Recombination of 2Fe-2S Ferredoxins Reveals Differences in the Inheritance of Thermostability and Midpoint Potential
Recombination of 2Fe-2S Ferredoxins Reveals Differences in the Inheritance of Thermostability and Midpoint Potential
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DOI：
10.1021/acssynbio.0c00303
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发表时间：
2020-12-18
影响因子：
4.7
通讯作者：
Silberg, Jonathan J.
中科院分区：
文献类型：
--
作者：
Campbell, Ian J.;Kahanda, Dimithree;Silberg, Jonathan J.
纯化可以在实验室中使用，以克服合成生物学中的组分限制，通过创建表现出与天然蛋白质不同的活性和稳定性的酶。为了研究重组如何影响在细胞中转移电子的氧化还原酶的性质，我们通过重组远亲蓝藻和蓝藻噬菌体Fds（53%的同一性）产生了铁氧还蛋白（Fd）嵌合体，它们具有相似的中点电位，但具有不同的热稳定性。FD嵌合体具有广泛的氨基酸取代保留的能力，以协调铁硫簇，虽然它们的热稳定性不同的分数从每个父母继承的残基。嵌合Fds的中点电位也不同。然而，所有合成的Fds都表现出在亲本蛋白质范围之外的中点电位。每个嵌合Fds也可以支持大肠杆菌中Fd-NADP还原酶和亚硫酸盐还原酶之间的电子转移，尽管嵌合Fds在相似水平的细胞电子转移所需的表达方面有所不同。这些结果显示了Fds如何通过重组而多样化，并揭示了热稳定性和电化学性质的遗传差异。此外，它们说明了如何使用合成代谢途径快速评估嵌合Fds的电子转移效率。
Recombination can be used in the laboratory to overcome component limitations in synthetic biology by creating enzymes that exhibit distinct activities and stabilities from native proteins. To investigate how recombination affects the properties of an oxidoreductase that transfers electrons in cells, we created ferredoxin (Fd) chimeras by recombining distantly related cyanobacterial and cyanomyophage Fds (53% identity) that present similar midpoint potentials but distinct thermostabilities. Fd chimeras having a wide range of amino acid substitutions retained the ability to coordinate an iron-sulfur cluster, although their thermostabilities varied with the fraction of residues inherited from each parent. The midpoint potentials of chimeric Fds also varied. However, all of the synthetic Fds exhibited midpoint potentials outside of the parental protein range. Each of the chimeric Fds could also support electron transfer between Fd-NADP reductase and sulfite reductase in Escherichia coli, although the chimeric Fds varied in the expression required for similar levels of cellular electron transfer. These results show how Fds can be diversified through recombination and reveal differences in the inheritance of thermostability and electrochemical properties. Furthermore, they illustrate how electron transfer efficiencies of chimeric Fds can be rapidly evaluated using a synthetic metabolic pathway.