric protein between soluble farnesyl diphosphate synthase whose crystal structure was elucidated and the membrane-bound LePGT, to make a soluble prenyltransferase, which accepts p-hydroxybenzoate as the substrate. This trial was, however, unsuccessful because the chimeric proteins did not show clear prenyltn … More rasferase activity, although some of them were highly expressed in E.coli.After many divergent approaches, we finally succeeded to express LePGT of Lithospermum erythrorhizon in the Baculovirus system in an appreciable amount. This protein, which has 9 transmembrane α-helices, was not influenced in the enzymatic activity if His-tag was attached at the C-termimus, and the fusion protein expressed in Sf9 cells were recovered from the microsomal fraction, which was then efficiently solubilized by sodium deoxycholate. This solubilized recombinant protein was affinity-purified with Ni-agarose column to be a single band in the SDS PAGE. We have been improving this protocol for the supply to crystallization experiment of the membrane protein.We have been trying to determine critical amino acids for the enzymatic function by introducing point mutation into LePGT protein. This site-directed mutagenesis study showed that the conserved sequences containing either NDxxD motif or YAHQD motif among several hydrophilic loops are important for the enzymatic activity, and some critical amino acids for the enzymatic function have been determined. It is presumed that the former motif is responsible for prenyl substrate recognition and the latter is for aromatic substrate. Now, the Km values of mutant enzymes are analyzed in order to identify the substrate binding sites.In the process of analyzing the LePGT ortholog, yeast Coq2, it was fond that the high expression of this prenyltransferase increased the accumulation of ubiquinone (Coenzyme Q) to tow fold both in yeast and in plant. Furthermore if the subcellular localization is altered from mitochondria, the native location, to endoplasmic reticulum, higher effect on the increase of ubiquinone production was observed. Less

接受芳香族底物的戊烯基转移酶广泛存在于从细菌到人类的各种生物体内，在介导电子转移的醌类化合物的生物合成中以及许多植物次生代谢产物中发挥着重要作用。然而，编码这些酶的基因的发现和它们的分子分析几乎没有报道。因为它们大多数是膜结合蛋白。在本研究中，我们使用对羟基苯甲酸：以异戊烯基转移酶为模型，如参与萘醌生物合成的LePGT和参与泛醌生物合成的Coq 2，并试图阐明它们的生化和酶促反应机制。首先，我们试图制备可溶性法呢基二磷酸合酶（其晶体结构已被阐明）与膜结合的LePGT之间的嵌合蛋白，以制备可溶性异戊烯基转移酶，其接受对羟基苯甲酸酯作为底物。然而，这项试验是不成功的，因为嵌合蛋白没有显示出明确的异戊二烯基。 ...更多信息经过多次尝试，我们最终成功地在杆状病毒系统中表达了紫草LePGT。该蛋白具有9个跨膜α-螺旋，如果在C-末端连接His-标签，则酶活性不受影响，并且从微粒体组分中回收在Sf 9细胞中表达的融合蛋白，然后用脱氧胆酸钠有效地溶解。可溶性重组蛋白经Ni-琼脂糖柱亲和纯化，SDS PAGE显示单一条带。我们一直在改进该方案，以提供给膜蛋白的结晶实验。我们一直在尝试通过引入点突变LePGT蛋白来确定酶功能的关键氨基酸。该定点突变研究表明，在几个亲水环中含有NDxxD基序或YAHQD基序的保守序列对酶活性是重要的，并且已经确定了一些酶功能的关键氨基酸。据推测，前者的模体是负责异戊二烯基底物的识别，后者是芳香底物。在LePGT的直系同源物酵母Coq 2的分析过程中，发现这种异戊烯基转移酶的高表达使泛醌（辅酶Q）在酵母和植物中的积累增加了两倍。此外，如果亚细胞定位从线粒体（天然位置）改变到内质网，则观察到对泛醌产量增加的更高影响。少