Cloning and kinetic characterization of Arabidopsis thaliana solanesyl diphosphate synthase
Cloning and kinetic characterization of Arabidopsis thaliana solanesyl diphosphate synthase
复制标题
DOI：
10.1042/bj20021311
复制
发表时间：
2003-03-01
影响因子：
4.1
通讯作者：
Kobayashi, A
中科院分区：
文献类型：
--
作者：
Hirooka, K;Bamba, T;Kobayashi, A
反式长链异戊二烯基二磷酸合酶催化异戊烯基二磷酸 (C-5) 单元与烯丙基二磷酸的顺序缩合，产生 C-30-C-50 异戊二烯基二磷酸酯，它是异戊二烯基醌侧链的前体。基于迄今为止表征的反式异戊二烯基二磷酸合酶中第一个富含天冬氨酸的基序周围区域之间的关系，我们从拟南芥中分离出了反式长链异戊二烯基二磷酸合酶成员的cDNA。该cDNA在大肠杆菌中异源表达，并对重组His(6)标签蛋白进行纯化和表征。产品分析表明，该 cDNA 编码茄尼基二磷酸 (C-45) 合酶 (At-SPS)。 At-SPS 使用法呢基二磷酸（FPP；C-15）和香叶基香叶基二磷酸（GGPP；C-20），但不接受 C-5 或 C-10 烯丙基二磷酸作为引物底物。 FPP和GGPP的米氏常数分别为5.73μM和1.61μM。我们还对从拟南芥植物中提取的异戊二烯醌侧链进行了分析，结果表明其主要异戊二烯醌，即质体醌和泛醌，含有 C-45 异戊二烯基部分。这表明 At-SPS 可能致力于任一或两个异戊二烯醌侧链的生物合成。这是第一个从多细胞生物体中建立的反式长链异戊二烯基二磷酸合酶。
trans-Long-chain prenyl diphosphate synthases catalyse the sequential condensation of isopentenyl diphosphate (C-5) units with allylic diphosphate to produce the C-30-C-50 prenyl diphosphates, which are precursors of the side chains of prenyl-quinones. Based on the relationship between product specificity and the region around the first aspartate-rich motif in trans-prenyl diphosphate synthases characterized so far, we have isolated the cDNA for a member of trans-long-chain prenyl diphosphate synthases from Arabidopsis thaliana. The cDNA was heterologously expressed in Escherichia coli, and the recombinant His(6)-tagged protein was purified and characterized. Product analysis revealed that the cDNA encodes solanesyl diphosphate (C-45) synthase (At-SPS). At-SPS utilized farnesyl diphosphate (FPP; C-15) and geranylgeranyl diphosphate (GGPP; C-20), but did not accept either the C-5 or the C-10 allylic diphosphate as a primer substrate. The Michaelis constants for FPP and GGPP were 5.73 muM and 1.61 muM respectively. We also performed an analysis of the side chains of prenylquinones extracted from the A. thaliana plant, and showed that its major prenylquinones, i.e. plastoquinone and ubiquinone, contain the C-45 prenyl moiety. This suggests that At-SPS might be devoted to the biosynthesis of either or both of the prenylquinone side chains. This is the first established trans-long-chain prenyl diphosphate synthase from a multicellular organism.