Putidaredoxin reductase and putidaredoxin. Cloning, sequence determination, and heterologous expression of the proteins.
Putidaredoxin reductase and putidaredoxin. Cloning, sequence determination, and heterologous expression of the proteins.
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DOI：
10.1016/s0021-9258(19)39292-0
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发表时间：
1990-04
期刊：
影响因子：
--
通讯作者：
J. Peterson;M. Lorence;B. Amarneh
中科院分区：
文献类型：
--
作者：
J. Peterson;M. Lorence;B. Amarneh
樟脑被细胞色素P-450 cam氧化需要黄素蛋白putidaredoxin还原酶和铁硫蛋白putidaredoxin参与，以介导电子从NADH转移到P-450进行氧活化。从人工培养的恶臭假单胞菌（Pseudomonasputida）全细胞DNA中克隆了一个2.2-内切酶对BamHI-StuI片段，并进行了序列测定。序列的翻译揭示了两个开放的阅读框架，可以编码putidaredoxin还原酶和putidaredoxin。在putidaredoxin的情况下，翻译的序列与公开的序列匹配（Tanaka，M.，Haniu，M.，康信，K. T.，Dus，K.，Gunsalus，我。C.（1974）J.Biol.Chem.249，3689-3701），除了一个氨基酸。与其他假单胞菌的蛋白质一样，这些蛋白质中的密码子使用在第三个核苷酸中强烈偏向G + C。在putidaredoxin编码区的3'端发现一个潜在的转录终止位点。存在于其他黄素蛋白中的“FAD结合”氨基酸共有序列在putidaredoxin还原酶中发现，起始于残基11，并且发现该序列的第二次出现起始于氨基酸156。第二个序列可以代表NAD结合位点。编码putidaredoxin还原酶和putidaredoxin的区域被亚克隆，并在大肠杆菌中分别以0.4和4.8 mg酶活性蛋白/g湿重的水平独立表达。定点突变被用来改变罕见的起始密码子，GTG，putidaredoxin还原酶的ATG，这导致在一个18倍的增加，该蛋白质的表达水平为7.4毫克/克湿重的细胞。这两个克隆的构建，表达这些重要的蛋白质，将有助于研究它们之间的相互作用和与P-450 cam。
The oxidation of camphor by cytochrome P-450cam requires the participation of a flavoprotein, putidaredoxin reductase, and an iron-sulfur protein, putidaredoxin, to mediate the transfer of electrons from NADH to P-450 for oxygen activation. A 2.2-kilobase pair BamHI-StuI fragment from whole cell DNA of camphor-grown Pseudomonas putida has been cloned and sequenced. Translation of the sequence revealed two open reading frames that could code for putidaredoxin reductase and putidaredoxin. In the case of putidaredoxin, the translated sequence matched the published sequence (Tanaka, M., Haniu, M., Yasunobu, K. T., Dus, K., and Gunsalus, I. C. (1974) J. Biol. Chem. 249, 3689-3701) with the exception of one amino acid. Codon usage in these proteins, like the proteins of other Pseudomonads, is strongly biased to G + C in the third nucleotide. A potential transcription termination site was found 3' to the putidaredoxin coding region. The "FAD-binding" amino acid consensus sequence, present in other flavoproteins, was found in putidaredoxin reductase beginning at residue 11 and a second occurrence of this sequence was found beginning with amino acid 156. The second sequence could represent the NAD-binding site. The regions encoding putidaredoxin reductase and putidaredoxin were subcloned and independently expressed in Escherichia coli at the level of 0.4 and 4.8 mg of enzymatically active protein/g wet weight of cells, respectively. Site-directed mutagenesis was used to change the rare start codon, GTG, of putidaredoxin reductase to ATG which resulted in an 18-fold increase in the level of expression of this protein to 7.4 mg/g wet weight of cells. The construction of these two clones, which express these important proteins, will facilitate studies of their interaction with each other and with P-450cam.