Effects of selenium on the structure and function of recombinant human S-adenosyl- l-methionine dependent arsenic (+3 oxidation state) methyltransferase in E. coli.

The effects of SeIV on the structure and function of recombinant human arsenic (+3 oxidation state) methyltransferase (AS3MT) purified from the cytoplasm of Escherichia coli were studied. The coding region of human AS3MT complementary DNA was amplified from total RNA extracted from HepG2 cell by reverse transcription PCR. Soluble and active human AS3MT was expressed in the E. coli with a Trx fusion tag under a lower induction temperature of 25 °C. Spectra (UV–vis, circular dichroism, and fluorescence) were first used to probe the interaction of SeIV and recombinant human AS3MT and the structure–function relationship of the enzyme. The recombinant human AS3MT had a secondary structure of 29.0% α-helix, 23.9% β-pleated sheet, 17.9% β-turn, and 29.2% random coil. When SeIV was added, the content of the α-helix did not change, but that of the β-pleated sheet increased remarkably in the conformation of recombinant human AS3MT. SeIV inhibited the enzymatic methylation of inorganic AsIII in a concentration-dependent manner. The IC50 value for SeIV was 2.38 μM. Double-reciprocal (1/ V vs. 1/[inorganic AsIII]) plots showed SeIV to be a noncompetitive inhibitor of the methylation of inorganic AsIII by recombinant human AS3MT with a Ki value of 2.61 μM. We hypothesized that SeIV interacts with the sulfhydryl group of cysteine(s) in the structural residues rather than the cysteines of the active site (Cys156 and Cys206). When SeIV was combined with cysteine(s) in the structural residues, the conformation of recombinant human AS3MT changed and the enzymatic activity decreased. Considering the quenching of tryptophan fluorescence, Cys72 and/or Cys226 are deduced to be primary targets for SeIV. [ABSTRACT FROM AUTHOR]