Insights into Laccase Engineering from Molecular Simulations: Toward a Binding-Focused Strategy.
Understanding the molecular determinants of enzyme performance is of primary importance for the rational design of ad hoc mutants. A novel approach, which combines efficient conformational sampling and quick reactivity scoring, is used here to shed light on how substrate oxidation was improved during the directed evolution experiment of a fungal laccase (from Pycnoporus cinnabarinus), an industrially relevant class of oxidoreductases. It is found that the enhanced activity of the evolved enzyme is mainly the result of substrate arrangement in the active site, with no important change in the redox potential of the T1 copper. Mutations at the active site shift the binding mode into a more buried substrate position and provide a more favorable electrostatic environment for substrate oxidation. As a consequence, engineering the binding event seems to be a viable way to in silico evolution of oxidoreductases.
†Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, c/Jordi Girona 29, 08034 Barcelona, Spain.
Emanuele
E
Monza
†Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, c/Jordi Girona 29, 08034 Barcelona, Spain.
M Fatima
MF
Lucas
‡Centro de Investigacion Biológica, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Susana
S
Camarero
‡Centro de Investigacion Biológica, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Lorea C
LC
Alejaldre
‡Centro de Investigacion Biológica, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Angel T
AT
Martínez
†Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, c/Jordi Girona 29, 08034 Barcelona, Spain.|§Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys 23, 08010 Barcelona, Spain.
Victor
V
Guallar
D020134:Catalytic Domain; D003300:Copper / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry*; D005658:Fungi; D042845:Laccase / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D010084:Oxidation-Reduction; D011485:Protein Binding; D015202:Protein Engineering
D005656:Fungal Proteins; D003300:Copper; D042845:Laccase
PELE; copper oxidoreductases; directed evolution; in silico enzyme engineering; substrate binding; substrate oxidation