Interplay of stability and dynamics in the optimization of a highly proficient de novo enzyme
Bhattacharya, S.; Adornato, G. M.; Chen, Y.; Huang, X.; Mouloud, W. E. Y.; Jo, H.; Volkov, A. N.; Korendovych, I. V.; Yang, Y.; Beratan, D. N.; Liu, P.; DeGrado, W. F.
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The de novo design of enzymes critically tests our understanding of natural enzymes and enables design of novel catalysts. Here, we identify the features responsible for the catalytic efficiency of a highly proficient de novo enzyme generated through computational design and optimized by directed evolution. Computational, spectroscopic, and biochemical studies reveal successfully designed features, including precise alignment of catalytic residues, transition state stabilization, and environmental tuning. In the most evolved enzyme, the binding of a transition state analog also led to widespread increases in backbone rigidity and conformational stability throughout the protein, except within a helix near the active site entrance, where the introduction of Gly and Pro increased dynamics and catalytic activity. Thus, the entire protein contributes to catalysis in the most optimized enzyme. These studies provide principles for designing efficient enzymes.
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