title: Selenoprotein synthesis in E. coli. Purification and characterisation of the enzyme catalysing selenium activation.
abstract: 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.
authors.affiliation: Lehrstuhl für Mikrobiologie, Universität München, Federal Republic of Germany.
authors.forename: A
authors.initials: A
authors.lastname: Ehrenreich
authors.forename: K
authors.initials: K
authors.lastname: Forchhammer
authors.forename: P
authors.initials: P
authors.lastname: Tormay
authors.forename: B
authors.initials: B
authors.lastname: Veprek
authors.forename: A
authors.initials: A
authors.lastname: Böck
mesh_terms: 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
chemical_list: 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