Elucidation of the deficiency in two yeast coenzyme Q mutants. Characterization of the structural gene encoding hexaprenyl pyrophosphate synthetase.
Elucidation of the deficiency in two yeast coenzyme Q mutants. Characterization of the structural gene encoding hexaprenyl pyrophosphate synthetase.
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DOI：
10.1016/s0021-9258(19)38280-8
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发表时间：
1990-08
期刊：
影响因子：
--
通讯作者：
M N Ashby;P A Edwards
中科院分区：
文献类型：
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作者：
M N Ashby;P A Edwards
聚异戊二烯侧链的组装及其向对羟基苯甲酸酯的转移是辅酶 Q 生物合成的前两个步骤。在酵母中，这些反应分别由六异戊二烯焦磷酸合成酶和 PHB:聚异戊二烯基转移酶催化。我们筛选了九个互补组的酵母辅酶Q突变体的这两种酶的活性，并发现两个菌株缺乏这两种酶的活性。缺乏六异戊二烯焦磷酸合成酶活性的菌株 C296-LH3 由质粒 pG3/T1 补充。当用含有来自 pG3/T1 基因组插入片段的 2,187 个碱基对片段的穿梭载体转化 C296-LH3 时，甘油生长和六异戊二烯基焦磷酸合成酶活性均得到恢复。后一种酶的活性高于野生型酵母中观察到的活性。活性的增加可归因于多拷贝质粒的基因剂量效应。在基因组片段上发现了编码 52,560 道尔顿蛋白质的 1,419 个碱基对开放阅读框。 RNA 转录物的大小和转录起始位置表明整个开放阅读框包含在 mRNA 内。六异戊二烯焦磷酸合成酶氨基酸序列与来自相关酶法尼基焦磷酸合成酶的氨基酸序列的比较显示存在三个高度保守的结构域。在其中两个结构域内，在来自三个物种的法呢基焦磷酸合成酶的氨基酸序列和本文报道的己戊二烯基焦磷酸合成酶氨基酸序列中恒定地发现了富含天冬氨酸的基序。这些天冬氨酸基序可以包含烯丙基和同烯丙基底物的结合位点。六异戊二烯基焦磷酸合成酶序列和来自大鼠的法呢基焦磷酸合成酶序列的疏水性特征看起来相似。此外，这两个序列比较的疏水性相关系数以高度置信度（p小于0.001）表明这两种蛋白质将折叠成相似的三维结构。
The assembly of a polyisoprenoid side chain and its transfer to para-hydroxybenzoate are the first two steps of coenzyme Q biosynthesis. In yeast these reactions are catalyzed by hexaprenyl pyrophosphate synthetase and PHB:polyprenyltransferase, respectively. We have screened nine complementation groups of yeast coenzyme Q mutants for the activities of these two enzymes and found two strains deficient in either activity. The strain deficient in hexaprenyl pyrophosphate synthetase activity, C296-LH3, is complemented by the plasmid pG3/T1. When C296-LH3 was transformed with a shuttle vector containing a 2,187-base pair fragment from the genomic insert of pG3/T1, both glycerol growth and hexaprenyl pyrophosphate synthetase activity were restored. The activity of the latter enzyme was higher than that seen in wild-type yeast. The increase in activity could be attributed to a gene dosage effect of the multi-copy plasmid. A 1,419-base pair open reading frame encoding a 52,560-dalton protein was found on the genomic fragment. The size of the RNA transcript and the location of transcriptional initiation indicate that the entire open reading frame is contained within the mRNA. Comparison of the hexaprenyl pyrophosphate synthetase amino acid sequence with amino acid sequences from the related enzyme farnesyl pyrophosphate synthetase show the presence of three highly conserved domains. Within two of the domains is an aspartate-rich motif found invariantly in the amino acid sequences of farnesyl pyrophosphate synthetase from three species and the hexaprenyl pyrophosphate synthetase amino acid sequence reported here. These aspartic acid motifs may comprise binding sites for the allylic and homoallylic substrates. The hydrophobicity profiles of the hexaprenyl pyrophosphate synthetase sequence and the farnesyl pyrophosphate synthetase sequence from rat appear similar. Furthermore, the hydrophobicity correlation coefficient of the comparison of these two sequences indicate with a high degree of confidence (p less than 0.001) that the two proteins will fold into similar three-dimensional structures.