Complementation of coq3 mutant yeast by mitochondrial targeting of the Escherichia coli UbiG polypeptide: Evidence that UbiG catalyzes both O-methylation steps in ubiquinone biosynthesis
Complementation of coq3 mutant yeast by mitochondrial targeting of the Escherichia coli UbiG polypeptide: Evidence that UbiG catalyzes both O-methylation steps in ubiquinone biosynthesis
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DOI：
10.1021/bi9602932
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发表时间：
1996-07-30
期刊：
影响因子：
2.9
通讯作者：
Clarke, CF
中科院分区：
文献类型：
--
作者：
Hsu, AY;Poon, WW;Clarke, CF
泛醌在线粒体电子传递链中起作用，最近的证据表明，还原形式的泛醌（泛醇）也可能作为脂溶性抗氧化剂起作用。泛醌的生物合成需要两个o -甲基化步骤。在真核生物中，第一个o -甲基化步骤是由辅酶q3多肽进行的，它催化一个甲基从s -腺苷蛋氨酸转移到3,4-二羟基-5-聚丙烯基苯甲酸酯。在大肠杆菌中，2-聚戊烯基-6-羟基酚是预测的底物；然而，相应的o -甲基转移酶尚未确定。大肠杆菌的第二个o -甲基化步骤，即将去甲基双醌转化为泛醌，由UbiG甲基转移酶进行，该酶的氨基酸序列与酵母Coq3甲基转移酶相同40%。基于第一个和最后一个甲基受体底物的化学相似性以及Coq3p和UbiG之间氨基酸序列的高度一致性，研究了UbiG催化两个o -甲基化步骤的能力。目前的研究表明，在酵母coq3突变体中，如果ubiG存在，则ubiG基因能够恢复呼吸；被修改为包含线粒体前导序列。线粒体靶向o -甲基转移酶活性是恢复呼吸和体内泛素生物合成能力的基本特征。体外导入实验表明，Coq3p上存在的线粒体先导序列在体外指导Coq3p的线粒体导入，并且加工到成熟形式需要膜电位。用大肠杆菌细胞裂解物和合成的法尼基底物类似物进行的体外甲基转移酶实验表明，UbiG可以甲基化真核中间体3,4-二羟基-5-法尼基苯甲酸酯的衍生物，以及大肠杆菌中间体2-法尼基-6-羟基苯酚的衍生物。这些数据表明，酵母Coq3多肽位于线粒体中，大肠杆菌UbiG催化了大肠杆菌的两个o -甲基化步骤。
Ubiquinone functions in the mitochondrial electron transport chain, Recent evidence suggests that the reduced form of ubiquinone (ubiquinol) may also function as a lipid soluble antioxidant. The biosynthesis of ubiquinone requires two O-methylation steps. In eukaryotes, the first O-methylation step is carried out by the Coq3 polypeptide, which catalyzes the transfer of a methyl group from S-adenosylmethionine to 3,4-dihydroxy-5-polyprenylbenzoate. In Escherichia coli, 2-polyprenyl-6-hydroxyphenol is the predicted substrate; however, the corresponding O-methyltransferase has not been identified. The second O-methylation step in E. coli, the conversion of demethylubiquinone to ubiquinone, is carried out by the UbiG methyltransferase, which is 40% identical in amino acid sequence with the yeast Coq3 methyltransferase. On the basis of the chemical similarity of the first and last methyl-acceptor substrates and the high degree of amino acid sequence identity between Coq3p and UbiG, the ability of UbiG to catalyze both O-methylation steps was investigated. The current study shows that the ubiG gene is able to restore respiration in the yeast coq3 mutant, provided ubiG; is modified to contain a mitochondrial leader sequence. The mitochondrial targeting of O-methyltransferase activity is an essential feature of the ability to restore respiration and hence ubiquinone biosynthesis in vivo. In vitro import assays show the mitochondrial leader sequence present on Coq3p functions to direct mitochondrial import of Coq3p in vitro and that processing to the mature form requires a membrane potential. In vitro methyltransferase assays with E. coli cell lysates and synthetically prepared farnesylated-substrate analogs indicate that UbiG methylates both the derivative of the eukaryotic intermediate, 3,4-dihydroxy-5-farnesylbenzoate, as well as that of the E. coli intermediate, 2-farnesyl-6-hydroxyphenol. The data presented indicate that the yeast Coq3 polypeptide is located in the mitochondria and that E. coli UbiG catalyzes both O-methylation steps in E. coli.