Constructing catalytically proficient enzymes from de novo designed proteins

从头设计的蛋白质构建催化效率高的酶

• 批准号: BB/R016445/1
• 负责人: Ross Anderson
• 金额: $ 64.97万
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR016445%2F1
• 关键词: Constructing; catalytically; proficient; enzymes; de

Enzymes are fundamentally important biological molecules that perform the bulk of the chemical reactions in all living organisms. The are themselves proteins, made up of chains of amino acids, though what differentiates them from proteins is their ability to massively increase the rate of chemical reactions. These reactions power cellular life and are involved in a great number of essential processes that give cells their chemical and physical characteristics. Many enzymes perform chemical reactions which have substantial commercial or medical value, as the products of the transformations may be drugs, fuels or other useful substances or materials. It is often the case that for such important or useful reactions, there are no manmade substances available to catalyse the specific chemical transformations with the same degree of precision or efficiency as enzymes. There are also many chemical transformations for which no enzyme has yet been discovered. Therefore, there is a huge interest in building tailor-made enzymes capable of performing selected chemical reactions. While we have gained an incredibly powerful understanding of natural enzymatic catalysis over the past 100 years, there remains a shortfall in the capabilities of artificial, designed enzymes and those found in nature. We believe that this due, in part, to the prevalent use of naturally evolved proteins as the starting points for creating artificial enzymes. These natural proteins may be fragile, difficult and expensive to purify, inactive out of their cellular environment, chemically sensitive to organic molecules and solvents, and, most significantly, they invariably bring an evolutionary complexity with them that can hinder modification by the enzyme designer. We believe that this evolutionary baggage is not a necessary feature of proteins and enzymes and that in certain cases, it might be preferable to work with proteins untouched by natural selection. Our simple proteins, called maquettes, are small robust protein scaffolds that contain no natural protein sequences, and are therefore free from any complexity imposed by evolution. Typically, we design these maquettes to include a non-protein molecule that imparts its own reactivity onto the scaffold. The heme molecule is a particularly versatile molecule that we include in our designs, and it is present in a plethora of natural enzymes, many of which catalyse exceptionally challenging chemical reactions.With our most recent work, we have used an elementary design process to develop a heme-containing maquette into an active artificial enzyme that functions as well as many natural enzymes for the removal of electrons - oxidation - from a broad range of substrates. It can even perform the detoxification of a common pollutant with higher efficiency than a natural enzyme that has evolved specifically for this purpose. The artificial enzyme is relatively insensitive towards temperature and the presence of organic solvents, and is an excellent starting point for the design of new, cheap and highly efficient biocatalysts that have huge potential in industrial biotechnology. The work we propose here aims to exploit this recent success and develop a diverse range of maquettes that will act as robust artificial enzymes capable of catalysing several commercially valuable and challenging reactions. Informed by the structures and our functional understanding of natural enzymes, we will use powerful new computational methods alongside iterative, experimental approaches to achieve this. Crucially, these include reactions not observed in nature, whereby the resulting products contain unusual and highly strained ring structures, and have significant biological activities (e.g. drugs, insecticides). Since the maquettes are fully and functionally assembled in bacteria, we can also employ powerful, high throughput laboratory evolution strategies to improve catalytically activity in a semi random manner.

酶是极其重要的生物分子，在所有生物体中执行大部分化学反应。它们本身就是蛋白质，由氨基酸链组成，但它们与蛋白质的区别在于它们能够大幅提高化学反应速率。这些反应为细胞生命提供动力，并参与大量赋予细胞化学和物理特性的基本过程。许多酶进行具有重大商业或医学价值的化学反应，因为转化的产物可能是药物、燃料或其他有用的物质或材料。通常的情况是，对于如此重要​​或有用的反应，没有人造物质可以以与酶相同的精度或效率来催化特定的化学转化。还有许多化学转化尚未发现酶。因此，人们对构建能够执行选定化学反应的定制酶产生了巨大的兴趣。尽管过去 100 年来我们对天然酶催化有了极其深入的了解，但人工设计的酶和自然界中发现的酶的能力仍然存在不足。我们认为，这在一定程度上是由于普遍使用自然进化的蛋白质作为制造人工酶的起点。这些天然蛋白质可能是脆弱的、难以纯化且昂贵的、在细胞环境之外无活性、对有机分子和溶剂化学敏感，并且最重要的是，它们总是带来进化复杂性，这可能阻碍酶设计者的修饰。我们认为，这种进化包袱并不是蛋白质和酶的必要特征，在某些情况下，最好使用未受自然选择影响的蛋白质。我们的简单蛋白质（称为模型）是坚固的小蛋白质支架，不包含天然蛋白质序列，因此不受进化带来的任何复杂性的影响。通常，我们设计这些模型以包含非蛋白质分子，该分子将其自身的反应性传递到支架上。血红素分子是我们设计中包含的一种特别通用的分子，它存在于大量天然酶中，其中许多酶能够催化极具挑战性的化学反应。在我们最近的工作中，我们使用基本设计过程将含血红素的模型开发成一种活性人工酶，其功能与许多天然酶一样，可从广泛的范围内去除电子（氧化） 的基材。它甚至可以比专门为此目的而进化的天然酶更高效地对常见污染物进行解毒。人工酶对温度和有机溶剂的存在相对不敏感，是设计新型、廉价和高效生物催化剂的绝佳起点，在工业生物技术中具有巨大潜力。我们在此提出的工作旨在利用最近的成功并开发各种模型，这些模型将作为强大的人工酶，能够催化几种具有商业价值和挑战性的反应。根据对天然酶的结构和功能的理解，我们将使用强大的新计算方法以及迭代实验方法来实现这一目标。至关重要的是，这些包括在自然界中未观察到的反应，由此产生的产物包含不寻常且高度紧张的环结构，并具有显着的生物活性（例如药物、杀虫剂）。由于模型在细菌中完全且功能性地组装，我们还可以采用强大的、高通量的实验室进化策略以半随机的方式提高催化活性。

项目成果

An expandable, modular de novo protein platform for precision redox engineering.

• DOI: 10.1073/pnas.2306046120
• 发表时间: 2023-08
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Hutchins, George H.;Noble, Claire E. M.;Bunzel, H. Adrian;Williams, Christopher;Dubiel, Paulina;Yadav, Sathish K. N.;Molinaro, Paul M.;Barringer, Rob;Blackburn, Hector;Hardy, Benjamin J.;Parnell, Alice E.;Landau, Charles;Race, Paul R.;Oliver, Thomas A. A.;Koder, Ronald L.;Crump, Matthew P.;Schaffitzel, Christiane;Oliveira, A. Sofia F.;Mulholland, Adrian J.;Anderson, J. L. Ross
• 通讯作者: Anderson, J. L. Ross

Rigidifying a De Novo Enzyme Increases Activity and Induces a Negative Activation Heat Capacity.

• DOI: 10.1021/acscatal.1c01776
• 发表时间: 2021-09-17
• 期刊: ACS catalysis
• 影响因子: 12.9
• 作者: Hindson SA;Bunzel HA;Frank B;Svistunenko DA;Williams C;van der Kamp MW;Mulholland AJ;Pudney CR;Anderson JLR
• 通讯作者: Anderson JLR

Antibodies generated in vitro and in vivo elucidate design of a thermostable ADDomer COVID-19 nasal nanoparticle vaccine

• DOI: 10.1101/2023.03.17.533092
• 发表时间: 2023-03
• 期刊: bioRxiv
• 作者: Dora Buzas;H. Bunzel;Oskar Staufer;E. Milodowski;Grace Edmonds;beatriz Vidana Matteo;C. Schaffitzel;Sathish K. N. Yadav;K. Gupta;Charlotte Fletcher;M. Williamson;Alexandra Harrison;Ufuk Borucu;Julien Capin;Ore Francis;Georgia Balchin;Sophie Hall;Mirella Vivoli Vega;F. Durbesson;R. Vincentelli;Joe Roe;L. Wooldridge;R. Burt;Ross J L Anderson;A. Mulholland;J. Hare;Mick Bailey;A. Davidson;A. Finn;David Morgan;Jamie F S Mann;Joachim P. Spatz;F. Garzoni;J. Bufton;I. Berger

Expression and In Vivo Loading of De Novo Proteins with Tetrapyrrole Cofactors.

使用四吡咯辅因子表达和体内装载 De Novo 蛋白质。

• DOI: 10.1007/978-1-0716-1826-4_8
• 发表时间: 2022
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Curnow P