Structural and biochemical insights into an engineered high-redox potential laccase overproduced in Aspergillus.

ABSTRACT

Fungal laccases have great potential as biocatalysts oxidizing a variety of aromatic compounds using oxygen as co-substrate. Here, the crystal structure of 7D5 laccase (PDB 6H5Y), developed in Saccharomyces cerevisiae and overproduced in Aspergillus oryzae, is compared with that of the wild type produced by basidiomycete PM1 (Coriolopsis sp.), PDB 5ANH. SAXS showed both enzymes form monomers in solution, 7D5 laccase with a more oblate geometric structure due to heavier and more heterogeneous glycosylation. The enzyme presents superior catalytic constants towards all tested substrates, with no significant change in optimal pH or redox potential. It shows noticeable high catalytic efficiency with ABTS and dimethyl-4-phenylenediamine, 7 and 32 times better than the wild type, respectively. Computational simulations demonstrated a more favorable binding and electron transfer from the substrate to the T1 copper due to the introduced mutations. PM1 laccase is exceptionally stable to thermal inactivation (t1/2 70 °C = 1.2 h). Yet, both enzymes display outstanding structural robustness at high temperature. They keep folded during 2 h at 100 °C though, thereafter, 7D5 laccase unfolds faster. Rigidification of certain loops due to the mutations added on the protein surface would diminish the capability to absorb temperature fluctuations leading to earlier protein unfolding.