Selenoprotein synthesis in E. coli. Purification and characterisation of the enzyme catalysing selenium activation.
The product of the selD gene from Escherichia coli catalyses the formation of an activated selenium compound which is required for the synthesis of Sec-tRNA (Sec, selenocysteine) from Ser-tRNA and for the formation of the unusual nucleoside 5-methylaminomethyl-2-selenouridine in several tRNA species. selD was overexpressed in a T7 promoter/polymerase system and purified to apparent homogeneity. Purified SELD protein is a monomer of 37 kDa in its native state and catalyses a selenium-dependent ATP-cleavage reaction delivering AMP and releasing the beta-phosphate as orthophosphate. The gamma-phosphate group of ATP was not liberated in a form able to form a complex with molybdate. It was precluded that any putative covalent or non-covalent ligand of SELD not removed during purification participated in the reaction. In a double-labelling experiment employing [75Se]selenite plus dithiothreitol and [gamma-32P]ATP the 75Se and 32P radioactivities co-chromatographed on a poly(ethyleneimine)-cellulose column. No radioactivity originating from ATP eluted in this position when [alpha-32P]ATP or [beta-32P]ATP or [14C]ATP were offered as substrates. The results support the speculation that the product of SELD is a phosphoselenoate with the phosphate moiety derived phosphoselenoate from the gamma-phosphate group of ATP. The alpha,beta cleavage of ATP is also supported by the finding that neither adenosine 5'-[alpha,beta-methylene]triphosphate nor adenosine 5'-[beta,gamma-methylene]triphosphate served as substrates in the reaction.
Ehrenreich
A
A
Lehrstuhl für Mikrobiologie, Universität München, Federal Republic of Germany.
Forchhammer
K
K
Tormay
P
P
Veprek
B
B
Böck
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D029721:Drosophila Proteins; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D006868:Hydrolysis; D010710:Phosphates / Q000378:metabolism; D010770:Phosphotransferases; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis; D012643:Selenium / Q000378:metabolism* / Q000494:pharmacology; D051140:Selenoproteins; D014166:Transferases / Q000378:metabolism
D001426:Bacterial Proteins; D029721:Drosophila Proteins; D010710:Phosphates; D011506:Proteins; D012346:RNA, Transfer, Amino Acyl; D051140:Selenoproteins; C112705:selenocysteinyl-tRNA; D000255:Adenosine Triphosphate; D014166:Transferases; D010770:Phosphotransferases; C062762:selenophosphate synthetase; C060600:selenium transferase; D012643:Selenium; D004229:Dithiothreitol