Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering
- University of Groningen, Netherlands
- Cambridge Biomedical Campus, United Kingdom
- University of Pavia, Italy
- c-LEcta GmbH, Germany
Abstract
Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (ΔTm ≥ +3 °C). By combining mutations, a 10-fold mutant was generated with a Tm of 94 °C (+51 °C relative to wildtype), almost reaching water's boiling point, and the highest increase with FRESCO to date. The 10-fold mutant's structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wildtype, while displaying a Tm of 88 °C (+45 °C relative to wildtype). This work demonstrates the value of enzyme stabilization through computational library design.
Data availability
Diffraction data have been deposited in PDB under the accession codes 6TQ3, 6TQ5, and 6TQ8.Details on the structures, enzyme kinetic data and statistical analyses are included in the supplementary information.
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Alcohol dehydrogenase from Candida magnoliae DSMZ 70638 (ADHA)Protein Data Bank, 6TQ3.
Article and author information
Author details
Funding
European Commission (EU-H2020-ROBOX grant agreement nr. 635734)
- Friso S Aalbers
- Maximilian JLJ Fürst
- Stefano Rovida
- J Rubén Gómez Castellanos
- Sebastian Bartsch
- Andreas Vogel
- Andrea Mattevi
- Marco W Fraaije
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Aalbers et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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