Identification of the key residues responsible for the assembly of selenodeiodinases
Department of Cell and Molecular Physiology
Alanine; Amino Acid Sequence; Animals; Binding, Competitive; Dimerization; Green Fluorescent Proteins; Iodide Peroxidase; Molecular Sequence Data; Mutation; Protein Binding; Protein Structure, Tertiary; Rats; Recombinant Fusion Proteins; Selenium; Thermodynamics
Life Sciences | Medicine and Health Sciences
Type I deiodinase is the best characterized member of a small family of selenoenzymes catalyzing the bioactivation and disposal of thyroid hormone. This enzyme is an integral membrane protein composed of two 27-kDa subunits that assemble into a functional enzyme after translation using a highly conserved sequence of 16 amino acids in the C-terminal half of the polypeptide, (148)DFLXXYIXEAHXXDGW(163). In this study, we used alanine scanning mutagenesis to identify the key residues in this domain required for holoenzyme assembly. Overexpression of sequential alanine-substituted mutants of a dimerization domain-green fluorescent protein fusion showed that sequence (152)IYI(154) was required for type I enzyme assembly and that a catalytically active monomer was generated by a single I152A substitution. Overexpression of the sequential alanine-substituted dimerization domain mutants in type II selenodeiodinase-expressing cells showed that five residues ((153)FLIVY(157)) at the beginning and three residues ((164)SDG(166)) at the end of this region were required for the assembly of the type II enzyme. In vitro binding analysis revealed a free energy of association of -60 +/- 5 kJ/mol for the noncovalent interaction between dimerization domain monomers. These data identify and characterize the essential residues in the dimerization domain that are responsible for the post-translational assembly of selenodeiodinases.
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Citation: J Biol Chem. 2006 May 26;281(21):14615-21. Epub 2006 Mar 25. Link to article on publisher's site
Simpson, Gregory I. C.; Leonard, Deborah; and Leonard, Jack L., "Identification of the key residues responsible for the assembly of selenodeiodinases" (2006). Open Access Articles. 682.