The exact chemical identity of reactive intermediates in O2-dependent uric acid biodegradation

O2 依赖性尿酸生物降解反应中间体的确切化学特性

• 批准号: BB/P000169/1
• 负责人: Roberto Steiner
• 金额: $ 42.21万
• 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: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FP000169%2F1
• 关键词: exact; chemical; identity; reactive; intermediates

Differently from the majority of other animals humans cope with large quantities of uric acid in their bodies. This is because during evolution we have progressively silenced a gene responsible for the production of an enzyme called urate oxidase (UOX). This enzyme is able to break down uric acid into more soluble compounds. The reasons for human adaptation to high uric acid levels are not entirely clear and, interestingly, mice without a functional UOX enzyme die shortly after birth. Under certain pathological conditions that cause a further increase in uric acid UOX is administered to patients to help restore normal levels. Crystalline uric acid deposits are also the hallmark of gout disease.UOX requires molecular oxygen (O2) to perform its task of breaking down uric acid. O2 is a very interesting molecule as in its normal "resting" state (the form present in the air) does not want to react with the vast majority of organic 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. In this work we will investigate how UOX uses O2 to break down uric acid. Interestingly, UOX belongs to a small group of enzymes that can bring oxygen into reacting with their organic substrates and steer the reaction towards the desired products with limited chemical tools at its disposal. In fact, as oxygen activation is not an easy task, the vast majority of enzymes rely on special additional components like metal and/or organic co-factors to form "active oxygen". UOX does not require these additional helpers and therefore understanding how it works is particularly intriguing. Using a technique called X-ray crystallography which allows to 'see' at very high resolution the 3D structure of molecules as small as urate oxidase (10,000 times smaller that the thickness of a human's hair) we have been able to visualise snapshots of the enzyme along the process of uric acid degradation (reaction intermediates) including also the state in which O2 is trapped above the substrate. These snapshots led us to formulate some hypotheses on how urate oxidase works. We are now in an excellent position to study the most elusive and critically important properties of UOX chemistry. For this we will use a technique called neutron crystallography that can detect atoms (hydrogens) that cannot be typically observed even by X-ray crystallography. By combining neutron crystallography, X-ray crystallography, modern spectroscopic techniques and advanced quantum mechanical theoretical methods to probe states that are not experimentally accessible we will understand general rules of O2 biochemistry in the context of UOX function. This integrated approach will allow a deeper understanding not only of UOX but also of oxygen, an essential component of life on Earth.

与大多数其他动物不同的是，人类需要应对体内大量的尿酸。这是因为在进化过程中，我们逐渐沉默了一种基因，该基因负责产生一种名为尿酸氧化酶(UOX)的酶。这种酶能够将尿酸分解成更易溶的化合物。人类适应高尿酸水平的原因尚不完全清楚，有趣的是，没有功能UOX酶的小鼠在出生后不久就会死亡。在导致尿酸进一步增加的某些病理情况下，患者会被给予UOX以帮助恢复正常水平。结晶尿酸沉积也是痛风病的标志。UOX需要分子氧(O2)来完成分解尿酸的任务。O2是一种非常有趣的分子，因为由于与其电子结构有关的原因，O2处于正常的“静止”状态(空气中存在的形式)不想与绝大多数有机分子反应。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)是一种破坏性的“活性氧”形式，在衰老过程中起着重要作用。因此，氧气生物化学的另一个挑战，除了“活性氧”的产生，就是它的控制。在这项工作中，我们将研究UOX是如何利用O2分解尿酸的。有趣的是，UOX属于一小部分酶，它们可以携带氧气与有机底物反应，并在有限的化学工具下将反应导向所需的产品。事实上，由于氧气的活化并不是一件容易的事情，绝大多数的酶都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”。UOX不需要这些额外的助手，因此了解它的工作原理特别有趣。使用一种名为X射线结晶学的技术，可以在非常高的分辨率下‘看到’小到尿酸氧化酶(小于人类头发厚度的1万倍)的分子的3D结构，我们已经能够可视化该酶沿着尿酸降解(反应中间体)的过程的快照，包括O2被困在底物上方的状态。这些快照让我们对尿酸酸氧化酶的工作原理提出了一些假设。我们现在处于一个很好的位置来研究UOX化学中最难以捉摸和至关重要的性质。为此，我们将使用一种名为中子结晶学的技术，该技术可以检测到即使是X射线结晶学也无法观察到的原子(氢)。通过结合中子结晶学、X射线结晶学、现代光谱技术和先进的量子力学理论方法来探测实验上无法获得的态，我们将在UOX函数的背景下了解O2生物化学的一般规律。这种综合的方法将使人们不仅能够更深入地了解UOX，而且还可以更深入地了解氧气，氧气是地球上生命的基本成分。

项目成果

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

Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis.

• DOI: 10.1107/s2052252520013615
• 发表时间: 2021-01-01
• 期刊: IUCrJ
• 影响因子: 3.9
• 作者: McGregor L;Földes T;Bui S;Moulin M;Coquelle N;Blakeley MP;Rosta E;Steiner RA
• 通讯作者: Steiner RA

Neutron crystallographic refinement with REFMAC5 from the CCP4 suite.

• DOI: 10.1107/s2059798323008793
• 发表时间: 2023-12-01
• 期刊: Acta crystallographica. Section D, Structural biology
• 作者: Catapano L;Long F;Yamashita K;Nicholls RA;Steiner RA;Murshudov GN
• 通讯作者: Murshudov GN