BIOSYNTHESIS, PROPERTIES, AND FUNCTIONS OF SELENOENZYMES AND SELENO-TRNAS

硒磷酸合成酶从ATP和硒化物形成富含能量的化合物硒磷酸盐。大肠杆菌酶中的必需半胱氨酸残基被真核生物和其他细菌中存在的同工酶形式的硒代半胱氨酸、苏氨酸或精氨酸取代。游离硒化物作为某些同工酶形式的底物是无活性的。通过酶过硫化物中间体递送活性硫烷硫的硫转移酶也可以用作体外硒递送系统，并且当Secys作为Se供体提供时，偶联系统有效地替代游离硒化物。然而，在体内，其中Se：S比率非常低，Se特异性递送酶连同其Se底物应该是必需的。为了确定是否身份不明的33 kDa硒结合蛋白纯化甲烷球菌vannielii是一个Secys特异性裂解酶或硒结合亚基的黄嘌呤脱氢酶，后者的纯化进行。催化活性的酶纯化从梭菌在没有DTT，硒结合亚基的分离正在进行中。此外，还发现了M.将监测vannieli提取物。对人胎盘硫氧还蛋白还原酶基因进行改造，使其在大肠杆菌中表达。杆菌通过插入E. coli fdhF SECIS C-末端甘氨酸密码子和UAA终止子之间的茎环结构。将钙调磷酸酶或聚组氨酸载体置于N-末端，以允许分别通过钙调蛋白或Ni柱分离。在大肠杆菌中表达的野生型（TGA密码子）和Cys突变体（TGT密码子）酶的活性。大肠杆菌中的黄色素含量较低，但有显著性差异，并呈现典型的黄色素谱。待确定表达的酶的C-末端结构。- 硒代磷酸盐，硒化物，硒转运蛋白，硒代半胱氨酸

Selenophosphate synthetase forms the energy-rich compound, selenophosphate, from ATP and selenide. An essential cysteine residue in the Escherichia coli enzyme is replaced by selenocysteine, threonine, or arginine in isoenzyme forms present in eukaryotes and other bacteria. Free selenide is inactive as substrate for some isoenzyme forms. Sulfur transferases that deliver active sulfane sulfur via enzyme persulfide intermediates also can serve as selenium delivery systems in vitro and, when Secys is supplied as Se donor, the coupled system effectively replaces free selenide. However, in vivo, where Se:S ratios are very low, a Se-specific delivery enzyme together with its Se substrate should be essential. To determine whether the unidentified 33 kDa selenium binding protein purified from Methanococcus vannielii is a Secys-specific lyase or the Se-binding subunit of xanthine dehydrogenase, purification of the latter was undertaken. Catalytically active enzyme was purified from Clostridium purinolyticum in the absence of DTT, and isolation of the selenium-binding subunit is in progress. Also, possible coenrichment of Secys lyase activity and 33 kDa [75Se]protein from M. vannielii extracts will be monitored. The thiroedoxin reductase gene from human placenta was modified for expression in E. coli. by inserting the E. coli fdhF SECIS stem-loop structure between the C-terminal glycine codon and the UAA stop. Either a calcineurin or a poly his vector was placed at the N-terminus to allow isolation via a calmodulin or a Ni column, respectively. Activities of wild type (TGA codon) and Cys mutant (TGT codon) enzymes expressed in E. coli were low but significant and typical flavin spectra were exhibited. C-terminal structures of the expressed enzyme are to be determined. - selenophosphate, selenide, Se-transfer protein, selenocysteine