Effects of selenium on the structure and function of recombinant human S-adenosyl-L-methionine dependent arsenic (+3 oxidation state) methyltransferase in E-coli
Effects of selenium on the structure and function of recombinant human S-adenosyl-L-methionine dependent arsenic (+3 oxidation state) methyltransferase in E-coli
复制标题
硒对大肠杆菌重组人S-腺苷-L-蛋氨酸依赖性砷（3氧化态）甲基转移酶结构和功能的影响
DOI：
10.1007/s00775-008-0464-6
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发表时间：
2009-05-01
影响因子：
3
通讯作者：
Wang, Zhilin
中科院分区：
文献类型：
--
作者：
Geng, Zhirong;Song, Xiaoli;Wang, Zhilin
研究了Se-IV对从大肠杆菌细胞质中纯化的重组人砷（+3氧化态）甲基转移酶（AS3MT）结构和功能的影响。通过逆转录PCR从HepG2细胞提取的总RNA中扩增人AS3MT互补DNA的编码区。在 25 摄氏度的较低诱导温度下，可溶性且有活性的人 AS3MT 在带有 Trx 融合标签的大肠杆菌中表达。首先使用光谱（UV-vis、圆二色性和荧光）探测 Se-IV 与重组人 AS3MT 的相互作用以及酶的结构功能关系。重组人 AS3MT 的二级结构为 29.0% α 螺旋、23.9% β 折叠片、17.9% β 转角和 29.2% 无规卷曲。当添加Se-IV时，重组人AS3MT构象中α螺旋的含量没有变化，但β折叠片的含量显着增加。 Se-IV 以浓度依赖性方式抑制无机 As-III 的酶促甲基化。 Se-IV 的 IC50 值为 2.38 μM。双倒数（1/V vs. 1/[无机 As-III]）图显示 Se-IV 是重组人 AS3MT 无机 As-III 甲基化的非竞争性抑制剂，K-i 值为 2.61 μM。我们假设 Se-IV 与结构残基中的半胱氨酸的巯基相互作用，而不是与结构残基中的半胱氨酸的巯基相互作用。活性位点的半胱氨酸（Cys156 和 Cys206）。当Se-IV与结构残基中的半胱氨酸结合时，重组人AS3MT的构象发生变化，酶活性降低。考虑到色氨酸荧光的猝灭，Cys72 和/或 Cys226 被推断为 Se-IV 的主要靶标。
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 IC50 value for Se-IV was 2.38 mu M. 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 mu M. 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.