Structure-Function Relationships in the Oligomeric NADPH-Dependent Assimilatory Sulfite Reductase
Structure-Function Relationships in the Oligomeric NADPH-Dependent Assimilatory Sulfite Reductase
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DOI：
10.1021/acs.biochem.8b00446
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发表时间：
2018-07-03
期刊：
影响因子：
2.9
通讯作者：
Stroupe, M. Elizabeth
中科院分区：
文献类型：
--
作者：
Askenasy, Isabel;Murray, Daniel T.;Stroupe, M. Elizabeth
硫同化的中心步骤是亚硫酸盐的六电子还原为硫化物，由氧化还原酶nadph依赖的同化亚硫酸盐还原酶（SiR）催化。SiR由两个亚基组成。一个是多结构域黄素结合还原酶（SiRFP），另一个是含铁氧化酶（SiRHP）。这两种酶主要是球形的，正如它们作为氧化还原酶的功能所期望的那样。因此，我们对它们的结构有相当多的了解，但不知道它们是如何组装的。奇怪的是，这两个结构都有明显的结构未定义的区域，这让人们对它们的功能产生了疑问，并提出了它们在形成更大的复合体中起关键作用的可能性。在这里，我们使用紫外可见光谱和圆二色光谱、等温滴定量热法、蛋白水解敏感性试验、电喷雾电离质谱法和活性分析来探索改变‘ ’中特定氨基酸对其在全酶复合物中功能的影响。此外，我们使用计算分析来预测这两个亚基内禀紊乱的倾向，发现SiRHP的n端被预测具有与内禀紊乱相关的特性。这两种蛋白质还含有内部区域，其性质表明内在紊乱。我们发现SiRHP的n端无序区对复合物的形成至关重要。结合我们对SiRFP氨基酸变异的分析，我们展示了每个SiR球形酶核心外的分子相互作用如何驱动这种原型氧化还原酶的复合体组装。
The central step m the assimilation of sulfur is a six-electron reduction of sulfite to sulfide, catalyzed by the oxidoreductase NADPH-dependent assimilatory sulfite reductase (SiR). SiR is composed of two subunits. One is a multidomain flavin binding reductase (SiRFP) and the other an iron-containing oxidase (SiRHP). Both enzymes are primarily globular, as expected from their functions as redox enzymes. Consequently, we know a fair amount about their structures but not how they assemble. Curiously, both structures have conspicuous regions that are structurally undefined, leaving questions about their functions and raising the possibility that they are critical in forming the larger complex. Here, we used ultraviolet-visible and circular dichroism spectroscopy, isothermal titration calorimetry, proteolytic sensitivity tests, electrospray ionization mass spectrometry, and activity assays to explore the effect of altering specific amino acids in `` on their function in the holoenzyme complex. Additionally, we used computational analysis to predict the propensity for intrinsic disorder within both subunits and found that SiRHP's N-terminus is predicted to have properties associated with intrinsic disorder. Both proteins also contained internal regions with properties indicative of intrinsic disorder. We showed that SiRHP's N-terminal disordered region is critical for complex formation. Together with our analysis of SiRFP amino acid variants, we show how molecular interactions outside the core of each SiR globular enzyme drive complex assembly of this prototypical oxidoreductase.