Identification and characteristics of the structural gene for the Drosophila eye colour mutant sepia, encoding PDA synthase, a member of the Omega class glutathione S-transferases
Identification and characteristics of the structural gene for the Drosophila eye colour mutant sepia, encoding PDA synthase, a member of the Omega class glutathione S-transferases
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DOI：
10.1042/bj20060424
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发表时间：
2006-09-15
影响因子：
4.1
通讯作者：
Yim, Jeongbin
中科院分区：
文献类型：
--
作者：
Kim, Jaekwang;Suh, Hyunsuk;Yim, Jeongbin
眼睛颜色突变体乌贼墨（se '）在PDA {6-乙酰基-2-氨基-3-7，8，9-四氢-4H-嘧啶并[4，5-B]-[1，4]二氮杂-4-酮或嘧啶二氮杂}合酶中有缺陷，该合酶参与6-PTP（2-氨基-4-氧代-6-异戊酰基-5，6，7，8-四氢蝶啶;也称为6-异戊酰基四氢蝶啶）转化为PDA，PDA是屈蝶呤生物合成中的关键中间体。然而，编码这种酶的基因的身份以及其分子特性尚未确定。在这里，我们确定和表征的基因编码PDA合酶，并表明它是乌贼的结构基因。基于先前报道的信息[Wiederrecht，Paton和Brown（1984）J.Biol.Chem.259，2195 - 2200; Wiederrecht和Brown（1984）J.Biol.Chem.259，14121-14127; Andres（1945）Drosoph.信息服务19，45; Ingham，Pinchin，霍华德和Ish-Horowicz（1985）Genetics 111，463486;霍华德，Ingham和Rushlow（1988）Genes Dev. 2，10371046]，我们从推测的乌贼基因座（染色体3L上的区域66135）中分离出5个预测编码GST（谷胱甘肽S-转移酶）的候选基因。所有克隆和表达的候选物都表现出相对较高的巯基转移酶和脱氢抗坏血酸还原酶活性，而对1-氯-2，4-二硝基苯的活性较低，这是Omega类GST的特征，而只有CG 6781在体外催化PDA的合成。通过SDS/PAGE估计重组CG 6781的分子量为28 kDa，通过凝胶过滤估计其分子量为56 kDa，表明其在天然条件下为同源二聚体。对CG 6781的基因组区域进行测序，发现se'等位基因在nt 190-194处有一个从'AAGAA'到'GTG'的移码突变，这产生了一个提前终止密码子。CG 6781开放阅读框在se'背景中的表达挽救了眼睛颜色缺陷以及PDA合酶活性和毛蝶呤含量。拯救的程度取决于转基因CG 6781的剂量。总之。我们已经发现了Ω类GST的新催化活性，并且CG 6781是编码PDA合酶的乌贼属的结构基因。
The eye colour mutant sepia (se') is defective in PDA {6-acetyl-2-amino-3-7,8,9-tetrahydro-4H-pyrimido[4,5-b]-[1,4]diazepin-4-one or pyrimidodiazepine} synthase involved in the conversion of 6-PTP (2-amino-4-oxo-6-pyruvoyl-5,6,7,8-tetrahydropteridine; also known as 6-pyruvoyltetrahydropterin) into PDA, a key intermediate in drosopterin biosynthesis. However, the identity of the gene encoding this enzyme, as well as its molecular properties, have not yet been established. Here, we identify and characterize the gene encoding PDA synthase and show that it is the structural gene for sepia. Based on previously reported information [Wiederrecht, Paton and Brown (1984) J. Biol. Chem. 259,21952200; Wiederrecht and Brown (1984) J. Biol. Chem. 259, 14121-14127; Andres (1945) Drosoph. Inf. Serv. 19, 45; Ingham, Pinchin, Howard and Ish-Horowicz (1985) Genetics 111, 463486; Howard, Ingham and Rushlow (1988) Genes Dev. 2, 10371046], we isolated five candidate genes predicted to encode GSTs (glutathione S-transferases) from the presumed sepia locus (region 66135 on chromosome 3L). All cloned and expressed candidates exhibited relatively high thiol transferase and dehydro-ascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro. The molecular mass of recombinant CG6781 was estimated to be 28 kDa by SDS/PAGE and 56 kDa by gel filtration, indicating that it is a homodimer under native conditions. Sequencing of the genomic region spanning CG6781 revealed that the se' allele has a frameshift mutation from 'AAGAA' to 'GTG' at nt 190-194, and that this generates a premature stop codon. Expression of the CG6781 open reading frame in an se' background rescued the eye colour defect as well as PDA synthase activity and drosopterins content. The extent of rescue was dependent on the dosage of transgenic CG6781. In conclusion. we have discovered a new catalytic activity for an Omega class GST and that CG6781 is the structural gene for sepia which encodes PDA synthase.