Journal
ACS CATALYSIS
Volume 12, Issue 9, Pages 5022-5035Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acscatal.2c00336
Keywords
multicopper oxidases; hyperthermophiles; enzyme speci fi city; epistasis; enzyme dynamics; allosteric regulation; Aquifex aeolicus
Categories
Funding
- Fundacao para a Ciencia e Tecnologia (FCT), Portugal [PTDC/BBBEBB/0122/2014, PTDC/BII-BBF/29564/2017, UIDB/04612/2020, UIDP/04612/2020]
- Ministerio de Ciencia, Innovacion y Universidades, Spain [PGC2018-098592-B-100]
- European Union [GA 824017]
- [CEECIND/01443/2017]
- Fundação para a Ciência e a Tecnologia [PTDC/BII-BBF/29564/2017] Funding Source: FCT
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This study characterizes the molecular features of the evolution of a hyperthermostable metallo-oxidase from the multicopper oxidase family into a laccase by combining mutagenesis with structural, kinetic, and in silico analyses. The results show that residue replacements and distal mutations modulate substrate binding and catalysis, and allosterically coupled, long-range dynamic networks favor catalytically competent conformational states.
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 (kcat 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, longrange 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.
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