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

亚硒酸盐合成酶由三磷酸腺苷和硒化物形成能量丰富的化合物--亚硒酸盐。在真核生物和其他细菌中，必需的半胱氨酸残基被硒半胱氨酸、苏氨酸或精氨酸以同工酶形式取代。对于某些同工酶来说，游离硒不能作为底物。硫转移酶通过酶过硫化物中间体传递活性硫烷硫，在体外也可以作为硒的传递系统，当SECYS作为硒供体时，该偶联体系有效地取代了游离的硒。然而，在体内，当Se：S的比率非常低时，Se特异性递送酶及其Se底物应该是必不可少的。为了确定从凡纳氏甲烷球菌中纯化的未知的33 kDa硒结合蛋白是Secys专一性裂解酶还是黄嘌呤脱氢酶的硒结合亚基，对后者进行了纯化。在没有DTT的情况下，从嘌呤溶血梭菌中纯化了具有催化活性的酶，并正在分离硒结合亚基。此外，还将监测vannielii分枝杆菌提取物中可能共浓缩的分泌物裂解酶活性和33 kDa[75Se]蛋白质。对人胎盘硫氧还蛋白还原酶基因进行了改造，使其在大肠杆菌中表达。通过在C-末端甘氨酸密码子和UAA终止之间插入E.ColiFdhF SECIS茎环结构。将钙调神经磷酸酶或聚His载体分别放置在N-末端，以允许通过钙调素或镍柱进行分离。在大肠杆菌中表达的野生型(TGA密码子)和半胱氨酸突变型(TGT密码子)酶活性较低，但表现出明显的典型的黄素谱。表达的酶的C-末端结构有待确定。--硒磷酸盐、硒化物、硒转移蛋白、硒半胱氨酸

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