Interplay of stability and dynamics in the optimization of a highly proficient de novo enzyme
- 1University of California, San Francisco
Abstract
The development of highly active de novo enzymes that catalyze new-to-nature reactions is becoming increasingly possible. Here, we identify the features underlying the catalytic efficiency of a highly proficient de novo enzyme, from the original computational design to an optimized catalyst obtained through two rounds of 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, binding of a transition state analog also led to widespread increases in backbone 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. Also, the initial design considered only the transition state but not substrate binding, leading to a dynamic Michaelis complex prior to optimization. These studies show the multiple features that need to be optimized to achieve high activity in a designed enzyme.
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BibTeX
@misc{bhattacharya2026interplay,
title = {{Interplay of stability and dynamics in the optimization of a highly proficient de novo enzyme}},
author = {S. Bhattacharya and G. M. Adornato and Y. Chen and X. Huang and W. E. Y. Mouloud and H. Jo and A. N. Volkov and I. V. Korendovych and Y. Yang and D. N. Beratan and P. Liu and W. F. DeGrado},
howpublished = {bioRxiv},
year = {2026},
month = may,
doi = {10.64898/2026.05.29.728685},
url = {https://www.biorxiv.org/content/10.64898/2026.05.29.728685v3},
note = {CC BY},
}
RIS
TY - UNPB
TI - Interplay of stability and dynamics in the optimization of a highly proficient de novo enzyme
AU - S. Bhattacharya
AU - G. M. Adornato
AU - Y. Chen
AU - X. Huang
AU - W. E. Y. Mouloud
AU - H. Jo
AU - A. N. Volkov
AU - I. V. Korendovych
AU - Y. Yang
AU - D. N. Beratan
AU - P. Liu
AU - W. F. DeGrado
PY - 2026
DA - 2026/05/29
PB - bioRxiv
DO - 10.64898/2026.05.29.728685
UR - https://www.biorxiv.org/content/10.64898/2026.05.29.728685v3
AB - The development of highly active de novo enzymes that catalyze new-to-nature reactions is becoming increasingly possible. Here, we identify the features underlying the catalytic efficiency of a highly proficient de novo enzyme, from the original computational design to an optimized catalyst obtained through two rounds of 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, binding of a transition state analog also led to widespread increases in backbone 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. Also, the initial design considered only the transition state but not substrate binding, leading to a dynamic Michaelis complex prior to optimization. These studies show the multiple features that need to be optimized to achieve high activity in a designed enzyme.
ER -