Dependence of Helicobacter pylori urease activity on the nickel-sequestering ability of the UreE accessory protein
Dependence of Helicobacter pylori urease activity on the nickel-sequestering ability of the UreE accessory protein
复制标题
DOI：
10.1128/jb.185.16.4787-4795.2003
复制
发表时间：
2003-08-01
影响因子：
3.2
通讯作者：
Maier, RJ
中科院分区：
文献类型：
--
作者：
Benoit, S;Maier, RJ
幽门螺杆菌ureE基因产物以前被证明是表达尿素酶所必需的，但其特征和作用尚未确定。UreE蛋白现在已经在大肠杆菌中过表达、纯化和鉴定，并表达了三个改变的版本来解决UreE的镍封存作用。纯化的尿素E在溶液中形成二聚体，每个二聚体能结合一个镍离子。在携带镍成熟蛋白HYPA和HypB突变的突变体的染色体中引入额外的ureE拷贝，导致部分尿素酶活性恢复(高达野生型水平的24%)。通过将富含组氨酸的序列(His-6或His-10位于C末端，His-10作为三明治融合)添加到UreE蛋白中，构建了与镍结合能力增强的UreE融合蛋白。每种融合蛋白在大肠杆菌中高效表达和纯化，并测定其与镍的结合能力和亲和力。每个构建物也在野生型幽门螺杆菌和HYPA和hiB突变株中表达，用于测定体内尿素酶活性。通过引入所有的工程版本，尿素酶活性都得到了提高，其中最大的镍隔离版本(His-6版本)也赋予了HYPA和hiB突变体最高的尿素酶活性。尿素酶活性的差异不是由于尿素酶多肽的数量不同造成的。将His-6加入到另一种表达的蛋白(磷酸三糖异构酶)中，不会刺激尿素酶，因此，尿素酶的激活与非特异性蛋白结合镍的水平无关。结果表明，幽门螺杆菌尿素酶活性与UreE辅助蛋白的固镍能力之间存在相关性。
The Helicobacter pylori ureE gene product was previously shown to be required for urease expression, but its characteristics and role have not been determined. The UreE protein has now been overexpressed in Escherichia coli, purified, and characterized, and three altered versions were expressed to address a nickel-sequestering role of UreE. Purified UreE formed a dimer in solution and was capable of binding one nickel ion per dimer. Introduction of an extra copy of ureE into the chromosome of mutants carrying mutations in the Ni maturation proteins HypA and HypB resulted in partial restoration of urease activity (up to 24% of the wild-type levels). Fusion proteins of UreE with increased ability to bind nickel were constructed by adding histidine-rich sequences (His-6 or His-10 to the C terminus and His-10 as a sandwich fusion) to the UreE protein. Each fusion protein was overexpressed in E. coli and purified, and its nickel-binding capacity and affinity were determined. Each construct was also expressed in wild-type H. pylori and in hypA and hypB mutant strains for determining in vivo urease activities. The urease activity was increased by introduction of all the engineered versions, with the greatest Ni-sequestering version (the His-6 version) also conferring the greatest urease activity on both the hypA and hypB mutants. The differences in urease activities were not due to differences in the amounts of urease peptides. Addition of His-6 to another expressed protein (triose phosphate isomerase) did not result in stimulation of urease, so urease activation is not related to the level of nonspecific protein-bound nickel. The results indicate a correlation between H. pylori urease activity and the nickel-sequestering ability of the UreE accessory protein.