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

在新兴合成生物学领域的核心，有一个基本目标是构建新的功能和生物相容的部件和设备，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在体内或体外设计和开发增强的甚至新的生化途径提供一个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模型——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能够在体内完全组装，利用大肠杆菌细胞色素加工机制插入催化所需的c型血红素辅因子。除了c型细胞色素外，另一组具有重大工业意义的酶是细胞色素P450家族。它们能够进行极其强大的化学反应，包括单氧反应。与c型细胞色素中发现的共价连接的c型血红素不同，p450含有b型血红素辅助因子。安德森实验室以前的工作已经使用计算设计来生产稳定的b型含血红素模型（BTMs）。与之前在实验室开发的CTMs不同，CTMs具有高度动态的“熔融球状”结构，这些b型血红素蛋白具有足够的稳定性，可以进一步进行结构表征，其中一个晶体结构正在解决。这种结构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都没有用于催化。在这个项目中，我们将尝试开发新的含有b型血红素的酶，目的是获得细胞色素p450样活性。结合Anderson和Mulholland实验室的优势，该项目将整合实验和计算方法来设计和表征新生酶。对不同配体和氧化还原电位调制的研究将允许设计大范围的btm。纯化蛋白将功能表征，催化活性测量，活性中间体光谱鉴定，为控制多步骤从头酶机制提供见解。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，最终构建将在体内发挥作用的酶，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。

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.