The fundamental question how enzymes work, which has continuously fascinated researchers for almost 140 years, remains partly unsolved. We do not today fully understand the impact of enzyme motion and dynamics in driving biocatalysis, and the evolution of novel catalytic functions[1], which hampers the full potential of enzyme design. Herein, our fundamental understanding of how protein and solvent dynamics facilitates biocatalysis is advanced through a simplified, hypothetical model that explains how water movement on the ps-ns timescale can be of high relevance for enzyme catalysis. The hypothesis[2] that water networks, and their reconfiguration when moving from the ground state to the transition state (as schematically shown in Figure 1), significantly impact the function of proteins is verified by an interdisciplinary approach; one that merges computational enzyme design, bioinformatics, experimental biocatalysis and biophysics with state-of-the art protein mass spectrometry. We explore the untapped opportunity to relocate water molecules in solvated binding pockets by protein design to afford biocatalysts with extended catalytic versatilities and improved properties (Figure 1). Based on an enhanced understanding of the fundamental role of water networks inside proteins, recent results from our enzyme engineering and synthetic biology programs centered on expanding the catalytic scope of biocatalysts beyond nature’s current capabilities for applications in textile recycling, material science and fine chemical synthesis will be discussed. The results herein high-light an unprecedented role of entropy associated with water in biomacromolecules.

[1] H. Renata, Z. J. Wang and F. H. Arnold. Expanding the Enzyme Universe: Accessing Non-Natural Reactions by Mechanism-Guided Directed Evolution. Angew. Chem., Int. Ed., 2015, 54, 3351-3367.

[2] M. J. Fink and P.-O. Syrén. Redesign of water networks for efficient biocatalysis. Curr. Opin. Chem. Biol. 2017, 37, 107-114.