Cuevas Zuviría Bruno, Detemple Franka, Amritkar Kaustubh, Garcia Amanda K, Seefeldt Lance, Einsle Oliver, Kaçar Betül
Department of Bacteriology, University of Wisconsin-Madison, Madison, United States.
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM)-Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria-CSIC (INIA/CSIC), Campus de Montegancedo, Madrid, Spain.
Elife. 2025 Sep 11;14:RP105613. doi: 10.7554/eLife.105613.
Previously, we identified the only dinitrogen reduction mechanism known to date as an ancient feature conserved from nitrogenase ancestors, which we directly tested by resurrecting and integrating synthetic ancestral nitrogenases into the genome of (Garcia et al., 2023), a genetically tractable, nitrogen-fixing model bacterium. Here, we extend this paleomolecular approach to investigate the structural evolution of nitrogenase over billions of years of evolution by combining phylogenetics, ancestral sequence reconstruction, protein crystallography, and deep-learning based predictions. This study reveals that nitrogenase, while maintaining a conserved multimeric core, evolved novel modular features aligned with major environmental transitions, suggesting that subtle distal changes and transient regulatory adaptations were key to its long-term persistence and to shaping protein evolution over geologic time. The framework established here provides a foundation for identifying structural constraints that governed ancient proteins and for situating their sequences and structures within phylogenetic and environmental contexts across time.
此前，我们确定了迄今为止已知的唯一一种二氮还原机制，它是从固氮酶祖先那里保留下来的一种古老特征，我们通过复活合成的祖先固氮酶并将其整合到一种基因易于操作的固氮模式细菌（加西亚等人，2023年）的基因组中，对此进行了直接测试。在这里，我们扩展了这种古分子方法，通过结合系统发育学、祖先序列重建、蛋白质晶体学和基于深度学习的预测，来研究数十亿年进化过程中固氮酶的结构演变。这项研究表明，固氮酶在保持保守的多聚体核心的同时，进化出了与主要环境转变相一致的新型模块化特征，这表明细微的远端变化和短暂的调节适应是其长期存续以及在地质时间内塑造蛋白质进化的关键。这里建立的框架为识别控制古代蛋白质的结构限制以及将它们的序列和结构置于跨越时间的系统发育和环境背景中奠定了基础。
