BEORHN: Bacterial Enzymatic Oxidation of Reactive Hydroxylamine in Nitrification via Combined Structural Biology and Molecular Simulation

BEORHN：通过结合结构生物学和分子模拟进行硝化反应中活性羟胺的细菌酶氧化

• 批准号: BB/V016660/1
• 负责人: Thomas Keal
• 金额: $ 46.95万
• 依托单位: Science and Technology Facilities Council
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV016660%2F1
• 关键词: BEORHN; Bacterial; Enzymatic; Oxidation; Reactive

The nitrogen cycle is critical to the environment and global health. The majority of nitrogen used in modern agriculture comes from artificial fertiliser comprised primarily of ammonia or ammonium compounds. This is converted into nitrogen-containing chemicals that are useful to plants (e.g. nitrate) by the action of nitrifying bacteria in soils and water and is then returned to nitrogen gas in the atmosphere through further bacterial action. Losses or imbalances in these processes lead to the release of the pollutant and greenhouse gas nitrous oxide (N2O), the pollutant nitric oxide (NO), the toxic intermediate hydroxylamine (NH2OH), or nitrites/nitrates into freshwater, resulting in algal blooms. Understanding the nitrification process is therefore critically important for agriculture, food security, the environment and human health. In the nitrification process, the second step involves the oxidation of the reactive compound hydroxylamine, catalysed by metal-containing proteins which contain a highly unusual iron-heme structure where the heme contains an additional bond or 'cross-link' to the protein. Two families of structurally very different proteins, hydroxylamine oxidoreductase (HAO) and cytochrome P460 (CytP460), carry out this chemical reaction to yield different reaction products (NO for HAO and N2O for CytP460). Each functional unit of HAO contains seven iron-heme units that function to transfer or 'shuttle' electrons and one P460 heme unit where the heme is further modified via cross-linking to a tyrosine amino acid residue and where the oxidation of hydroxylamine occurs. In CytP460s each functional unit contains one catalytic P460 unit but, in this case, cross linked to a different kind of amino acid (lysine). Furthermore, to add to the complexity, within the CytP460 family, the two proteins so far identified in different families of bacteria (N. europaea and M. capsulatus), have different heme environments despite carrying out exactly the same chemical reaction. Our project addresses this poorly understood second step in the nitrification process, namely the catalytic oxidation of hydroxylamine by HAO and CytP460. We will target these protein systems by combining integrated spectroscopic and structural biology approaches and computational chemistry using high performance computing. We will use X-ray crystallography with near-simultaneous measurement of spectroscopic data of the same crystal to assign correct electronic states to the enzyme's active site. We will use thousands of very small (micro)crystals to obtain structures of enzymes at room temperature and to produce structural movies of the enzymes in action (more traditional techniques produce an average structure more similar to a single movie frame). These spectroscopic and structural data will be combined with state-of-the-art computational methods (molecular dynamics and recently developed quantum mechanics/molecular mechanics approaches) to better understand at the atomic level how these enzymes work. Linking experiments and simulations in this way, we will obtain a fundamental understanding of the function of these enzymes, and why the reactions they catalyse result in different products. Our ultimate goal is to design new, mutated enzymes, using our knowledge of how their structure affects the reactions they catalyse, to change their products from NO to N2O and vice versa, so demonstrating the potential for control of catalysis in future biotechnological applications.

氮周期对环境和全球健康至关重要。现代农业中使用的大多数氮都来自人造肥料，主要由氨或铵化合物组成。这通过硝化细菌在土壤和水中的作用而转化为对植物（例如硝酸盐）有用的含氮化学物质，然后通过进一步的细菌作用将其返回到大气中的氮气中。这些过程中的损失或失衡导致污染物和温室气氮氧化物（N2O），污染物一氧化氮（NO），有毒的中间羟胺（NH2OH）或亚硝酸盐/硝酸盐释放到淡水中。因此，了解硝化过程对于农业，粮食安全，环境和人类健康至关重要。在硝化过程中，第二步涉及反应性化合物羟胺的氧化，该反应性化合物羟胺由含金属的蛋白质催化，其中包含高度不寻常的铁 - 血红素结构，其中血红素包含额外的键或与蛋白质的“交联”。两个结构上非常不同的蛋白质家族，羟胺氧化还原酶（HAO）和细胞色素P460（CYTP460）进行了这种化学反应，以产生不同的反应产物（对于Cytp460的HAO和N2O NO）。 HAO的每个功能单元都包含七个铁血红素单元，这些单元可传递或“穿梭”电子和一个P460血红素单元，其中血红素通过交联通过交联对酪氨酸氨基酸残基进行了进一步修饰，以及羟胺的氧化发生。在CYTP460中，每个功能单元包含一个催化P460单元，但在这种情况下，与不同种类的氨基酸（赖氨酸）链接的交叉。此外，为了增加Cytp460家族中的复杂性，尽管在不同的细菌（N. europaea和M. capsulatus）中鉴定出的两种蛋白质，尽管进行了完全相同的化学反应，但仍具有不同的血红素环境。我们的项目解决了硝化过程中的第二步，即HAO和CYTP460对羟胺的催化氧化。我们将通过使用高性能计算结合综合光谱和结构生物学方法和计算化学来瞄准这些蛋白质系统。我们将使用X射线晶体学对同一晶体的光谱数据进行近距离测量，以将正确的电子状态分配给酶的活性位点。我们将使用数千种非常小的（微型）晶体在室温下获得酶的结构，并在作用中生产酶的结构电影（更传统的技术产生的平均结构与单个电影框架更相似）。这些光谱和结构数据将与最先进的计算方法（分子动力学和最近开发的量子力学/分子力学方法）结合使用，以在原子水平上更好地理解这些酶的作用。通过这种方式将实验和模拟联系起来，我们将获得对这些酶功能的基本理解，以及为什么它们催化的反应会导致不同的产物。我们的最终目标是使用我们对结构如何影响其催化反应的知识来设计新的，突变的酶，将其产品从NO更改为N2O，反之亦然，因此证明了在未来生物技术应用中控制催化的潜力。

项目成果

Computational infrared and Raman spectra by hybrid QM/MM techniques: a study on molecular and catalytic material systems.

• DOI: 10.1098/rsta.2022.0234
• 发表时间: 2023-07-10
• 期刊: PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY A-MATHEMATICAL PHYSICAL AND ENGINEERING SCIENCES
• 影响因子: 5
• 作者: Guan, Jingcheng;Lu, You;Sen, Kakali;Nasir, Jamal Abdul;Desmoutier, Alec W. W.;Hou, Qing;Zhang, Xingfan;Logsdail, Andrew J. J.;Dutta, Gargi;Beale, Andrew M. M.;Strange, Richard W. W.;Yong, Chin;Sherwood, Paul;Senn, Hans M. M.;Catlow, C. Richard A.;Keal, Thomas W. W.;Sokol, Alexey A. A.
• 通讯作者: Sokol, Alexey A. A.

Multiscale QM/MM modelling of catalytic systems with ChemShell

• DOI: 10.1039/d3cp00648d
• 发表时间: 2023-04-20
• 期刊: PHYSICAL CHEMISTRY CHEMICAL PHYSICS
• 影响因子: 3.3
• 作者: Lu，You;Sen，Kakali;Keal，Thomas W.
• 通讯作者: Keal，Thomas W.