Structural and biochemical implications of single amino acid substitutions in the nucleotide-dependent switch regions of the nitrogenase Fe protein from Azotobacter vinelandii.
Structural and biochemical implications of single amino acid substitutions in the nucleotide-dependent switch regions of the nitrogenase Fe protein from Azotobacter vinelandii.
复制标题
维氏固氮菌固氮酶 Fe 蛋白的核苷酸依赖性开关区域中单个氨基酸取代的结构和生化意义。
DOI：
10.1007/s00775-004-0605-5
复制
发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Peters,JohnW
中科院分区：
文献类型：
--
作者：
Jang,SeBok;Jeong,MiSuk;Seefeldt,LanceC;Peters,JohnW
用X射线衍射法测定了在先前所涉及的核苷酸依赖的信号转导通路中具有特定氨基酸取代的固氮酶铁蛋白的结构，称为Switch I和Switch II。在固氮酶的铁蛋白中，核苷酸依赖的开关区负责负责核苷酸结合和水解的位点与铁蛋白的[4Fe-4S]簇之间的通信，以及在形成大分子复合体时与MoFe蛋白相互作用的对接界面。在这项研究中，棕色固氮菌二氨氮固氮酶铁蛋白的结构特征是在镁ADP结合和无核苷酸状态下，39位天冬氨酸被天冬氨酸取代，这为实验观察到改变的铁蛋白在单一电子转移事件后形成捕获复合体提供了解释。结构表明，取代允许在开关I Asn39和开关II Asp125之间形成氢键。在天然酶的结构中，由于静电排斥，Asp39和Asp125的侧链之间不存在类似的相互作用。这些结果表明，Asp39和Asp125之间的静电斥力是催化过程中Fe蛋白：MoFe蛋白复合体解离的重要原因。在另一项研究中，用谷氨酸取代Asp129的铁蛋白的结构特征为观察到在没有结合核苷酸的情况下Glu129取代的变体具有与结合了镁ADP的天然铁蛋白相同的生化性质提供了结构基础。较长的Glu侧链与磷酸结合环(P-loop)的相互作用导致Switch II区的构象类似于ADP的磷酸与P-环结合所产生的构象。
The structures of nitrogenase Fe proteins with defined amino acid substitutions in the previously implicated nucleotide-dependent signal transduction pathways termed switch I and switch II have been determined by X-ray diffraction methods. In the Fe protein of nitrogenase the nucleotide-dependent switch regions are responsible for communication between the sites responsible for nucleotide binding and hydrolysis and the [4Fe-4S] cluster of the Fe protein and the docking interface that interacts with the MoFe protein upon macromolecular complex formation. In this study the structural characterization of theAzotobacter vinelandiinitrogenase Fe protein with Asp at position 39 substituted by Asn in MgADP-bound and nucleotide-free states provides an explanation for the experimental observation that the altered Fe proteins form a trapped complex subsequent to a single electron transfer event. The structures reveal that the substitution allows the formation of a hydrogen bond between the switch I Asn39 and the switch II Asp125. In the structure of the native enzyme the analogous interaction between the side chains of Asp39 and Asp125 is precluded due to electrostatic repulsion. These results suggest that the electrostatic repulsion between Asp39 and Asp125 is important for dissociation of the Fe protein:MoFe protein complex during catalysis. In a separate study, the structural characterization of the Fe protein with Asp129 substituted by Glu provides the structural basis for the observation that the Glu129-substituted variant in the absence of bound nucleotides has biochemical properties in common with the native Fe protein with bound MgADP. Interactions of the longer Glu side chain with the phosphate binding loop (P-loop) results in a similar conformation of the switch II region as the conformation that results from the binding of the phosphate of ADP to the P-loop.