Crystal structure of Myxococcus xanthus nucleoside diphosphate kinase and its interaction with a nucleotide substrate at 2.0 A resolution.
Crystal structure of Myxococcus xanthus nucleoside diphosphate kinase and its interaction with a nucleotide substrate at 2.0 A resolution.
复制标题
DOI：
10.1006/jmbi.1993.1673
复制
发表时间：
1994-01
影响因子：
5.6
通讯作者：
Roger L. Williams;D. Oren;J. Muñoz-Dorado;S. Inouye;M. Inouye;Edward A Arnold
中科院分区：
文献类型：
--
作者：
Roger L. Williams;D. Oren;J. Muñoz-Dorado;S. Inouye;M. Inouye;Edward A Arnold
已确定的X-射线晶体结构的核苷二磷酸（NDP）激酶从粘球菌xanthus使用多个同晶置换技术，并在2.0 A分辨率的结晶R-因子为0.17的改进。这是第一次报告的结构的酶活性NDP激酶和酶的结合核苷酸。结构已确定为P4（3）2（1）2和I222晶型。酶单体由四链反平行β折叠组成。片的表面部分地覆盖有五个螺旋段。在四聚体晶体形式的不对称单元中有两个蛋白质分子。它们形成具有广泛界面的二聚体，其中每个单体掩埋1092 A2。二聚体界面中的大部分接触面积在疏水或芳族残基之间。两个二聚体通过结晶学2重轴相关以产生四聚体。这种四聚体也存在于斜方晶体中;然而，在这种情况下，222对称性完全是晶体学的。在四聚体形成时，来自每个单体的额外的473 A2的溶剂可及表面积被掩埋。四聚体中二聚体之间的界面通过盐桥稳定。平衡沉降研究与溶液中的酶为四聚体一致。腺苷二磷酸（ADP）与酶的复合物的结构进行了测定，并揭示了大多数的核苷酸与蛋白质的相互作用是与焦磷酸和核糖基团，而基地没有与蛋白质的氢键和相互作用，只有通过堆叠与侧链的Phe 59。Mg 2+与ADP的焦磷酸盐相互作用，并通过溶剂分子与保守的Asp 120残基的侧链相互作用。与核苷酸的相互作用模式是新颖的，核苷酸结合在β折叠的一侧。在存在或不存在Mg 2+的情况下生长的晶体中的核苷酸的结构基本上相同。此外，从三磷酸腺苷（ATP）到酶的磷酸转移反应可以在没有Mg 2+的情况下发生。这表明，只有第二步的反应，其中酶转移磷酸核苷二磷酸受体显着催化的金属。
The X-ray crystallographic structure of nucleoside diphosphate (NDP) kinase from Myxococcus xanthus has been determined using multiple isomorphous replacement techniques and refined at 2.0 A resolution to a crystallographic R-factor of 0.17. This is the first report of the structure of an enzymatically active NDP kinase and of the enzyme with a bound nucleotide. The structure has been determined in P4(3)2(1)2 and I222 crystal forms. The enzyme monomer consists of a four-stranded antiparallel beta-sheet. The surfaces of the sheet are partially covered with five helical segments. There are two protein molecules in the asymmetric unit of the tetragonal crystal form. They form a dimer with an extensive interface in which 1092 A2 per monomer is buried. The majority of the contact area in the dimer interface is between hydrophobic or aromatic residues. Two dimers are related by a crystallographic 2-fold axis to yield a tetramer. This tetramer is also present in the orthorhombic crystals; however, in this case, the 222 symmetry is entirely crystallographic. Upon tetramer formation, an additional 473 A2 of solvent-accessible surface area from each monomer becomes buried. The interface between dimers in the tetramer is stabilized by salt bridges. Equilibrium sedimentation studies are consistent with the enzyme being a tetramer in solution. The structure of a complex of adenosine diphosphate (ADP) with the enzyme was determined and reveals that most of the nucleotide interactions with the protein are with the pyrophosphate and ribose groups, while the base has no hydrogen bonds with the protein and interacts only by stacking with the side chain of Phe59. The Mg2+ interacts with the pyrophosphate of the ADP and via a solvent molecule with the side chain of the conserved Asp120 residue. The mode of interaction with the nucleotide is novel, with the nucleotide binding at the side of the beta-sheet. The structures of the nucleotide in crystals grown in the presence or absence of Mg2+ are essentially identical. In addition, the phosphotransfer reaction from adenosine triphosphate (ATP) to the enzyme can occur without Mg2+. This suggests that only the second step of the reaction in which the enzyme transfers the phosphate to a nucleoside diphosphate acceptor is significantly catalyzed by the metal.