Beyond directed evolution--semi-rational protein engineering and design

Abstract

Over the past two decades, directed evolution has transformed the field of protein engineering. The advances in understanding protein structure and function, in no insignificant part a result of directed evolution studies, are increasingly empowering scientists and engineers to device more effective methods for manipulating and tailoring biocatalysts. Abandoning large combinatorial libraries, the focus has shifted to small, functionally rich libraries and rational design. A critical component to the success of these emerging engineering strategies are computational tools for the evaluation of protein sequence datasets and the analysis of conformational variations of amino acids in proteins. Highlighting the opportunities and limitations of such approaches, this review focuses on recent engineering and design examples that require screening or selection of small libraries.

Figure 1

Mutations at or near the substrate access tunnels can restrict water accessibility in haloalkane dehalogenase which benefits catalysis by shielding the substrate complex from bulk solvent.

Figure 2

Structure-based enzyme redesign. A) Engineering of an ω-transaminase for the industrial production of silagliptin (figure adapted from [40]). Mutations in the initial redesign (shown in red) concentrate on the active site while changes by directed evolution (in yellow) are skewed towards dimer stabilization. B) SCHEMA analysis of three parental cellobiohydrolases optimizes generation of functional chimera by minimizing recombination of protein fragments with incompatible topologies.

Figure 3

Computational enzyme redesign. A) Mutations (shown in red) change the substrate specificity of phenylalanine adenylation domain to amino acids with small hydrophobic (leucine) and charged side chains (arginine). B) Engineering of loop region (red) in the active site of human guanosine deaminase creates an ammelide-specific deaminase.

Figure 4

De novo design of a Diels-Alderase. An ensemble of theozymes (computational models of the reaction’s presumptive transition state including key amino acids) were matched against a library of protein scaffolds to identify suitable combinations. Subsequently, design solutions were refined in silico and tested experimentally (figure adapted from [40]).

Figure 5

Converting myoglobin into nitric oxide reductase. Remodeling of a hydrophobic pocket into a ferrous binding site transforms the oxygen-binding protein into a biocatalyst.