Molecular discection and structural biology of aromatic substrate prenyltransferase

芳香族底物丙基转移酶广泛存在于细菌和人体中，在参与电子传递的苯醌类化合物的生物合成以及许多植物次生代谢物的生物合成中起着重要的作用。然而，编码这些酶的基因的发现和分子分析几乎没有报道，因为它们大多是膜结合蛋白。在本研究中，我们以对羟基苯甲酸：异丙苯基转移酶为模型，例如参与萘醌生物合成的LePGT和参与泛醌生物合成的Coq2，并试图阐明它们的生化和酶反应机理。首先，我们尝试在晶体结构已明确的可溶性法尼基二磷酸合成酶和膜结合的LePGT之间构建嵌合蛋白，以制备可接受对羟基苯甲酸酯为底物的可溶性异丙烯基转移酶。然而，这项试验没有成功，因为嵌合蛋白没有显示明显的prenyltn…。经过多种不同的方法，我们最终成功地在杆状病毒系统中大量表达了紫草LePGT。该蛋白有9个跨膜α-螺旋，如果将His-Tag连接在C-TERIMUS上，其酶活性不受影响，并从微粒体部分回收表达的融合蛋白，然后用脱氧胆酸钠有效地溶解该融合蛋白。重组蛋白经Ni-琼脂糖柱亲和纯化后，在SDS-PAGE中呈单一条带。我们一直在改进这一方法，用于膜蛋白的结晶实验。我们一直试图通过在LePGT蛋白中引入点突变来确定酶功能的关键氨基酸。这种定点突变研究表明，在几个亲水环之间含有NDxxD基序或YAHQD基序的保守序列对酶活性是重要的，并确定了一些对酶功能至关重要的氨基酸。推测前者负责识别异戊烯基底物，后者负责识别芳香族底物。现在，为了确定底物结合部位，对突变酶的Km值进行了分析。在分析LePGT同源基因酵母Coq2的过程中，发现这种异戊烯基转移酶的高表达使辅酶Q在酵母和植物中的积累增加了一倍。此外，如果将亚细胞定位从线粒体的天然定位改变为内质网定位，则可以观察到更大的增加泛醌产量的效果。较少

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