Selenophosphate synthetase genes from lung adenocarcinoma cells: Sps1 for recycling L-selenocysteine and Sps2 for selenite assimilation.
A labile selenium donor compound monoselenophosphate is synthesized from selenide and ATP by selenophosphate synthetase (SPS). In the present study, Sps1 and Sps2 were cloned from a cDNA library prepared from human lung adenocarcinoma cells (NCIH441). The human lung Sps1 has been cloned as an ORF of 1,179 bp, identical in sequence to that of the recently revised human liver Sps1. The in-frame TGA codon of the lung Sps2 was genetically altered to TGT (Cys) to obtain the Sps2Cys gene. Expression of the recombinant plasmids containing Sps1 or Sps2Cys was highly toxic to Escherichia coli host cells grown aerobically. Accordingly, the human lung Sps homologs were characterized by an in vivo complementation assay using a selD mutant strain. An added selenium source and a low salt concentration (0.1-0.25% NaCl) in the medium were required for reproducible and sensitive in vivo complementation. Sps2Cys effectively complemented the selD mutant, and the resulting formate dehydrogenase H activity was as high as that of WT E. coli MC4100. In contrast, only a weak complementation of the selD mutant by the Sps1 gene was observed when cells were grown in selenite media. Better complementation with added l-selenocysteine suggested involvement of a selenocysteine lyase for mobilization of selenium. Based on this apparent substrate specificity of the Sps1 and Sps2 gene products we suggest that the Sps1-encoded enzyme depends on a selenium salvage system that recycles l-selenocysteine, whereas the Sps2 enzyme can function with a selenite assimilation system.
Tamura
Takashi
T
Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan. tktamura@cc.okayama-u.ac.jp
Yamamoto
Shinpei
S
Takahata
Muneaki
M
Sakaguchi
Hiromich
H
Tanaka
Hidehiko
H
Stadtman
Thressa C
TC
Inagaki
Kenji
K
D000230:Adenocarcinoma / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology* / Q000235:genetics*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009857:Oncogenes; D010770:Phosphotransferases / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017279:Selenocysteine / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018038:Sodium Selenite / Q000378:metabolism
D011994:Recombinant Proteins; D017279:Selenocysteine; D010770:Phosphotransferases; C062762:selenophosphate synthetase; D018038:Sodium Selenite