Acceleration and control of spin-restricted oxygenation by cofactor-independent dioxygeanses

不依赖辅因子的双加氧酶加速和控制自旋限制氧合

• 批准号: BB/I020411/1
• 负责人: Roberto Steiner
• 金额: $ 45.01万
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI020411%2F1
• 关键词: Acceleration; control; spin; restricted; oxygenation

Humans like all other highly evolved organisms strictly depend on atmospheric oxygen for survival. Oxygen obtained via the respiration process is essential for the production of energy required to carry out our physiological functions as well as for the defence against various kinds of infections. Oxygen is also used for the degradation of various organic compounds and some bacteria use it to help breakdown molecules that are environmental pollutants. This is thanks to the action of particular enzymes, called oxygenases, which are able to promote reactions in which oxygen atoms are incorporated into molecules otherwise difficult to dispose of. The task of oxygenases is a difficult one because oxygen in its normal 'resting' state (the form present in the air) does not want to react with the vast majority of molecules for reasons related to its electronic structure. Oxygen needs activation to react. A major problem, however, is that once 'activated' oxygen can react indiscriminately with many biological molecules with detrimental consequences. For example, reactive oxygen species (ROS) are damaging forms of 'active oxygen' that play an important role in aging. Therefore, besides the generation of 'active oxygen', another challenge in oxygen biochemistry, is its control. ACTIVATION and CONTROL are critical keywords in oxygen-dependent biological processes. In this work we will investigate two bacterial oxygenases called with the acronyms of HOD and QDO which constitute a separate family from other oxygenases. Interestingly, they can bring oxygen into reactions with their organic substrates (ACTIVATION) and steer the reaction towards the desired products (CONTROL) with limited tools at their disposals. In fact, as oxygen activation is not an easy task, the vast majority of oxygenases rely on special additional components like metal and/or organic co-factors to form 'active oxygen'. HOD and QDO don't possess these additional features and therefore understanding how they work is particularly intriguing. Using a technique called X-ray crystallography which allows us to visualise at very high resolution the 3D structure of molecules as small as HOD and QDO (they are about ten thousand times smaller that the thickness of a human's hair) we now know in detail the shape of these enzymes. They do not look like other known dioxygenases; rather they have an architecture of another enzyme family which typically catalyses reactions not involving oxygen. Using the same X-ray technique we have also seen where the substrate binds to HOD when oxygen is not around and what specific interactions it makes with the enzyme. Similarly, we have seen how the reaction product is bound before leaving the enzyme for a new reaction cycle. These snapshots led us to formulate some hypotheses on how HOD/QDO work. We are now in an excellent position to study the most interesting aspects of how HOD/QDO work. These are on one hand the steps in which oxygen gets ACTIVATED and CONTROLLED to convert the substrate into products and on the other hand the reasons which allow a protein scaffold used typically for different reactions to be used here to host oxygen biochemistry. We will again use X-ray crystallography to visualise oxygen bound to the these enzymes, modern spectroscopic techniques to study important electronic properties at different stages of the reaction cycle, and advanced quantum mechanical theoretical methods to probe states that are not experimentally accessible. This multi-angle approach will allow novel insights into the biology of oxygen, an essential component of life on Earth.

像所有其他高度进化的生物一样，人类严格依赖大气中的氧气生存。通过呼吸过程获得的氧气对于产生执行我们的生理功能所需的能量以及防御各种感染是必不可少的。氧也被用来降解各种有机化合物，一些细菌用它来帮助分解作为环境污染物的分子。这要归功于被称为加氧酶的特殊酶的作用，加氧酶能够促进氧原子被结合到分子中的反应，否则很难处理。氧合酶的任务是一项困难的任务，因为由于与其电子结构有关的原因，处于正常休息状态(空气中存在的形式)的氧气不想与绝大多数分子反应。氧气需要活化才能反应。然而，一个主要的问题是，一旦被激活，氧气就会不分青红皂白地与许多生物分子发生反应，造成有害的后果。例如，活性氧物种(ROS)是一种有害的“活性氧”形式，在衰老过程中起着重要作用。因此，除了“活性氧”的产生，氧气生物化学的另一个挑战是它的控制。激活和控制是氧依赖生物过程中的关键关键字。在这项工作中，我们将研究两种细菌加氧酶，它们被称为HOD和QDO，它们构成了一个独立于其他加氧酶的家族。有趣的是，它们可以将氧气带入与其有机底物的反应中(活化)，并使用有限的工具将反应引导到所需的产品(控制)。事实上，由于氧活化并不是一件容易的事情，绝大多数加氧酶依赖于特殊的附加成分，如金属和/或有机辅助因子来形成“活性氧”。HOD和QDO不具备这些附加功能，因此了解它们的工作原理特别有趣。使用一种名为X射线结晶学的技术，我们可以以非常高的分辨率可视化小到HOD和QDO(它们的厚度大约是人类头发厚度的一万倍)的分子的3D结构，我们现在详细地知道了这些酶的形状。它们看起来不像其他已知的双加氧酶；相反，它们具有另一个酶家族的结构，通常催化不涉及氧气的反应。使用同样的X射线技术，我们还看到了当氧气不在身边时，底物与HOD结合的位置，以及它与酶发生了什么特定的相互作用。同样，我们已经看到了在离开酶进入新的反应周期之前，反应产物是如何结合的。这些快照让我们对HOD/QDO如何工作提出了一些假设。我们现在处于一个很好的位置来研究HOD/QDO如何工作的最有趣的方面。一方面是氧气被激活和控制以将底物转化为产物的步骤，另一方面是允许在这里使用通常用于不同反应的蛋白质支架来承载氧气生物化学的原因。我们将再次使用X射线结晶学来显示与这些酶结合的氧，用现代光谱技术来研究反应周期不同阶段的重要电子性质，以及用先进的量子力学理论方法来探测实验上无法达到的状态。这种多角度的方法将使人们能够对氧气的生物学有新的见解，氧气是地球上生命的重要组成部分。

项目成果

Origin of the proton-transfer step in the cofactor-free (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase: effect of the basicity of an active site His residue.

• DOI: 10.1074/jbc.m113.543033
• 发表时间: 2014-03-21
• 期刊: The Journal of biological chemistry
• 作者: Hernandez-Ortega A;Quesne MG;Bui S;Heuts DP;Steiner RA;Heyes DJ;de Visser SP;Scrutton NS
• 通讯作者: Scrutton NS

Rapid and efficient room-temperature serial synchrotron crystallography using the CFEL TapeDrive.

• DOI: 10.1107/s2052252522010193
• 发表时间: 2022-11-01
• 期刊: IUCrJ
• 影响因子: 3.9

Direct evidence for a peroxide intermediate and a reactive enzyme-substrate-dioxygen configuration in a cofactor-free oxidase.

• DOI: 10.1002/anie.201405485
• 发表时间: 2014-12-08
• 期刊: Angewandte Chemie (International ed. in English)
• 作者: Bui S;von Stetten D;Jambrina PG;Prangé T;Colloc'h N;de Sanctis D;Royant A;Rosta E;Steiner RA
• 通讯作者: Steiner RA

Online Raman spectroscopy for structural biology on beamline ID29 of the ESRF.

ESRF 光束线 ID29 上的结构生物学在线拉曼光谱。

• DOI: 10.1016/j.jsb.2017.10.004
• 发表时间: 2017
• 期刊: Journal of structural biology
• 影响因子: 3
• 作者: Von Stetten D