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

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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.