Distal Mutations Shape Substrate-Binding Sites during Evolution of a Metallo-Oxidase into a Laccase
Brissos, Vânia (Universidade Nova de Lisboa)
Borges, Patrícia T. (Universidade Nova de Lisboa)
Núñez-Franco, Reyes (Zymvol Biomodeling)
Lucas, Maria Fátima (Zymvol Biomodeling)
Frazão, Carlos (Universidade Nova de Lisboa)
Monza, Emanuele (Zymvol Biomodeling)
Masgrau, Laura (Universitat Autònoma de Barcelona. Departament de Química)
Cordeiro, Tiago N. (Universidade Nova de Lisboa)
Martins, Lígia O. (Universidade Nova de Lisboa)

Date:	2022
Abstract:	Laccases are in increasing demand as innovative solutions in the biorefinery fields. Here, we combine mutagenesis with structural, kinetic, and in silico analyses to characterize the molecular features that cause the evolution of a hyperthermostable metallo-oxidase from the multicopper oxidase family into a laccase (k 273 s -1 for a bulky aromatic substrate). We show that six mutations scattered across the enzyme collectively modulate dynamics to improve the binding and catalysis of a bulky aromatic substrate. The replacement of residues during the early stages of evolution is a stepping stone for altering the shape and size of substrate-binding sites. Binding sites are then fine-tuned through high-order epistasis interactions by inserting distal mutations during later stages of evolution. Allosterically coupled, long-range dynamic networks favor catalytically competent conformational states that are more suitable for recognizing and stabilizing the aromatic substrate. This work provides mechanistic insight into enzymatic and evolutionary molecular mechanisms and spots the importance of iterative experimental and computational analyses to understand local-to-global changes.
Grants:	European Commission 824017 Agencia Estatal de Investigación PGC2018-098592-B-100
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:	Multicopper oxidases ; Hyperthermophiles ; Enzyme specificity ; Epistasis ; Enzyme dynamics ; Allosteric regulation ; Aquifex aeolicus
Published in:	ACS catalysis, Vol. 12, Issue 9 (April 2022) , p. 5022-5035, ISSN 2155-5435

DOI: 10.1021/acscatal.2c00336
PMID: 36567772

14 p, 7.3 MB

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