%0 Journal Article
%T Binding Stoichiometry of a Recombinant Selenophosphate Synthetase with One Synonymic Substitution E197D to a Fluorescent Nucleotide Analog of ATP, TNP-ATP
%A Y. V. Preobrazhenskaya
%A A. I. Stenko
%A M. V. Shvarts
%A V. Y. Lugovtsev
%J Journal of Amino Acids
%D 2013
%I Hindawi Publishing Corporation
%R 10.1155/2013/983565
%X The transformation of the strain - with plasmid vector pET11a containing the cloned gene of bacterial selenophosphate synthetase (SPS), selD, from the E. coli BL21-Gold (DE3) strain gives an overproducing strain of SPS with one synonymic substitution, E197D. The transformation efficiency was estimated as 8 ¡Á 108 CFU/¦Ìg plasmid DNA. 28£¿mg of highly purified preparation of recombinant SPS capable of binding TNP-ATP was eluted from DEAE-Sephadex column in amount of 15 % from the total soluble protein in crude extract. The fluorescent derivative of ATP, 2¡ä(3¡ä)-O-(2,4,6-trinitrophenyl)adenosine-5¡ä-triphosphate (TNP-ATP), was used as a synthetic analog of the substrate for the monitoring and quantitative analysis of the functional activity of SPS. The non-linear regression analysis of the saturation curve of TNP-ATP binding to D197 SPS with GraphPad Prism software fits to a model with 2 distinct binding sites with different in order. The SPS existence in a form of tetramer in given reaction conditions, in accordance with the concentration stoichiometry of 4 moles of TNP-ATP to 1 mole of recombinant protein, is being discussed. The tetramer structure was predicted with molecular modelling software YASARA and modelled in vacuum using steepest descent minimization energy method. We hypothesize here the recombinant SPS exists as a dimer in solution with two active sites capable of ATP binding in each subunit. 1. Introduction Selenium may be incorporated into a protein body as an aminoacid selenocysteine. Selenophosphate synthetase (EC 2.7.9.3.), a product of selD gene in bacteria, catalyzes selenophosphate formation with the help of ATP and Mg2+ ions [1]. This reaction appears common for all living organisms and serves to transform selenium into biologically active form¡ªselenophosphate. Then, in bacteria selenoprotein synthesis is performed during a few definite steps. First, selenocysteine-specific tRNA ( ) is aminoacylated with serine¡ªand this tRNA is unique for this aminoacid! It means that selenocysteine has its own tRNA¡ªit is a aminoacid in the genetic code. After that, the formated product is easily converted to selenocysteine¡ª with selenophosphate made at the previous step [2]. Further, selenocysteine formed could be incorporated into protein molecule that is encoded with UGA codon [3]. Thus, in contrary to the other aminoacids, the selenocysteine biosynthesis is carried out on its tRNA [4, 5]. In eubacteria, monoselenophosphate could be used as a selenium donor as for incorporation of selenium into the protein body as far as for conversion of thiouridine
%U http://www.hindawi.com/journals/jaa/2013/983565/