Ashby M N, Kutsunai S Y, Ackerman S, Tzagoloff A, Edwards P A
Department of Biological Chemistry, UCLA School of Medicine 90024.
J Biol Chem. 1992 Feb 25;267(6):4128-36.
Coenzyme Q functions as a lipid-soluble electron carrier in eukaryotes. In Saccharomyces cerevisiae, the enzymes responsible for the assembly of the polyisoprenoid side chain and subsequent transfer to para-hydroxybenzoate (PHB) are encoded by the nuclear genes COQ1 and COQ2, respectively. Yeast mutants defective in coenzyme Q biosynthesis are respiratory defective and provide a useful tool to study this non-sterol branch of the isoprenoid biosynthetic pathway. We isolated a 5.5-kilobase genomic DNA fragment that was able to functionally complement a coq2 strain. Additional complementation analyses located the COQ2 gene within a 2.1-kilobase HindIII-BglII restriction fragment. Sequence analyses revealed the presence of a 1,116-base pair open reading frame coding for a predicted protein of 372 amino acids and a molecular mass of 41,001 daltons. The amino acid sequence exhibits a typical amino-terminal mitochondrial leader sequence and six potential membrane-spanning domains. Primer extension and Northern analyses indicate the gene is transcriptionally active. Transformation of a coq2 strain with the 2.1-kilobase HindIII-BglII genomic restriction fragment on a multicopy plasmid restores PHB:polyprenyltransferase activity to wild-type levels. Disruption of the chromosomal COQ2 gene indicates the gene is not essential for viability, yet is required for PHB:polyprenyltransferase activity and respiratory function. In addition, the deduced amino acid sequence of PHB:polyprenyltransferase contains a putative allylic polyprenyl diphosphate-binding site. The presence of this aspartate-rich domain in a number of functionally distinct proteins which utilize polyprenyl diphosphate substrates is reported.
辅酶Q在真核生物中作为脂溶性电子载体发挥作用。在酿酒酵母中，负责多聚异戊二烯侧链组装并随后转移至对羟基苯甲酸（PHB）的酶分别由核基因COQ1和COQ2编码。辅酶Q生物合成缺陷的酵母突变体存在呼吸缺陷，为研究类异戊二烯生物合成途径的这一非甾醇分支提供了有用的工具。我们分离出一个5.5千碱基的基因组DNA片段，它能够在功能上互补coq2菌株。进一步的互补分析将COQ2基因定位在一个2.1千碱基的HindIII - BglII限制性片段内。序列分析揭示存在一个1116碱基对的开放阅读框，编码一个预测的由372个氨基酸组成、分子量为41,001道尔顿的蛋白质。氨基酸序列具有典型的氨基端线粒体前导序列和六个潜在的跨膜结构域。引物延伸和Northern分析表明该基因具有转录活性。用多拷贝质粒上的2.1千碱基HindIII - BglII基因组限制性片段转化coq2菌株可将PHB：聚异戊二烯基转移酶活性恢复到野生型水平。染色体COQ2基因的破坏表明该基因对于生存力并非必需，但对于PHB：聚异戊二烯基转移酶活性和呼吸功能是必需的。此外，PHB：聚异戊二烯基转移酶的推导氨基酸序列包含一个假定的烯丙基聚异戊二烯基二磷酸结合位点。据报道，在许多利用聚异戊二烯基二磷酸底物的功能不同的蛋白质中都存在这个富含天冬氨酸的结构域。
