Substrate Promotes Productive Gas Binding in the α-Ketoglutarate-Dependent Oxygenase FIH.
Substrate Promotes Productive Gas Binding in the α-Ketoglutarate-Dependent Oxygenase FIH.
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DOI：
10.1021/acs.biochem.5b01003
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发表时间：
2016-01-19
期刊：
影响因子：
2.9
通讯作者：
Knapp MJ
中科院分区：
文献类型：
--
作者：
Taabazuing CY;Fermann J;Garman S;Knapp MJ
Fe2+/α kg依赖性加氧酶利用分子氧进行多种具有重要生物学意义的反应，如DNA碱基切除修复、组蛋白去甲基化和细胞缺氧反应。这些酶遵循一个顺序的机制，在这个机制中，O2结合并在主要底物结合后发生反应，使得那些促进O2结合的结构因素成为它们化学反应的中心。这一领域的一大挑战是确定产生生产性更替的战略。因子抑制HIF （FIH）是一种依赖Fe2+/α kg的加氧酶，通过羟基化HIF-1α蛋白中的c端反激活结构域（CTAD），形成人类细胞中氧传感机制的一部分。FIH的结构是通过O2类似物NO与Fe结合来解决的，这为该酶的气体结合几何结构提供了第一个直接的见解。通过DFT计算、{FeNO} EPR光谱和UV-Vis吸收光谱的结合，我们证明了CTAD结合通过改变结合气体分子的取向来刺激O2的反应性。虽然FIH对NO具有中等的亲和力，但结合气体可以采用两种取向中的任何一种，稳定性相似；在CTAD结合后，NO采用适合支持氧化脱羧的单一首选取向。结合其他相关酶的研究，我们的数据表明，αKG加氧酶利用底物诱导结合O2的重定向机制将O2活化与底物羟基化紧密结合。
The Fe2+/αKG-dependent oxygenases use molecular oxygen to carry out a wide variety of reactions with important biological implications, such as DNA base-excision repair, histone demethylation, and the cellular hypoxia response. These enzymes follow a sequential mechanism in which O2 binds and reacts after the primary substrate binds, making those structural factors that promote productive O2 binding central to their chemistry. A large challenge in this field is to identify strategies that engender productive turnover. Factor Inhibiting HIF (FIH), is a Fe2+/αKG-dependent oxygenase that forms part of the O2 sensing machinery in human cells by hydroxylating the c-terminal transactivation domain (CTAD) found within the HIF-1α protein. The structure of FIH was solved with the O2 analogue NO bound to Fe, offering the first direct insight into the gas binding geometry in this enzyme. Through a combination of DFT calculations, {FeNO} EPR spectroscopy, and UV-Vis absorption spectroscopy we demonstrate that CTAD binding stimulates O2 reactivity by altering the orientation of the bound gas molecule. Although FIH binds NO with moderate affinity, the bound gas can adopt either of two orientations with similar stability; upon CTAD binding, NO adopts a single preferred orientation that is appropriate to support oxidative decarboxylation. Combined with other studies on related enzymes, our data suggests that substrate induced reorientation of bound O2 is the mechanism utilized by the αKG oxygenases to tightly couple O2 activation to substrate hydroxylation.