Evolutionary Specialization of a Promiscuous Designer Enzyme
Leveson-Gower, Reuben (University of Groningen)
Tiessler-Sala, Laura (Universitat Autònoma de Barcelona. Departament de Química)
Rozeboom, Henriette (University of Groningen)
Thunnissen, Andy-Mark (University of Groningen)
Maréchal, Jean-Didier (Universitat Autònoma de Barcelona. Departament de Química)
Roelfes, Gerard (University of Groningen)

Date:	2025
Abstract:	The evolution of a promiscuous enzyme for its various activities often results in catalytically specialized variants. This is an important natural mechanism to ensure the proper functioning of natural metabolic networks. It also acts as both a curse and blessing for enzyme engineers, where enzymes that have undergone directed evolution may exhibit exquisite selectivity at the expense of a diminished overall catalytic repertoire. We previously performed two independent directed evolution campaigns on a promiscuous designer enzyme that leverages the unique properties of a noncanonical amino acid (ncAA) para -aminophenylalanine (pAF) as catalytic residue, resulting in two evolved variants which are both catalytically specialized. Here, we combine mutagenesis, crystallography, and computation to reveal the molecular basis of the specialization phenomenon. In one evolved variant, an unexpected change in quaternary structure biases substrate dynamics to promote enantioselective catalysis, while the other demonstrates synergistic cooperation between natural side chains and the pAF residue to form semisynthetic catalytic machinery.
Grants:	European Commission 885396 Agència de Gestió d'Ajuts Universitaris i de Recerca 2021/SGR-866 Agència de Gestió d'Ajuts Universitaris i de Recerca 2017/SGR-1323 Agencia Estatal de Investigación PID2020-116861GB-I00 Agencia Estatal de Investigación PID2023-149492NB-I00
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Subject:	Biocatalysis ; Designer Enzymes ; Noncanonical-Amino Acids ; Directed Evolution ; Quantum Chemistry ; Molecular Dynamics ; X-ray Crystallography
Published in:	ACS catalysis, Vol. 15, Issue 3 (January 2025) , p. 1544-1552, ISSN 2155-5435

DOI: 10.1021/acscatal.4c06409
PMID: 39944761

9 p, 7.8 MB

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