Functional characterization of OsPPT1, which encodes p-hydroxybenzoate polyprenyltransferase involved in ubiquinone biosynthesis in Oryza sativa
Functional characterization of OsPPT1, which encodes p-hydroxybenzoate polyprenyltransferase involved in ubiquinone biosynthesis in Oryza sativa
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DOI：
10.1093/pcp/pcj025
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发表时间：
2006-05-01
影响因子：
4.9
通讯作者：
Yazaki, Kazufumi
中科院分区：
文献类型：
--
作者：
Ohara, Kazuaki;Yamamoto, Kyoko;Yazaki, Kazufumi
芳香族中间体对羟基苯甲酸酯(PHB)的预酯化反应是生物合成泛醌(UQ)的关键步骤。催化这种戊烯基化反应的酶是对羟基苯甲酸聚戊烯基转移酶(PPT)，它用聚戊烯基二磷酸合成酶提供的戊烯基链取代PHB上的芳香质子。水稻基因组包含3个与酵母PPT(COQ2基因)高度相似的PPT候选基因，通过逆转录-聚合酶链式反应(RT-PCR)分离到与COQ2相似性最高的水稻基因，命名为OsPPT1a。OsPPT1a推导的氨基酸序列在N端含有一个可能的线粒体分选信号，并含有PPT蛋白家族成员典型的底物结合位点的保守结构域。基于使用绿色荧光蛋白-PPT融合的研究，OsPPT1a蛋白的亚细胞定位主要在线粒体中。酵母互补研究表明，OsPPT1a的表达成功地恢复了coq2突变体的生长缺陷。利用重组蛋白进行的戊烯基转移酶检测表明，OsPPT1a可以接受不同链长的异戊烯基二磷酸作为异戊烯基供体，而芳香底物PHB作为异戊烯基受体具有严格的底物专一性。二磷酸香叶酯和PHB的表观K值分别为59.7和6.04亩M。还研究了OsPPT1a和COQ2对二价阳离子的需求，发现Mg2+产生的酶活力最高。Northern分析表明，OsPPT1a基因在水稻各组织中均有表达。这些结果表明，OsPPT1a是一种参与水稻UQ生物合成的功能性PPT。
Prenylation of the aromatic intermediate p-hydroxybenzoate (PHB) is a critical step in ubiquinone (UQ) biosynthesis. The enzyme that catalyzes this prenylation reaction is p-hydroxybenzoate polyprenyltransferase (PPT), which substitutes an aromatic proton at the inposition of PHB with a prenyl chain provided by polyprenyl diphosphate synthase. The rice genome contains three PPT candidates that share significant similarity with the yeast PPT (COQ2 gene), and the rice gene showing the highest similarity to COQ2 was isolated by reverse transcription-PCR and designated OsPPT1a. The deduced amino acid sequence of OsPPT1a contained a putative mitochondrial sorting signal at the N-terminus and conserved domains for putative substrate-binding sites typical of PPT protein family members. The subcellular localization of OsPPT1a protein was shown to be mainly in mitochondria based on studies using a green fluorescent protein-PPT fusion. A yeast complementation study revealed that OsPPT1a expression successfully recovered the growth defect of the coq2 mutant. A prenyltransferase assay using recombinant protein showed that OsPPT1a accepted prenyl diphosphates of various chain lengths as prenyl donors, whereas it showed strict substrate specificity for the aromatic substrate PHB as a prenyl acceptor. The apparent K. values for geranyl diphosphate and PHB were 59.7 and 6.04 mu M, respectively. The requirement by OsPPT1a and COQ2 for divalent cations was also studied, with Mg2+ found to produce the highest enzyme activity. Northern analysis showed that OsPPT1a mRNA was accumulated in all tissues of O. sativa. These results suggest that OsPPT1a is a functional PPT involved in UQ biosynthesis in O. sativa.