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 fulllength 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.Selenocysteine residue plays an essential catalytic role in various selenoproteins such as glutathione peroxidases from mammals (1) and formate dehydrogenase from Escherichia coli (2). Selenocysteine is co-translationally incorporated into a nascent polypeptide chain as encoded by the UGA-stop codon (2, 3). The initial step of selenoprotein biosynthesis involves selenophosphate synthetase (SPS), 1 which catalyzes the formation of selenophosphate from selenide and ATP (4): selenophosphate is a precursor molecule of selenocysteyl-tRNA (5), which decodes the UGA codon.Selenocysteine lyase (SCL) is a pyridoxal 5Ј-phosphate (PLP) enzyme that catalyzes the elimination of elemental selenium from L-selenocysteine to yield L-alanine. SCL has been purified from pig liver (6) and Citrobacter freundii (7) as the first enzyme that specifically acts on a selenium-containing compound and not on the sulfur analog. The mammalian enzyme is a homodimer with the subunit M r of 48,000, whereas the bacterial enzyme is monomeric with the M r of 64,000. SCL was proposed to cooperate with SPS in selenophosphate biosynthesis based on the following observations. 1) Selenium derived from L-selenocysteine added to Clostridium sticklandii cultures is more efficiently incorporated into se...

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