Gursinsky T, Jäger J, Andreesen J R, Söhling B
Institut für Mikrobiologie, Martin Luther Universität Halle, Saale, Germany.
Arch Microbiol. 2000 Sep;174(3):200-12. doi: 10.1007/s002030000196.
The four genes required for selenocysteine incorporation were isolated from the gram-positive, amino acid-fermenting anaerobe Eubacterium acidaminophilum, which expresses various selenoproteins of different functions. The sel genes were located in an unique organization on a continuous fragment of genomic DNA in the order selD1 (selenophosphate synthetase 1), selA (selenocysteine synthase), selB (selenocysteine-specific elongation factor), and selC (selenocysteine-specific tRNA). A second gene copy, encoding selenophosphate synthetase 2 (selD2), was present on a separate fragment of genomic DNA. SelD1 and SelD2 were only 62.9% identical, but the two encoding genes, selD1 and selD2, contained an in-frame UGA codon encoding selenocysteine, which corresponds to Cys-17 of Escherichia coli SelD. The function of selA, selB, and selC from E. acidaminophilum was investigated by complementation of the respective E. coli deletion mutant strains and determined as the benzyl viologen-dependent formate dehydrogenase activity in these strains after anaerobic growth in the presence of formate. selA and selC from E. acidaminophilum were functional and complemented the respective mutant strains to 83% (selA) and 57% (selC) compared to a wild-type strain harboring the same plasmid. Complementation of the E. coli selB mutant was only observed when both selB and selC from E. acidaminophilum were present. Under these conditions, the specific activity of formate dehydrogenase was 55% of that of the wild type. Transformation of this selB mutant with selB alone was not sufficient to restore formate dehydrogenase activity.
参与硒代半胱氨酸掺入的四个基因是从革兰氏阳性、氨基酸发酵厌氧菌嗜酸真杆菌中分离出来的，该菌表达多种具有不同功能的硒蛋白。硒基因以独特的排列方式位于基因组DNA的一个连续片段上，顺序为selD1（硒磷酸合成酶1）、selA（硒代半胱氨酸合成酶）、selB（硒代半胱氨酸特异性延伸因子）和selC（硒代半胱氨酸特异性tRNA）。编码硒磷酸合成酶2（selD2）的第二个基因拷贝存在于基因组DNA的一个单独片段上。SelD1和SelD2的同源性仅为62.9%，但两个编码基因selD1和selD2都含有一个编码硒代半胱氨酸的框内UGA密码子，该密码子对应于大肠杆菌SelD的第17位半胱氨酸。通过对相应的大肠杆菌缺失突变株进行互补，研究了嗜酸真杆菌中selA、selB和selC的功能，并在甲酸盐存在下厌氧生长后测定了这些菌株中依赖苄基紫精的甲酸脱氢酶活性。与携带相同质粒的野生型菌株相比，嗜酸真杆菌的selA和selC具有功能，并使相应的突变株分别恢复到83%（selA）和57%（selC）的活性。只有当嗜酸真杆菌的selB和selC同时存在时，才能观察到对大肠杆菌selB突变株的互补作用。在这些条件下，甲酸脱氢酶的比活性为野生型的55%。仅用selB转化该selB突变株不足以恢复甲酸脱氢酶活性。
