Identification of the high affinity Mn2+ binding site of bacteriophage lambda phosphoprotein phosphatase: effects of metal ligand mutations on electron paramagnetic resonance spectra and phosphatase activities.
Identification of the high affinity Mn2+ binding site of bacteriophage lambda phosphoprotein phosphatase: effects of metal ligand mutations on electron paramagnetic resonance spectra and phosphatase activities.
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DOI：
10.1021/bi010637a
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发表时间：
2001-07
期刊：
影响因子：
2.9
通讯作者：
D. J. White;N. J. Reiter;R. Sikkink;L. Yu;F. Rusnak
中科院分区：
文献类型：
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作者：
D. J. White;N. J. Reiter;R. Sikkink;L. Yu;F. Rusnak
噬菌体 lambda 磷蛋白磷酸酶 (lambdaPP) 与哺乳动物 Ser/Thr 磷蛋白磷酸酶 (PPP) 具有结构相似性，包括免疫抑制剂药物靶点钙调神经磷酸酶。 PPP 拥有一个包含双核金属簇的保守活性位点，其金属配体由磷酸酯酶基序加上 C 末端的两个额外组氨酸残基提供。 lambdaPP 与 28 种真细菌和古菌磷酸酯酶的多重序列比对鉴定了磷酸酯酶基序的活性位点残基，并且在许多情况下还鉴定了 2 个额外的 C 端 His 金属配体。与 lambdaPP 最相似的是大肠杆菌 PrpA 和 PrpB。使用 lambdaPP 的晶体结构 [Voegtli, W. C., et al. (2000) Biochemistry 39, 15365-15374] 作为 PPP 和相关细菌磷酸酯酶的结构和活性位点模型，我们通过电子顺磁共振 (EPR) 光谱、Mn(2+) 结合分析和磷酸酶动力学研究了用 Mn(2+) 重构的 lambdaPP 的突变形式。对 Mn(2+) 结合活性位点突变 lambdaPP 蛋白的分析表明，H22N、N75H 和 H186N 突变会降低磷酸酶活性，但仍允许单核 Mn(2+) 和 [(Mn(2+))(2)] 结合。高亲和力 Mn(2+) 结合位点由 M2 位点配体 H186 和 Asn75 组成，但不是来自 M1 位点的 H22，后者被认为是较低亲和力位点。
Bacteriophage lambda phosphoprotein phosphatase (lambdaPP) has structural similarity to the mammalian Ser/Thr phosphoprotein phosphatases (PPPs) including the immunosuppressant drug target calcineurin. PPPs possess a conserved active site containing a dinuclear metal cluster, with metal ligands provided by a phosphoesterase motif plus two additional histidine residues at the C-terminus. Multiple sequence alignment of lambdaPP with 28 eubacterial and archeal phosphoesterases identified active site residues from the phosphoesterase motif and in many cases 2 additional C-terminal His metal ligands. Most highly similar to lambdaPP are E. coli PrpA and PrpB. Using the crystal structure of lambdaPP [Voegtli, W. C., et al. (2000) Biochemistry 39, 15365-15374] as a structural and active site model for PPPs and related bacterial phosphoesterases, we have studied mutant forms of lambdaPP reconstituted with Mn(2+) by electron paramagnetic resonance (EPR) spectroscopy, Mn(2+) binding analysis, and phosphatase kinetics. Analysis of Mn(2+)-bound active site mutant lambdaPP proteins shows that H22N, N75H, and H186N mutations decrease phosphatase activity but still allow mononuclear Mn(2+) and [(Mn(2+))(2)] binding. The high affinity Mn(2+) binding site is shown to consist of M2 site ligands H186 and Asn75, but not H22 from the M1 site which is ascribed as the lower affinity site.