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Understanding activity-stability tradeoffs in biocatalysts by enzyme proximity sequencing.
Vanella, Rosario; Küng, Christoph; Schoepfer, Alexandre A; Doffini, Vanni; Ren, Jin; Nash, Michael A.
Afiliación
  • Vanella R; Institute of Physical Chemistry, Department of Chemistry, University of Basel, 4058, Basel, Switzerland. rosario.vanella@unibas.ch.
  • Küng C; Department of Biosystems Science and Engineering, ETH Zurich, 4058, Basel, Switzerland. rosario.vanella@unibas.ch.
  • Schoepfer AA; Institute of Physical Chemistry, Department of Chemistry, University of Basel, 4058, Basel, Switzerland.
  • Doffini V; 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.
  • Nash MA; Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne (EPFL), 1015, Lausanne, Switzerland.
Nat Commun ; 15(1): 1807, 2024 Feb 28.
Article en En | MEDLINE | ID: mdl-38418512
ABSTRACT
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Mutación Missense Idioma: En Revista: Nat Commun Asunto de la revista: BIOLOGIA / CIENCIA Año: 2024 Tipo del documento: Article País de afiliación: Suiza Pais de publicación: Reino Unido
Texto completo
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XML
PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Mutación Missense Idioma: En Revista: Nat Commun Asunto de la revista: BIOLOGIA / CIENCIA Año: 2024 Tipo del documento: Article País de afiliación: Suiza Pais de publicación: Reino Unido