Constructing catalytically proficient enzymes from de novo designed proteins

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.

The overarching goal of this project is to design and construct manmade oxidoreductase enzymes that are capable of catalysing a diverse range of useful, valuable and synthetically challenging chemical transformations. This will provide expressible, versatile and biocompatible components that can be integrated into metabolic pathways in living cells, or exploited as green biocatalysts in industrial biotechnology.

We will achieve this using a combined computational and iterative approach to de novo protein design, informed by the engineering principles that underpin catalytic function in natural oxidoreductases and our successful design of a hyperthermostable de novo peroxidase. The protein maquettes used in this work - simple 4-helix bundle proteins assembled from first principles - are expressed and translocated to the E. coli periplasm, where heme is covalently appended to the protein backbone with high efficiency, and are therefore fully assembled in vivo.

Aided by computational design and directed evolution methodologies, we will construct functional de novo peroxidases and peroxygenases that are capable of coupling hydrogen peroxide reduction to a variety of substrate oxidations and oxygenations. We will improve the structural resolution of our designs, facilitating a design process with a precision that enables the accurate placement of peroxide-activating amino acid side chains and the creation of a bona fide substrate binding site within the maquette.

Using non-natural substrates, we will access unusual carbene-heme adducts that act as powerful agents for stereoselective cyclopropanation and carbene insertion reactions, focussing on commercially valuable and synthetically challenging chemical transformations. We will focus on gaining a better mechanistic understanding of this process through computational and experimental means, and will encapsulate our de novo enzymes in hydrogels facilitating the creation of recoverable heterogeneous catalysts.

Scientific discovery is integral to the international competitiveness of the UK. Through the construction of new de novo enzymes capable of catalysing oxidations, oxygenations and carbene transfers, this project will deliver an unprecedented advance in the 'Synthetic Biology' and 'New Strategic Approaches to Industrial Biotechnology' BBSRC Strategic Priority Areas, while delivering vital information that will further our fundamental understanding of natural protein design and engineering. These advances will contribute significantly to the UK's position as a world leader in these areas.

We anticipate that this project will deliver significant impact on the commercial sector. The construction of cheap, expressible, chemically resistant and green biocatalysts will be of particular interest in industrial biotechnology, especially with activity established under whole cell biotransformation conditions. Furthermore, since several of the carbene transfer reactions are targeted towards commercially valuable products (e.g. GSK-LSD1, pyrethroid insecticides) we predict there may be significant interest from biotechnological and pharmaceutical companies. To fully maximise 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 has been the focus of significant public concern and since our work is directly related to this field, 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 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.