Assembly of Artificial Oxidoreductases

Proteins are biological molecules constructed from linear chains of amino acids that adopt complex 3D structures informed by their amino acid sequence. Each protein typically has a unique structure that is indelibly linked to the function it performs in nature. Enzymes are proteins that catalyze the chemical reactions that occur in the cell, examples of which facilitate the capture and storage of chemical energy from respiration and photosynthesis. The design of new artificial proteins and enzymes remains one of the great challenges in biochemistry, testing our fundamental understanding of the nature of protein as a material. Unlocking the exceptionally powerful array of chemistries that natural enzymes perform promises routes to new drugs, therapies and sources of renewable green energy. Most attempts to construct new enzymes have focussed on modifying natural proteins and enzymes to introduce new catalytic function with modest degrees of success. The problems associated with redesigning natural proteins are due to the layers of complexity that nature incorporates through natural selection into a protein's complicated 3D structure. This complexity serves to complicate functional deconstruction of naturally evolved proteins and enzymes, rendering their redesign intrinsically difficult. We believe that this complexity is not a necessary feature of proteins and enzymes. Our method to effectively avoid such complexity is to work with proteins that have been untouched by natural selection. These simple proteins, neoproteins, are small, robust protein scaffolds with generic amino acid sequences that serve as templates onto which natural protein functions can be added. Non-protein components of certain proteins and enzymes, such as the heme molecule of the protein hemoglobin, can be effectively supported in neoproteins and the various functions that these molecules perform in natural proteins can be exploited. An example of how this method can be effectively used is the creation of a heme-binding neoprotein capable of reversibly binding oxygen, a function common to myoglobin, hemoglobin and the recently discovered neuroglobin. Functional elements of engineering are added step-by-step and the requirements to form such a protein are surprisingly few in number. And, as E. coli produces the artificial protein in large quantities, the oxygen-binding neoprotein is exceptionally cheap to produce and easy to alter through standard molecular biology techniques. Since the oxygen bound state in heme proteins is a pre-requisite for a multitude of catalytic processes in natural proteins, we plan to take inspiration from nature to further the development of these proteins into artificial enzymes. We have developed the oxygen-binding neoprotein to include hemes rigidly attached to the protein backbone. This alleviates problems associated with heme loss from previous designs and allows for an unprecedented control of neoprotein properties and function. Since natural oxygen-dependent catalysis requires that oxygen be 'activated' by the controlled addition of electrons, we will explore this reaction in our oxygen binding neoproteins, gaining valuable information about the generation and stability of intermediates capable of powerful oxygenic catalysis. Ultimately, we plan to combine the oxygen binding and electron delivery functions into either a single protein or a combination of associated protein subunits with discrete functions. Much as modular furniture design uses combinations of smaller functionally independent subunits such as legs, drawers, shelves and assembles them to particular specifications, we think an analogous approach can be applied to the construction of new proteins and enzymes whose functions are dictated by the designer. An advantage of this approach is that through the reproduction of enzyme and protein function in artificial proteins a deep fundamental understanding of the workings of their natural counterparts is gained.

Through our recent successful design and assembly of an artificial oxygen-binding protein (published in Nature), we have shown that functional elements present in natural oxidoreductase enzymes can be simply assembled in proteins free from the complexity that has hindered de novo enzyme design. These simplified proteins, neoproteins, have shown that it may be possible to integrate these functional elements into artificial protein assemblies to create the first artificial oxidoreductase enzymes. This represents a unique solution to the problem of building artificial enzymes and will provide valuable information to this end, as well as establishing the engineering principles guiding natural protein and enzyme construction. We will achieve this goal by first taking advantage of recent preliminary work in which we demonstrated that it is possible to express neoproteins with c-type heme cofactors covalently attached to the protein backbone. These covalent linkages allow a level of fine-tuning of heme redox potentials and chemistries that until now was unobtainable. C-type neoproteins will undergo full biophysical characterization and will be designed for roles in solely oxygen binding or electron transfer. We will study the activation of molecular oxygen in neoproteins proven to support the binding of oxygen. Electrons will be delivered to the oxygen bound state and the generation and stability of high valent oxo-iron species will be studied and evaluated using a suite of spectroscopies and biophysical techniques. These intermediates are key to accessing the chemistry performed by natural monooxygenases such as the cytochromes P450. We will combine oxygen binding and electron transfer functionalities into single neoproteins, assemblies of neoproteins and complexes of neoproteins with natural electron transfer proteins to create the first artificial oxidoreductases capable of binding and activating molecular oxygen.

Scientific discovery is integral to the international competitiveness of the UK. Through the assembly of the first functional oxidoreductase enzymes, this project will deliver an unprecedented advance in the BBSRC Strategic Priority Areas of Bionanotechnology and Synthetic Biology, while delivering vital information that will further our fundamental understanding of natural enzymes. These advances will contribute significantly to the UK's position as a world leader in these areas. Since natural oxidoreductases catalyze chemical transformations key to realizing efficient green energy production and vital to the synthesis of drugs and natural products we anticipate the assembly of artificial oxidoreductases and artificial proteins that support functional elements of oxidoreductases to deliver significant impact upon the commercial sector. There has been significant interest in the preliminary work behind this proposal - we have applied for a patent in the USA for the design of an oxygen binding protein and have conducted preliminary talks with a company regarding potential commercialization. To fully maximize impact on the commercial sector we plan to undertake training in science business and innovation, establish close ties with the University Research and Development Office and establish and maintain contacts with industry. Synthetic biology and bionanotechnology have been the focus of significant public concern and since our work is directly related to both these fields, we plan to allay such concern by regularly engaging and educating the public through University public outreach schemes and the media. JLRA will attend courses in communication skills and media training, continue participating in public outreach schemes run by the University and the Synthetic Components Network and maintain accessible websites displaying information about our current research. Press offices of the BBSRC, Royal Society and the University of Bristol will be contacted when high profile research papers are accepted. We anticipate that this fundamental research will significantly impact upon the third sector. We will maximize impact on policy-makers, funding bodies and academic institutions by providing clear evidence of the value of synthetic biology research and raising its profile within the UK. This research will be actively promoted through the scientific community and within the University of Bristol itself, with the aim of establishing links and new collaborations with other departments and disciplines. Training and expertise in this field will be offered to those involved in the project (PDRAs, PhD students, etc.), providing them with the skills to succeed in a future career in academia or industry.