Molecular Mechanism of the Catalytic Function of prenyl Chain Elongating Enzymes Participating in the biosynthesis of Isoprenoid Compounds

在类异戊二烯化合物的生物合成中，所有的碳骨架都是由戊烯基转移酶合成的二磷酸戊烯基。近几十年来，许多催化戊烯基转移酶(E)型戊烯基链延伸的结构基因已被鉴定和表征。然而，直到1998年我们分离出黄体微球菌（Micrococcus luteus）脱戊烯基二磷酸合成酶基因之前，对(Z)-二磷酸戊烯基合成酶的结构一直没有任何了解。(Z)-戊烯基二磷酸合酶的氨基酸序列与(E)-戊烯基链延长酶的氨基酸序列完全不同。在2.2 A分辨率下确定了十一戊烯基二磷酸合酶的晶体结构，是顺戊烯基链延长酶中的第一个三维结构。该酶具有一种与类异戊二烯生物合成酶完全不同的新型蛋白质折叠，具有常见的“类异戊二烯合成酶折叠”结构。根据精确的三维结构和定点诱变实验积累的数据，推导出了该酶的合理底物结合模型。我们分离并鉴定了橡胶树橡胶转移基因的cDNA。在大肠杆菌细胞中过表达重组酶的体外橡胶生产中发现，Hevea橡胶转移酶可能需要在Hevea胶乳的洗底颗粒部分中加入一些活化因子来生产高分子量的橡胶分子。醇在高等生物中作为糖基载体脂类，在n -连接糖蛋白和糖基磷脂酰肌醇锚定蛋白的生物合成中具有重要作用。通过数据库搜索一个编码顺-戊烯基链延长酶高度保守区域的基因，我们确定了一个人类脱氢多酚二磷酸合成酶。通过Northern blotting分析人体组织中脱氢多酚二磷酸合成酶mRNA的表达水平，发现睾丸和肾脏组织中脱氢多酚二磷酸合成酶mRNA表达水平较高。少

In the biosynthesis of isoprenoid compounds all of the carbon backbones are derived from prenyl diphosphates, which are synthesized by the action of prenyltransferases.During the last decades the structural genes for many kinds of prenyltransferases catalyzing (E)-type prenyl chain elongation have been identified and characterized. However, no information has been available about the structures of (Z)-prenyl diphosphate syntheses until our isolation of the gene for Micrococcus luteus undecaprenyl diphosphate synthase in 1998. The amino acid sequence of the (Z)-prenyl diphosphate synthase is totally different from those of (E)-prenyl chain elongating enzymes.The crystal structure of the undecaprenyl diphosphate synthase has been determined at 2.2 A resolution as the first three dimensional structure among cis-prenyl chain elongating enzymes. This enzyme shows a novel protein fold which is completely different from those for the enzymes relating to isoprenoid biosynthesis, having a commo … More n structure "isoprenoid synthase fold". A plausible substrate-binding model of the enzyme has been deduced from the precise three-dimensional structure as well as the data accumulated from site-directed mutagenesis experiments.We have isolated and characterized the cDNA for the rubber transferaes from Hevea brasiliensis. In viro rubber production using the recombinant enzyme overexpressed in E. coli cells revealed that the Hevea rubber transferase might require some activation factors in the washed bottom particle fraction of Hevea latex for the production of high molecular weight rubber molecules.Dolichol has an essential role in the biosynthesis of N-linked glycoproteins and glycosylphosphatidylinositol-anchored proteins as the glycosyl carrier lipid in higher organisms. Through the database search for a gene encoding highly conserved regions for cis-prenyl chain elongating enzymes, we have identified a human dehydrodolichyl diphosphate synthase. The mRNA expression levels of the dehydrodolichyl diphosphate synthase in human tissues were analyzed by Northern blotting to show high expressions in testis as well as in kidney. Less