In silico enzyme design


02/12/2010, Szeged, Biological Research Center


The role of reorganization energy in rational enzyme design
M Fuxreiter and L Mones
2014, Current Opinion in Chemical Biology 21, 34-41
Abstract: Computational design is becoming an integral component in developing novel enzymatic activities. Catalytic efficiencies of man-made enzymes however are far behind their natural counterparts. The discrepancy between laboratory and naturally evolved enzymes suggests that a major catalytic factor is still missing in the computational process. Reorganization energy, which is the origin of catalytic power of natural enzymes, has not been exploited yet for design. As exemplified in case of KE07 Kemp eliminase, this quantity is optimized by directed evolution. Mutations beneficial for evolution, but without direct impact on catalysis can be identified based on contributions to reorganization energy. We propose to incorporate the reorganization energy in scaffold selection to provide highly evolvable initial designs.

Optimization of reorganization energy drives evolution of the designed Kemp eliminase KE07
A Labas, E Szabo, L Mones and M Fuxreiter
2013, Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 1834 (5), 908-917
Abstract: Understanding enzymatic evolution is essential to engineer enzymes with improved activities or to generate enzymes with tailor-made activities. The computationally designed Kemp eliminase KE07 carries out an unnatural reaction by converting of 5-nitrobenzisoxazole to cyanophenol, but its catalytic efficiency is significantly lower than those of natural enzymes. Three series of designed Kemp eliminases (KE07, KE70, KE59) were shown to be evolvable with considerable improvement in catalytic efficiency. Here we use the KE07 enzyme as a model system to reveal those forces, which govern enzymatic evolution and elucidate the key factors for improving activity. We applied the Empirical Valence Bond (EVB) method to construct the free energy pathway of the reaction in the original KE07 design and the evolved R7 1/3H variant. We analyzed catalytic effect of residues and demonstrated that not all mutations in evolution are favorable for activity. In contrast to the small decrease in the activation barrier, in vitro evolution significantly reduced the reorganization energy. We developed an algorithm to evaluate group contributions to the reorganization energy and used this approach to screen for KE07 variants with potential for improvement. We aimed to identify those mutations that facilitate enzymatic evolution, but might not directly increase catalytic efficiency. Computational results in accord with experimental data show that all mutations, which appear during in vitro evolution were either neutral or favorable for the reorganization energy. These results underscore that distant mutations can also play role in optimizing efficiency via their contribution to the reorganization energy. Exploiting this principle could be a promising strategy for computer-aided enzyme design. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.

Book chapters

The Empirical Valence Bond Approach as a Tool for Designing Artificial Catalysts
M Fuxreiter and L Mones
2017, Theory and Applications of the Empirical Valence Bond Approach: From Physical Chemistry to Chemical Biology
Abstract: At the molecular level, enzymatic catalysis is a complex phenomenon involving a fine-tuned set of interactions between the substrate and the enzyme all along the chemical event, which results in several orders of magnitude increase of the catalytic rate constant (kcat) as compared to the corresponding reference reaction in solution. The active site of the enzyme includes residues, which are involved in the chemical reaction either directly by forming covalent bonds or indirectly via electrostatic interactions. Although the active site residues stand out in catalysis due to their geometric proximity to the substrate, other distant regions of the enzyme can also provide significant contributions. The impact of the whole enzymatic environment–including residues that are located further from the active center–are less trivial to assess. The proposal, that the enzyme acts as a “super solvent” of the reaction has gradually gained recognition and is now widely accepted.[1] Therefore, atomistic simulations of reactions should not be limited to the sole investigation of the active site using gas phase simulations or cluster models;[2] instead, adequate models will need to consider the effect of the entire enzymatic/solvent dynamics.