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首页> 外文期刊>Biochemistry >Structural and catalytic properties of the expressed and purified NAD(H)- and NADP(H)-binding domains of proton-pumping transhydrogenase from Escherichia coli
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Structural and catalytic properties of the expressed and purified NAD(H)- and NADP(H)-binding domains of proton-pumping transhydrogenase from Escherichia coli

机译：大肠杆菌质子泵转氢酶表达和纯化的NAD（H）-和NADP（H）-结合域的结构和催化特性

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摘要

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains three domains: the hydrophilic domains I and III harbor the binding sites for NAD(H) and NADP(H), respectively, and domain II represents the membrane-spanning region. Proton translocation involves primarily domain II but possibly also domain III, which contains the essential betaAsp392 residue. In the present investigation, the major portions of domain I (EcTHSalpha1 and EcTHSalpha2) and domain III (EcTHSbeta1) were overexpressed in E. coli and purified therefrom. EcTHSbeta1 was purified mainly in its holoform containing approximately 95% NADP+ and 5% NADPH. When combined, EcTHSalpha1/EcTHSalpha2 and EcTHSbeta1 were catalytically active, indicating native-like structures. Due to the lack of structural information and its possible role in proton pumping, EcTHSbeta1 was primarily characterized. Substrate-binding characteristics and conformational changes were investigated by fluorescence and CD. Fluorescence arising from the single betaTrp415 of EcTHSbeta1 was quenched upon binding of NADPH by resonance energy transfer, an effect that provides an important tool for investigating substrate interactions with this domain and the determination of Kd values. The apparent relative binding affinity for NADPH was found to be about 50 times higher than that for NADP+. Circular dichroism was used to estimate secondary structure content and for conformational analysis of EcTHSbeta1 in the absence and presence of added substrates at various temperatures. Results show that domain III complexed with NADPH or NADP+ adopts different conformations. Isoelectric focusing and native gel electrophoresis experiments support this finding. It is proposed that these structural differences play a central role in a conformationally-driven proton pump mechanism of the intact enzyme.
机译：来自大肠杆菌的质子泵浦烟酰胺核苷酸转氢酶包含三个结构域：亲水性结构域I和III分别具有NAD（H）和NADP（H）的结合位点，结构域II代表跨膜区域。质子易位主要涉及结构域II，但也可能涉及结构域III，该结构域包含必需的betaAsp392残基。在本研究中，结构域I（EcTHSalpha1和EcTHSalpha2）和结构域III（EcTHSbeta1）的主要部分在大肠杆菌中过表达并从中纯化。 EcTHSbeta1主要是在其含有约95％NADP +和5％NADPH的全盐形式中纯化的。结合使用时，EcTHSalpha1 / EcTHSalpha2和EcTHSbeta1具有催化活性，表明具有天然结构。由于缺乏结构信息及其在质子泵浦中的可能作用，EcTHSbeta1的主要特征。通过荧光和CD研究底物结合特征和构象变化。当NADPH结合时，通过共振能量转移猝灭EcTHSbeta1的单个betaTrp415产生的荧光，该作用为研究底物与该结构域的相互作用和确定Kd值提供了重要的工具。发现对NADPH的表观相对结合亲和力比对NADP +的表观相对结合亲和力高约50倍。在不存在和存在各种温度下添加底物的情况下，圆二色性用于估算二级结构含量并用于EcTHSbeta1的构象分析。结果表明，与NADPH或NADP +复合的结构域III具有不同的构象。等电聚焦和天然凝胶电泳实验支持这一发现。提出这些结构差异在完整酶的构象驱动的质子泵机制中起中心作用。

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