Hirooka Kazutake, Bamba Takeshi, Fukusaki Ei-ichiro, Kobayashi Akio
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita Yamadaoka 2-1, Osaka 565-0871, Japan.
Biochem J. 2003 Mar 1;370(Pt 2):679-86. doi: 10.1042/BJ20021311.
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 prenylquinones. 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 microM and 1.61 microM 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.
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反应，生成C(30)-C(50)异戊二烯基二磷酸，这些产物是异戊二烯醌侧链的前体。基于目前已鉴定的反式异戊二烯基二磷酸合酶的产物特异性与第一个富含天冬氨酸基序周围区域之间的关系，我们从拟南芥中分离出了一种反式长链异戊二烯基二磷酸合酶成员的cDNA。该cDNA在大肠杆菌中进行了异源表达，对重组的His(6)标签蛋白进行了纯化和特性鉴定。产物分析表明，该cDNA编码茄尼基二磷酸（C(45)）合酶（At-SPS）。At-SPS利用法尼基二磷酸（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)烯丙基二磷酸作为起始底物。FPP和GGPP的米氏常数分别为5.73 microM和1.61 microM。我们还对从拟南芥植物中提取的异戊二烯醌的侧链进行了分析，结果表明其主要的异戊二烯醌，即质体醌和泛醌，含有C(45)异戊二烯基部分。这表明At-SPS可能参与了异戊二烯醌侧链中一种或两种的生物合成。这是首个从多细胞生物体中鉴定出的反式长链异戊二烯基二磷酸合酶。
