Structure and Mutational Analysis of a Plant Mitochondrial Nucleoside Diphosphate Kinase. Identification of Residues Involved in Serine Phosphorylation and Oligomerization1
Structure and Mutational Analysis of a Plant Mitochondrial Nucleoside Diphosphate Kinase. Identification of Residues Involved in Serine Phosphorylation and Oligomerization1
复制标题
植物线粒体核苷二磷酸激酶的结构和突变分析。
作者：
M. Johansson;Alasdair K. MacKenzie;I. Andersson;C. Knorpp
我们首次报道了一种植物(豌豆属植物，俄勒冈州糖荚)线粒体核苷二磷酸的晶体结构。与其他真核生物核苷二磷酸酶类似，植物酶是一个六角体；不对称单元中的六个单体以二聚体的三聚体的形式排列。不同的功能与寡聚体结构和丝氨酸残基的磷酸化有关。我们发现，丝氨酸自身磷酸化的发生依赖于酶的活性。严格保守的Ser-119突变为Ala，使Ser的磷酸化程度降低到野生型的一半左右，酶活性只有轻微的变化。我们还表明，将另一个严格保守的Ser，Ser-69突变为Ala后，酶活性分别降低到以dCDP和dTDP为受体的野生型的6%和14%。通过交联实验观察到S69A突变体的寡聚模式发生了变化。随着四聚体的增加，可以检测到三聚体形成的减少和二聚体相互作用的变化。我们认为S69突变体参与了该植物核苷二磷酸激酶低聚体状态的稳定。
We report the first crystal structure of a plant (Pisum sativum L. cv Oregon sugarpod) mitochondrial nucleoside diphosphate kinase. Similar to other eukaryotic nucleoside diphosphate kinases, the plant enzyme is a hexamer; the six monomers in the asymmetric unit are arranged as trimers of dimers. Different functions of the kinase have been correlated with the oligomeric structure and the phosphorylation of Ser residues. We show that the occurrence of Ser autophosphorylation depends on enzymatic activity. The mutation of the strictly conserved Ser-119 to Ala reduced the Ser phosphorylation to about one-half of that observed in wild type with only a modest change of enzyme activity. We also show that mutating another strictly conserved Ser, Ser-69, to Ala reduces the enzyme activity to 6% and 14% of wild-type using dCDP and dTDP as acceptors, respectively. Changes in the oligomerization pattern of the S69A mutant were observed by cross-linking experiments. A reduction in trimer formation and a change in the dimer interaction could be detected with a concomitant increase of tetramers. We conclude that the S69 mutant is involved in the stabilization of the oligomeric state of this plant nucleoside diphosphate kinase.
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DOI：
10.1073/pnas.97.26.14194
发表时间：
2000-12-19
影响因子：
11.1
作者：
Postel, EH;Abramczyk, BM;Kyin, S
通讯作者：
Kyin, S
影响因子：
5.6
作者：
Philip A. Webb;O. Perisic;Chairmaine E. Mendola;J. Backer;Roger L. Williams
通讯作者：
Philip A. Webb;O. Perisic;Chairmaine E. Mendola;J. Backer;Roger L. Williams
影响因子：
5.6
作者：
Roger L. Williams;D. Oren;J. Muñoz-Dorado;S. Inouye;M. Inouye;Edward A Arnold
通讯作者：
Roger L. Williams;D. Oren;J. Muñoz-Dorado;S. Inouye;M. Inouye;Edward A Arnold
影响因子：
10.3
作者：
STEEG, PS;BEVILACQUA, G;SOBEL, ME
通讯作者：
SOBEL, ME