Rhizobium leguminosarum HupE is a highly-specific diffusion facilitator for nickel uptake
Rhizobium leguminosarum HupE is a highly-specific diffusion facilitator for nickel uptake
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DOI：
10.1039/c4mt00298a
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发表时间：
2015-01-01
期刊：
影响因子：
3.4
通讯作者：
Palacios, Jose
中科院分区：
文献类型：
--
作者：
Albareda, Marta;Rodrigue, Agnes;Palacios, Jose
在天然的低镍生境中，细菌需要镍转运蛋白来合成含镍金属酶。在这项工作中，我们进行了功能和拓扑特性的豆科根瘤菌HupE，镍渗透酶所需的[NiFe]氢化酶合成提供这个元素。在大肠杆菌nikABCDE突变株HYD 723中的表达研究表明，HupE是一种中等亲和力的通透酶（表观Km 227 +/- 21 nM; V-max 49 +/- 21 pmol Ni 2 + min（-1）mg（-1）细菌干重），其功能是吸收Ni（II）离子的能量非依赖性扩散促进剂。这种Ni 2+转运不受类似阳离子如Mn 2+、Zn 2+或Co 2+的抑制，但被Cu 2+阻断。定点HupE突变体的分析允许鉴定几个残基（H36、D42、H43、F69、E90、H130和E133），这些残基对于在E. coli细胞。通过使用翻译融合报告基因，我们证明了存在五个跨膜结构域与周质N-末端结构域和C-末端结构域埋在脂质双层。周质N-末端结构域有助于蛋白质的稳定性和功能性。
Bacteria require nickel transporters for the synthesis of Ni-containing metalloenzymes in natural, low nickel habitats. In this work we carry out functional and topological characterization of Rhizobium leguminosarum HupE, a nickel permease required for the provision of this element for [NiFe] hydrogenase synthesis. Expression studies in the Escherichia coli nikABCDE mutant strain HYD723 revealed that HupE is a medium-affinity permease (apparent K-m 227 +/- 21 nM; V-max 49 +/- 21 pmol Ni2+ min(-1) mg(-1) bacterial dry weight) that functions as an energy-independent diffusion facilitator for the uptake of Ni(II) ions. This Ni2+ transport is not inhibited by similar cations such as Mn2+, Zn2+, or Co2+, but is blocked by Cu2+. Analysis of site-directed HupE mutants allowed the identification of several residues (H36, D42, H43, F69, E90, H130, and E133) that are essential for HupE-mediated Ni uptake in E. coli cells. By using translational fusions to reporter genes we demonstrated the presence of five transmembrane domains with a periplasmic N-terminal domain and a C-terminal domain buried in the lipid bilayer. The periplasmic N-terminal domain contributes to stability and functionality of the protein.