1. Computational and Systems Biology
  2. Structural Biology and Molecular Biophysics
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Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering

  1. Friso S Aalbers
  2. Maximilian JLJ Fürst
  3. Stefano Rovida
  4. Milos Trajkovic
  5. J Rubén Gómez Castellanos
  6. Sebastian Bartsch
  7. Andreas Vogel
  8. Andrea Mattevi
  9. Marco W Fraaije  Is a corresponding author
  1. University of Groningen, Netherlands
  2. Cambridge Biomedical Campus, United Kingdom
  3. University of Pavia, Italy
  4. c-LEcta GmbH, Germany
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Cite this article as: eLife 2020;9:e54639 doi: 10.7554/eLife.54639

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.

The following data sets were generated

Article and author information

Author details

  1. Friso S Aalbers

    Molecular Enzymology Group, University of Groningen, Groningen, Netherlands
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2142-9661
  2. Maximilian JLJ Fürst

    MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.
  3. Stefano Rovida

    Department of Biology and Biotechnology L. Spallanzani"", University of Pavia, Pavia, Italy
    Competing interests
    No competing interests declared.
  4. Milos Trajkovic

    Molecular Enzymology group, University of Groningen, Groningen, Netherlands
    Competing interests
    No competing interests declared.
  5. J Rubén Gómez Castellanos

    Department of Biology and Biotechnology, University of Pavia, Pavia, Italy
    Competing interests
    No competing interests declared.
  6. Sebastian Bartsch

    R&D, c-LEcta GmbH, Leipzig, Germany
    Competing interests
    Sebastian Bartsch, A patent application on the original ADH was filed by c-LEcta (WO 2019/012095).
  7. Andreas Vogel

    R&D, c-LEcta GmbH, Leipzig, Germany
    Competing interests
    No competing interests declared.
  8. Andrea Mattevi

    Dept. Biology and Biotechnology, University of Pavia, Pavia, Italy
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9523-7128
  9. Marco W Fraaije

    Molecular Enzymology group, University of Groningen, Groningen, Netherlands
    For correspondence
    m.w.fraaije@rug.nl
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6346-5014

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.

Reviewing Editor

  1. Philip A Cole, Harvard Medical School, United States

Publication history

  1. Received: December 20, 2019
  2. Accepted: March 30, 2020
  3. Accepted Manuscript published: March 31, 2020 (version 1)
  4. Version of Record published: April 17, 2020 (version 2)

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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