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

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Abstract

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 K i 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.

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Scheme 1

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Abbreviations

AS3MT:

Arsenic (+3 oxidation state) methyltransferase

CD:

Circular dichroism

cDNA:

Complementary DNA

DMAV :

Dimethylarsinic acid

DTT:

1,4-Dithiothreitol

GSH:

Glutathione

iAsIII :

Inorganic arsenite

iAsV :

Inorganic arsenate

IPTG:

Isopropyl β-d-thiogalactopyranoside

MALDI:

Matrix-assisted laser desorption/ionization

2-ME:

2-Mercaptoethanol

MMAV :

Monomethylarsonic acid

MS:

Mass spectrometry

Ni-NTA:

Nickel nitrilotriacetic acid

SAM:

S-Adenosyl-l-methionine

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SeIV :

Selenite

TOF:

Time of flight

Tris–HCl:

Tris(hydroxymethyl)aminomethane hydrochloride

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (20535020, 20671051, 20721002, and 90813020) and the National Basic Research Program of China (2007CB925102).

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Authors and Affiliations

  1. State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, People’s Republic of China

    Zhirong Geng, Xiaoli Song & Zhilin Wang

  2. Key Laboratory for Atomic and Molecular Nanosciences of Education Ministry, Department of Chemistry, Tsinghua University, Beijing, 100084, People’s Republic of China

    Zhi Xing, Sichun Zhang & Xinrong Zhang

  3. School of Science, Nanjing Agricultural University, Nanjing, 210095, People’s Republic of China

    Jinlong Geng

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  1. Zhirong Geng
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Correspondence to Zhilin Wang.

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Geng, Z., Song, X., Xing, Z. et al. Effects of selenium on the structure and function of recombinant human S-adenosyl-l-methionine dependent arsenic (+3 oxidation state) methyltransferase in E. coli . J Biol Inorg Chem 14, 485–496 (2009). https://doi.org/10.1007/s00775-008-0464-6

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