Selenoprotein synthesis in E. coli. Purification and characterisation of the enzyme catalysing selenium activation.
Eur J Biochem
; 206(3): 767-73, 1992 Jun 15.
Article
em En
| MEDLINE
| ID: mdl-1606960
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.
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Base de dados:
MEDLINE
Assunto principal:
Fosfotransferases
/
Selênio
/
Proteínas de Bactérias
/
Biossíntese de Proteínas
/
Proteínas de Drosophila
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Escherichia coli
Idioma:
En
Ano de publicação:
1992
Tipo de documento:
Article