Constructing catalytically proficient enzymes from de novo designed proteins

点击翻译按钮获取中文摘要

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.