Multistate Enzyme Design Enables Efficient and Stereoselective Multistep Catalysis
- 1University of Ottawa
Abstract
Enzymes catalyze multistep reactions by stabilizing successive transition states within well organized, yet dynamic active sites. However, computational enzyme design typically targets a single transition state using rigid active-site models. Here, we introduce multistate enzyme design, which uses conformational ensembles to optimize active sites across an entire reaction coordinate. Applied to a de novo Morita--Baylis--Hillmanase, multistate enzyme design outperformed conventional single-state design, with the most active variant achieving >100-fold higher bi-substrate catalytic efficiency and surpassing an extensively optimized enzyme from directed evolution in both efficiency and enantioselectivity. Structural and kinetic analyses revealed that multistate design preserved catalytic preorganization and conformational plasticity, distributed stabilization across the reaction coordinate and avoided kinetic bottlenecks created by single-state optimization. By contrast, single-state design compromised preorganization, destabilized upstream states and shifted rate limitation away from the targeted transition state. Multistate enzyme design provides a framework for designing catalytic landscapes rather than static active sites, opening a route to efficient de novo enzymes for complex multistep chemistry.
Related
- Sequential Design of Experiments enables accessible and efficient multidimensional mapping of single- and multi-protein condensates
- Volumetric denoising enables high-throughput volume electron microscopy and efficient downstream analysis
- Direct measurement of PET hydrolase interfacial kinetics reveals catalysis-independent surface remodelling
- Enhancing ω-transaminase catalysis of bulky substrates guided by binding mechanism
- Catalysis tuned for metabolic sensing: dynamics and mechanism of the RNA methyltransferase METTL16
BibTeX
@misc{pham2026multistate,
title = {{Multistate Enzyme Design Enables Efficient and Stereoselective Multistep Catalysis}},
author = {N. T. H. Pham and R. Guo and R. P. Garcia Jimenez and A. E. Hutton and L. O. Johannissen and J. A. Wehrstedt and B. Seifinoferest and Z. Birch-Price and J. Berreur and S. Hay and M. C. Thompson and A. P. Green and R. A. Chica},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.11.750617},
url = {https://www.biorxiv.org/content/10.64898/2026.09.11.750617v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Multistate Enzyme Design Enables Efficient and Stereoselective Multistep Catalysis
AU - N. T. H. Pham
AU - R. Guo
AU - R. P. Garcia Jimenez
AU - A. E. Hutton
AU - L. O. Johannissen
AU - J. A. Wehrstedt
AU - B. Seifinoferest
AU - Z. Birch-Price
AU - J. Berreur
AU - S. Hay
AU - M. C. Thompson
AU - A. P. Green
AU - R. A. Chica
PY - 2026
DA - 2026/09/11
PB - bioRxiv
DO - 10.64898/2026.09.11.750617
UR - https://www.biorxiv.org/content/10.64898/2026.09.11.750617v1
AB - Enzymes catalyze multistep reactions by stabilizing successive transition states within well organized, yet dynamic active sites. However, computational enzyme design typically targets a single transition state using rigid active-site models. Here, we introduce multistate enzyme design, which uses conformational ensembles to optimize active sites across an entire reaction coordinate. Applied to a de novo Morita--Baylis--Hillmanase, multistate enzyme design outperformed conventional single-state design, with the most active variant achieving >100-fold higher bi-substrate catalytic efficiency and surpassing an extensively optimized enzyme from directed evolution in both efficiency and enantioselectivity. Structural and kinetic analyses revealed that multistate design preserved catalytic preorganization and conformational plasticity, distributed stabilization across the reaction coordinate and avoided kinetic bottlenecks created by single-state optimization. By contrast, single-state design compromised preorganization, destabilized upstream states and shifted rate limitation away from the targeted transition state. Multistate enzyme design provides a framework for designing catalytic landscapes rather than static active sites, opening a route to efficient de novo enzymes for complex multistep chemistry.
ER -