De Novo Enzymes: From Computational Design to mRNA DisplayReview
Trends Biotechnol. 2010, 28, 340-345, 10.1016/j.tibtech.2010.04.003
Enzymes offer cheap, environmentally responsible and highly efficient alternatives to chemical catalysts. The past two decades have seen a significant rise in the use of enzymes in industrial settings. Although many natural enzymes have been modified through protein engineering to better suit practical applications, these approaches are often insufficient. A key goal of enzyme engineers is to build enzymes de novo – or, ‘from scratch’. To date, several technologies have been developed to achieve this goal: namely, computational design, catalytic antibodies and mRNA display. These methods rely on different principles, trading off rational protein design against an entirely combinatorial approach of directed evolution of vast protein libraries. The aim of this article is to review and compare these methods and their potential for generating truly de novo biocatalysts.
Selection and Evolution of Enzymes from a Partially Randomized Non-Catalytic Scaffold
Nature 2007, 448, 828-831, 10.1038/nature06032
Enzymes are exceptional catalysts that facilitate a wide variety of reactions under mild conditions, achieving high rate-enhancements with excellent chemo-, regio- and stereoselectivities. There is considerable interest in developing new enzymes for the synthesis of chemicals and pharmaceuticals1,2,3 and as tools for molecular biology. Methods have been developed for modifying and improving existing enzymes through screening, selection and directed evolution4,5. However, the design and evolution of truly novel enzymes has relied on extensive knowledge of the mechanism of the reaction6,7,8,9,10. Here we show that genuinely new enzymatic activities can be created de novo without the need for prior mechanistic information by selection from a naive protein library of very high diversity, with product formation as the sole selection criterion. We used messenger RNA display, in which proteins are covalently linked to their encoding mRNA11, to select for functional proteins from an in vitro translated protein library of >1012independent sequences without the constraints imposed by any in vivo step. This technique has been used to evolve new peptides and proteins that can bind a specific ligand12,13,14,15,16,17,18, from both random-sequence libraries12,14,15,16 and libraries based on a known protein fold17,18. We now describe the isolation of novel RNA ligases from a library that is based on a zinc finger scaffold18,19, followed by in vitro directed evolution to further optimize these enzymes. The resulting ligases exhibit multiple turnover with rate enhancements of more than two-million-fold.
Metal: ZnLigand type: Amino acidHost protein: Human retinoid-X-receptor (hRXRa)Anchoring strategy: DativeOptimization: GeneticReaction: RNA ligationMax TON: >7ee: ---PDB: ---Notes: ---
Structure and Dynamics of a Primordial Catalytic fold Generated by In Vitro Evolution
Nat. Chem. Biol. 2013, 9, 81-83, 10.1038/nchembio.1138
Engineering functional protein scaffolds capable of carrying out chemical catalysis is a major challenge in enzyme design. Starting from a noncatalytic protein scaffold, we recently generated a new RNA ligase by in vitro directed evolution. This artificial enzyme lost its original fold and adopted an entirely new structure with substantially enhanced conformational dynamics, demonstrating that a primordial fold with suitable flexibility is sufficient to carry out enzymatic function.
Metal: ZnLigand type: Amino acidHost protein: Human retinoid-X-receptor (hRXRa)Anchoring strategy: DativeOptimization: GeneticReaction: RNA ligationMax TON: ---ee: ---PDB: 2LZENotes: ---