Understanding activity-stability tradeoffs in biocatalysts by enzyme proximity sequencing.

Autor: Vanella R; Institute of Physical Chemistry, Department of Chemistry, University of Basel, 4058, Basel, Switzerland. rosario.vanella@unibas.ch.; Department of Biosystems Science and Engineering, ETH Zurich, 4058, Basel, Switzerland. rosario.vanella@unibas.ch., Küng C; Institute of Physical Chemistry, Department of Chemistry, University of Basel, 4058, Basel, Switzerland.; Department of Biosystems Science and Engineering, ETH Zurich, 4058, Basel, Switzerland., Schoepfer AA; Institute of Physical Chemistry, Department of Chemistry, University of Basel, 4058, Basel, Switzerland.; Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne (EPFL), 1015, Lausanne, Switzerland.; National Center for Competence in Research (NCCR), Catalysis, École Polytechnique Fédérale de Lausanne (EPFL), 1015, Lausanne, Switzerland., Doffini V; Institute of Physical Chemistry, Department of Chemistry, University of Basel, 4058, Basel, Switzerland.; Department of Biosystems Science and Engineering, ETH Zurich, 4058, Basel, Switzerland., Ren J; Institute of Physical Chemistry, Department of Chemistry, University of Basel, 4058, Basel, Switzerland.; Department of Biosystems Science and Engineering, ETH Zurich, 4058, Basel, Switzerland., Nash MA; Institute of Physical Chemistry, Department of Chemistry, University of Basel, 4058, Basel, Switzerland. michael.nash@unibas.ch.; Department of Biosystems Science and Engineering, ETH Zurich, 4058, Basel, Switzerland. michael.nash@unibas.ch.; National Center for Competence in Research (NCCR), Molecular Systems Engineering, 4058, Basel, Switzerland. michael.nash@unibas.ch.; Swiss Nanoscience Institute, 4056, Basel, Switzerland. michael.nash@unibas.ch.
Jazyk: angličtina
Zdroj: Nature communications [Nat Commun] 2024 Feb 28; Vol. 15 (1), pp. 1807. Date of Electronic Publication: 2024 Feb 28.
DOI: 10.1038/s41467-024-45630-3
Abstrakt: Understanding the complex relationships between enzyme sequence, folding stability and catalytic activity is crucial for applications in industry and biomedicine. However, current enzyme assay technologies are limited by an inability to simultaneously resolve both stability and activity phenotypes and to couple these to gene sequences at large scale. Here we present the development of enzyme proximity sequencing, a deep mutational scanning method that leverages peroxidase-mediated radical labeling with single cell fidelity to dissect the effects of thousands of mutations on stability and catalytic activity of oxidoreductase enzymes in a single experiment. We use enzyme proximity sequencing to analyze how 6399 missense mutations influence folding stability and catalytic activity in a D-amino acid oxidase from Rhodotorula gracilis. The resulting datasets demonstrate activity-based constraints that limit folding stability during natural evolution, and identify hotspots distant from the active site as candidates for mutations that improve catalytic activity without sacrificing stability. Enzyme proximity sequencing can be extended to other enzyme classes and provides valuable insights into biophysical principles governing enzyme structure and function.
(© 2024. The Author(s).)
Databáze: MEDLINE
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