Department of Drug Science and Technology, University of Turin, Turin, Italy.
Department of Basic and Clinical Neuroscience, The Maurice Wohl Institute, King's College London, Denmark Hill Campus, London, UK.
IUBMB Life. 2022 Jul;74(7):715-722. doi: 10.1002/iub.2618. Epub 2022 Apr 27.
Iron-sulfur clusters are prosthetic groups that are assembled on their acceptor proteins through a complex machine centered on a desulfurase enzyme and a transient scaffold protein. Studies to establish the mechanism of cluster formation have so far used either in vitro or in vivo methods, which have often resulted in contrasting or non-comparable results. We suggest, here, an alternative approach to study the enzymatic reaction, that is based on the combination of genetically engineered bacterial strains depleted of specific components, and the detection of the enzymatic kinetics in cellular extracts through metabolomics. Our data prove that this ex vivo approach closely reproduces the in vitro results while retaining the full complexity of the system. We demonstrate that co-presence of bacterial frataxin and iron is necessary to observe an inhibitory effect of the enzymatic activity of bacterial frataxin. Our approach provides a new powerful tool for the study of iron-sulfur cluster biogenesis.
铁硫簇是一种通过以脱硫酶和瞬态支架蛋白为中心的复杂机器在其受体蛋白上组装的辅基。目前，用于确定簇形成机制的研究使用了体外或体内方法，但这些方法通常导致相互矛盾或不可比较的结果。我们在这里提出了一种替代方法来研究酶反应，该方法基于组合经基因工程改造的缺乏特定成分的细菌菌株，并通过代谢组学在细胞提取物中检测酶动力学。我们的数据证明，这种离体方法在保留系统的全部复杂性的同时，非常接近地再现了体外结果。我们证明，细菌 frataxin 和铁的共同存在对于观察细菌 frataxin 的酶活性的抑制作用是必要的。我们的方法为铁硫簇生物发生的研究提供了一种新的强大工具。
