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

Abstract

The effects of Se(IV) 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 degrees C. Spectra (UV-vis, circular dichroism, and fluorescence) were first used to probe the interaction of Se(IV) and recombinant human AS3MT and the structure-function relationship of the enzyme. The recombinant human AS3MT had a secondary structure of 29.0% alpha-helix, 23.9% beta-pleated sheet, 17.9% beta-turn, and 29.2% random coil. When Se(IV) was added, the content of the alpha-helix did not change, but that of the beta-pleated sheet increased remarkably in the conformation of recombinant human AS3MT. Se(IV) inhibited the enzymatic methylation of inorganic As(III) in a concentration-dependent manner. The IC(50) value for Se(IV) was 2.38 muM. Double-reciprocal (1/V vs. 1/[inorganic As(III)]) plots showed Se(IV) to be a noncompetitive inhibitor of the methylation of inorganic As(III) by recombinant human AS3MT with a K (i) value of 2.61 muM. We hypothesized that Se(IV) interacts with the sulfhydryl group of cysteine(s) in the structural residues rather than the cysteines of the active site (Cys156 and Cys206). When Se(IV) 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 Se(IV).

Rapid Equilibrium Kinetic Analysis of Arsenite Methylation Catalyzed by Recombinant Human Arsenic (+3 Oxidation State) Methyltransferase (hAS3MT)

“…Cys-156 and Cys-206 of hAS3MT were proved to be the cysteine residues in the active site that bind to iAs 3ϩ (20). The results of our MALDI-TOF mass spectroscopy experiment suggest that the disulfide bond reduced by the reductant is associated with Cys-250.…”

“…However, phenylalanine has a very low quantum yield, and the fluorescence of tyrosine is easily quenched when it is ionized or close to an amino group, a carboxyl group, or a tryptophan (35). In hAS3MT, there are three tryptophan residues (Trp-73, Trp-203, and Trp-213) close to the cysteine residues (Cys-156, Cys-206, Cys-72, and Cys-250) that are important to the enzymatic activity (20,26). Changes in intrinsic fluorescence intensity of hAS3MT reflect the perturbation of the active site.…”

“…The enzyme was thoroughly inactive in the GSH reaction system when any of the cysteine residues at positions 72, 156, 206, and 250 were mutated into serine residues (20,26). Cys-156 and Cys-206 of hAS3MT were proved to be the cysteine residues in the active site that bind to iAs 3ϩ (20).…”

“…Preparation of hAS3MT-The cloning, heterologous expression, and purification of recombinant hAS3MT were carried out as described previously (20). Details are shown in the supplemental Materials.…”

“…Both models start from the reduction of iAs 5ϩ to iAs 3ϩ and do not further describe the function of the enzyme in the reaction. To elucidate the detailed mechanism, we studied the involvement of the cysteine residue of human AS3MT (hAS3MT) in the methylation reaction by site-directed mutagenesis and found that the conserved Cys-156 and Cys-206 were essential for catalytic activity and that Cys-72 and Cys-250 also played a key role in the reaction (20,26). Similar to GSH, cysteine residues of hAS3MT also have a sulfhydryl group and can be involved in oxidation-reduction reactions.…”