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首页> 外文期刊>Biochemistry >Mn~(2+) is a native metal ion activator for bacteriophage lambda protein phosphatase
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Mn~(2+) is a native metal ion activator for bacteriophage lambda protein phosphatase

机译：Mn〜（2+）是噬菌体λ蛋白磷酸酶的天然金属离子活化剂

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摘要

Bacteriophage lambda protein phosphatase (lambdaPP) is a member of a large family of metal-containing phosphoesterases, including purple acid phosphatase, protein serine/threonine phosphatases, 5'-nucleotidase, and DNA repair enzymes such as Mre11. lambdaPP can be activated several-fold by various divalent metal ions, with Mn~(2+) and Ni~(2+) providing the most significant activatin. Despite the extensive characterization of purified lambdaPP in vitro, little is known about the identity and stoichiometry of metal ions used by lambdaPP in vivo. In this report, we describe the use of metal analysis, activity measurements, and whole cell EPR spectroscopy to investigate in vivo metal binding and activation of lambdaPP. Escherichia coli cells overexpressing lambdaPP show a 22.5-fold increase in intracellular Mn concentration and less dramatic changes in the intracellular concentration of other biologically relevant metal ions compared to control cells that do not express lambdaPP. Phosphatase activity assessed using para-nitrophenylphosphate as substrate is increased 850-fold in cells overexpressing lambdaPP, indicating the presence of metal-activated enzyme in cell lysate. EPR spectra of intact cells overexpressing lambdaPP exhibit resonances previously attributed to mononuclear Mn~(2+) and dinuclear [(Mn~(2+))_2] species bound to lambdaPP.Spin quantitation of EPR spectra of intact E.coli cells overexpressing lambdaPP indicates the presence of approximately 40 muM mononuclear Mn~(2+)-lambdaPP and 60 muM[(Mn~(2+))_2]-lambdaPP. The data suggest that overexpression of lambdaPP results in a mixture of apo-, mononuclear-Mn~(2+), and dinuclear-[(Mn~(2+))_2] metalloisoforms and that Mn~(2+) is a physiologically relevant activating metal ion in E. coli.
机译：噬菌体λ蛋白磷酸酶（lambdaPP）是一大类含金属的磷酸酯酶的成员，包括紫色酸性磷酸酶，蛋白质丝氨酸/苏氨酸磷酸酶，5'-核苷酸酶和DNA修复酶（例如Mre11）。 lambdaPP可以被各种二价金属离子活化数倍，其中Mn〜（2+）和Ni〜（2+）提供最显着的活化素。尽管在体外对纯化的lambdaPP进行了广泛的表征，但对于lambdaPP在体内使用的金属离子的身份和化学计量知之甚少。在此报告中，我们描述了使用金属分析，活性测量和全细胞EPR光谱研究体内金属结合和lambdaPP的激活。与不表达lambdaPP的对照细胞相比，过表达lambdaPP的大肠杆菌细胞的细胞内锰浓度增加了22.5倍，而其他生物学相关金属离子的细胞内浓度变化却不那么明显。在过表达lambdaPP的细胞中，使用对硝基苯基磷酸酯为底物评估的磷酸酶活性增加了850倍，表明细胞裂解物中存在金属激活的酶。过量表达lambdaPP的完整细胞的EPR谱显示出共振，该共振先前归因于与lambdaPP结合的单核Mn〜（2+）和双核[（Mn〜（2 +））_ 2]物种。旋转定量完整表达过的lambdaPP的大肠杆菌细胞的EPR谱表示存在约40μM单核Mn〜（2 +）-lambdaPP和60 muM [（Mn〜（2 +））_ 2] -lambdaPP。数据表明lambdaPP的过表达导致脱辅基-，单核-Mn〜（2+）和双核-[（Mn〜（2 +））_ 2]金属亚型的混合物，并且Mn〜（2+）在生理上是大肠杆菌中的相关活化金属离子。

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