Distal Mutations Shape Substrate-Binding Sites during Evolution of a Metallo-Oxidase into a Laccase

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.