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.

酶是从根本上重要的生物分子，它们在所有生物体中执行大部分化学反应。它们本身是由氨基酸链组成的蛋白质，尽管它们与蛋白质的区别在于它们大大提高化学反应速率的能力。这些反应为细胞寿命提供动力，并参与了许多基本过程，这些过程赋予细胞化学和物理特征。许多酶进行的化学反应具有大量的商业或医学价值，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，对于如此重要​​或有用的反应，没有人为的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量身定制的酶有很大的兴趣。在过去的100年中，我们对自然酶促催化的理解非常有力，但人工，设计酶和自然界中发现的酶的能力仍然存在不足。我们认为，这部分归因于普遍使用自然进化的蛋白作为创建人工酶的起点。这些天然蛋白质可能是脆弱的，难以纯化的，不活跃的，从其细胞环境中进行化学敏感，对有机分子和溶剂化学敏感，最重要的是，它们总是会带来与它们的进化复杂性，从而通过酶设计器来阻碍修饰。我们认为，这种进化行李不是蛋白质和酶的必要特征，在某些情况下，最好使用自然选择不受欢迎的蛋白质。我们的简单蛋白质（称为maquettes）是不包含天然蛋白质序列的小鲁棒蛋白支架，因此没有进化带来的任何复杂性。通常，我们设计了这些小块以包括一个非蛋白质分子，该分子将其自身反应性赋予脚手架。血红素分子是我们在设计中包含的特别用途的分子，它存在于许多天然酶中，其中许多酶催化了许多极具挑战性的化学反应。在我们的最新作品中，我们使用基本设计过程来将血红素conte的杂物造成自然式的杂货 - 以及一种自然的杂物 - 以及一种自然的启用 - 以及一种自然的启用 - 以及一种自然的作用 - 以及一种自然的作用 - 以及一种自然的作用 - 以及一种自然的作用 - 以及一种自然的作用 - 以及一种自然的作用 - 以及一种自然的作用。底物。它甚至可以比专门为此目的而发展的天然酶执行具有更高效率的常见污染物的排毒。人造酶对温度和有机溶剂的存在相对不敏感，并且是设计新型，廉价且高效的生物催化剂的绝佳起点，它们在工业生物技术方面具有巨大的潜力。我们在这里提出的工作旨在利用这一最近的成功，并开发出多种多样的小伙子，将充当强大的人工酶，能够催化几种商业上有价值且具有挑战性的反应。在结构和我们对天然酶的功能理解的情况下，我们将使用强大的新计算方法以及迭代的实验方法来实现这一目标。至关重要的是，这些反应在自然界中未观察到，因此所产生的产品包含异常和高度应变的环结构，并具有重要的生物学活性（例如药物，杀虫剂）。由于小块在细菌中完全且功能地组装，因此我们还可以采用强大的高吞吐量实验室进化策略，以半随机的方式改善催化活性。

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

Evolution of dynamical networks enhances catalysis in a designer enzyme

• DOI: 10.1101/2020.08.21.260885
• 发表时间: 2020-01-01
• 期刊: bioRxiv
• 作者: Bunzel, H. A.;Anderson, J. L. R.;Mulholland, A. J.
• 通讯作者: Mulholland, A. J.

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