Construction and laboratory evolution of de novo b-type heme containing oxidoreductases

含氧化还原酶的从头 B 型血红素的构建和实验室进化

• 批准号: 1945347
• 金额: --
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Studentship
• 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=studentship-1945347
• 关键词: Construction; laboratory; evolution; de; novo

At the core of the emergent Synthetic Biology field there is a fundamental goal to construct new functional and bio-compatible parts and devices for incorporation into explicitly biological organisms or systems. Such a synthesis of artificial and biological components will provide an incredibly powerful framework for the design and exploitation of augmented or even new biochemical pathways in or ex vivo. One approach to the design of novel proteins is through the use of maquettes - simplified protein scaffolds that avoid the complexity and evolutional redundancy of natural proteins. The Anderson group has previously designed de novo oxidoreductases by applying rational design rules developed through analysis of natural cytochromes. Of particular note is C45, a highly efficient manmade c-type cytochrome (CTM) designed using this approach, that is capable of a wide array of substrate oxidations using catalytic intermediates also used by natural heme-enzymes. C45 is capable of being assembled fully functionally in vivo, utilising the E. coli cytochrome processing machinery to insert the c-type heme cofactor required for catalysis. Alongside the c-type cytochromes, another group of enzymes of great industrial relevance is the cytochrome P450 family. They are capable of carrying out extremely powerful chemistry, including monooxygenation reactions. Instead of the covalently linked c-type heme found in c-type cytochromes, P450s contain a b-type heme cofactor. Previous work in the Anderson lab has used computational design to produce stable b-type heme-containing maquettes (BTMs). Unlike the CTMs previously developed in the lab, which have a highly dynamic 'molten globule like' structure, these b-type heme proteins have sufficient stability for further structural characterisation, with a crystal structure being solved for one. This structural insight allows for more rational changes to be made to the design. None of the BTMs developed in the Anderson lab so far have been engineer towards catalysis. In this project we will attempt to develop de novo b-type heme containing enzymes, with an aim towards cytochrome P450-like activity. Combining the strengths of the Anderson and Mulholland labs, the project will integrate experimental and computational methods to design and characterise de novo enzymes. Investigation into different ligands and modulation of redox potentials will allow for a wide range of BTMs to be designed. Purified proteins will functionally characterised, catalytic activities measured, and reactive intermediates spectroscopically identified, providing insight into controlling multi-step de novo enzyme mechanisms. I will use directed evolution strategies and high throughput screening methodologies alongside QM/MM and MD computational packages to ultimately construct enzymes that will be functional in vivo, and can catalyse reactions of high value, either therapeutically or industrially. This project falls within the EPSRC Synthetic Biology research area.

新兴合成生物学领域的核心是构建新的功能和生物相容性部件和设备，以纳入明确的生物有机体或系统。这种人工和生物成分的合成将为设计和开发体内或体外增强的甚至新的生化途径提供令人难以置信的强大框架。设计新蛋白质的一种方法是通过使用maquettes -简化的蛋白质支架，避免了天然蛋白质的复杂性和进化冗余。安德森小组先前通过应用通过分析天然细胞色素开发的合理设计规则设计了从头氧化还原酶。特别值得注意的是C45，一种使用这种方法设计的高效人造C型细胞色素（CTM），它能够使用天然血红素酶也使用的催化中间体进行广泛的底物氧化。C45能够利用E.大肠杆菌细胞色素加工机器插入的c型血红素辅因子所需的催化。除了c型细胞色素外，另一组具有重要工业相关性的酶是细胞色素P450家族。它们能够进行非常强大的化学反应，包括单氧化反应。而不是共价连接的c型血红素中发现的c型细胞色素，P450含有b型血红素辅因子。安德森实验室以前的工作已经使用计算设计来产生稳定的b型含血红素的模型（BTM）。与先前在实验室中开发的具有高度动态的“熔融球样”结构的CTM不同，这些b型血红素蛋白具有足够的稳定性以用于进一步的结构表征，其中一个晶体结构被解决。这种结构性的洞察力允许对设计进行更合理的更改。到目前为止，安德森实验室开发的BTM还没有一个是针对催化的。在这个项目中，我们将尝试开发从头b型血红素含酶，对细胞色素P450样活性的目标。结合安德森和穆赫兰实验室的优势，该项目将整合实验和计算方法来设计和重新构建酶。对不同配体和氧化还原电位的调节的研究将允许设计宽范围的BTM。纯化的蛋白质将进行功能表征，测量催化活性，并通过光谱鉴定反应中间体，从而深入了解控制多步从头酶机制。我将使用定向进化策略和高通量筛选方法以及QM/MM和MD计算包，最终构建在体内发挥功能的酶，并可以催化高价值的反应，无论是治疗还是工业。该项目属于EPSRC合成生物学研究领域的福尔斯。