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.

氮循环对环境和全球健康至关重要。现代农业中使用的大部分氮来自主要由氨或铵化合物组成的人造肥料。通过土壤和水中的硝化细菌的作用，将其转化为对植物有用的含氮化学物质（例如硝酸盐），然后通过进一步的细菌作用返回大气中的氮气。这些过程中的损失或不平衡导致污染物和温室气体一氧化二氮（N2 O）、污染物一氧化氮（NO）、有毒中间体羟胺（NH 2 OH）或亚硝酸盐/硝酸盐释放到淡水中，导致藻类大量繁殖。因此，了解硝化过程对农业、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，由含金属的蛋白质催化，所述含金属的蛋白质含有非常不寻常的铁-血红素结构，其中血红素含有与蛋白质的额外键或“交联”。两个家族的结构非常不同的蛋白质，羟胺氧化还原酶（HAO）和细胞色素P460（CytP 460），进行这种化学反应，产生不同的反应产物（NO的HAO和N2 O的CytP 460）。HAO的每个功能单元含有七个铁-血红素单元，其功能是转移或“穿梭”电子，和一个P460血红素单元，其中血红素通过交联进一步修饰为酪氨酸氨基酸残基，并且其中发生羟胺的氧化。在CytP 460中，每个功能单元包含一个催化P460单元，但在这种情况下，交联到不同种类的氨基酸（赖氨酸）。此外，为了增加复杂性，在CytP 460家族中，迄今为止在不同细菌家族中鉴定的两种蛋白质（N. europaea和M. capsulatus），尽管进行完全相同的化学反应，但具有不同的血红素环境。我们的项目解决了硝化过程中这一知之甚少的第二步，即HAO和CytP 460对羟胺的催化氧化。我们将通过结合集成光谱和结构生物学方法以及使用高性能计算的计算化学来针对这些蛋白质系统。我们将使用X射线晶体学与几乎同时测量同一晶体的光谱数据，以分配正确的电子状态的酶的活性位点。我们将使用数千个非常小的（微）晶体在室温下获得酶的结构，并制作酶的结构电影（更传统的技术产生的平均结构更类似于单个电影帧）。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）相结合，以更好地了解这些酶在原子水平上的工作方式。以这种方式连接实验和模拟，我们将获得对这些酶的功能的基本理解，以及为什么它们催化的反应会产生不同的产物。我们的最终目标是设计新的突变酶，利用我们对它们的结构如何影响它们催化的反应的知识，将它们的产物从NO变成N2 O，反之亦然，从而展示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.