cDNA cloning, purification, and characterization of mouse liver selenocysteine lyase - Candidate for selenium delivery protein in selenoprotein synthesis
cDNA cloning, purification, and characterization of mouse liver selenocysteine lyase - Candidate for selenium delivery protein in selenoprotein synthesis
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DOI：
10.1074/jbc.275.9.6195
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发表时间：
2000-03-03
影响因子：
4.8
通讯作者：
Esaki, N
中科院分区：
文献类型：
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作者：
Mihara, H;Kurihara, T;Esaki, N
硒半胱氨酸裂解酶(SCL)(EC 4.4.1.16)是一种5‘-磷酸吡哆醛依赖的酶，能特异性催化L-硒半胱氨酸分解为L-丙氨酸和元素硒。该酶被认为是硒蛋白生物合成中硒向磷酸硒合成酶的递送蛋白(Lacourciere，G.M.，和Stadtman，T.C.(1998)J.Biol)。化学。273、30921-30926)。我们从猪的肝脏中纯化了SCL，并测定了其部分氨基酸序列。在表达序列标签数据库中找到了编码猪SCL类似肽的小鼠c DNA克隆，并将其序列作为探针分离小鼠肝脏全长cDNA.小鼠SCL(MSCL)基因全长2,172个碱基对，含有一个开放阅读框，编码432个氨基酸残基(M-r47,201)的多肽链。我们还测定了推测的人SCL的N-末端区域的序列。这些酶在一级结构上与催化L-半胱氨酸脱硫为铁-硫簇提供硫的NIFS有较远的亲缘关系。在大肠杆菌中高效表达的重组MSCL为同源二聚体，亚基M-r为47,000。该酶是磷酸吡哆醛依赖性的，对L-硒半胱氨酸具有高度的特异性(L-硒半胱氨酸的k(CAT)/K-m值约为L-半胱氨酸的4,200倍)。逆转录聚合酶链式反应和Western印迹分析表明，MSCL是胞质的，主要存在于肝、肾和睾丸，其中小鼠的硒磷酸合成酶也很丰富，支持MSCL在硒蛋白合成中与硒磷酸合成酶协同作用的观点。这是首次报道哺乳动物SCL的一级结构。
Selenocysteine lyase (SCL) (EC 4.4.1.16) is a pyridoxal 5'-phosphate-dependent enzyme that specifically catalyzes the decomposition of L-selenocysteine to L-alanine and elemental selenium. The enzyme was proposed to function as a selenium delivery protein to selenophosphate synthetase in selenoprotein biosynthesis (Lacourciere, G. M., and Stadtman, T. C. (1998) J. Biol. Chem. 273, 30921-30926). We purified SCL from pig liver and determined its partial amino acid sequences. Mouse cDNA clones encoding peptides resembling pig SCL were found in the expressed sequence tag data base, and their sequences were used as probes to isolate full-length mouse liver cDNA. The cDNA for mouse SCL (mSCL) was determined to be 2,172 base pairs in length, containing an open reading frame encoding a polypeptide chain of 432 amino acid residues (M-r 47,201). We also determined the sequence of the N-terminal region of putative human SCL. These enzymes were shown to be distantly related in primary structure to NifS, which catalyzes the desulfurization of L-cysteine to provide sulfur for iron-sulfur clusters. The recombinant mSCL overproduced in Escherichia coli was a homodimer with the subunit M-r of 47,000. The enzyme was pyridoxal phosphate dependent and highly specific to L-selenocysteine (the k(cat)/K-m value for L-selenocysteine was about 4,200 times higher than that for L-cysteine). Reverse transcriptase-polymerase chain reaction and Western blot analyses revealed that mSCL is cytosolic and predominantly exists in the liver, kidney, and testis, where mouse selenophosphate synthetase is also abundant, supporting the view that mSCL functions in cooperation with selenophosphate synthetase in selenoprotein synthesis. This is the first report of the primary structure of mammalian SCL.