[SCAN Highlight] Turning a Hyperthermostable Metallo-Oxidase into a Laccase by Directed Evolution

SCAN - Paper Highlight

Title: Turning a Hyperthermostable Metallo-Oxidase into a Laccase by Directed Evolution

Speaker: Vânia Brissos

Affiliation: Microbial & Enzyme Technology Lab, ITQB

Abstract:

Multicopper oxidases are multifunctional enzymes that can be broadly divided into two functional classes: metallo-oxidases (with a robust activity towards metals, such as Cu⁺ or Fe²⁺) and laccases (with a superior catalytic efficiency towards organic compounds). Laccases are green catalysts with an outstanding redox capability over a wide range of aromatic substrates using O₂ as an electron acceptor and releasing H₂O as reduced product. Hyperthermostable laccases are highly in demand for their robustness in biotechnological applications. McoA from the hyperthermophilic bacterium Aquifex aeolicus shows a notable thermoactivity (T_opt = 75°C) and thermostability (temperature values at the mid-point (T_m) of 105, 110 and 114°C) and a remarkable specificity constant (k_cat/K_m) for the oxidation of cuprous and ferrous ions. A laboratory evolution approach was conducted to improve the metallo-oxidase McoA specificity for aromatic compounds. Four rounds of random mutagenesis of the mcoA-gene followed by high-throughput screening (~94,000 clones) led to the identification of the 2B3 variant featuring a 2-order of magnitude higher catalytic efficiency (k_cat/K_m) than the wild-type enzyme for the typical laccase substrate ABTS (2,2’-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid)) and displaying additionally a higher activity for phenolics and synthetic aromatic dyes. Notably, the recombinant 2B3 variant, unlike the wild-type, did not show temperature-dependent aggregation, exhibiting enhanced solubility and thus higher kinetic and thermodynamic thermostability. The structural basis of the altered substrate’s catalytic efficiency and increased solubility/thermostability of the 2B3 variant were discussed based on the biochemical analysis of single and double site-directed mutants and hit variants from each generation of in vitro evolution. The hyper-robustness of the evolved enzyme reported here shows clear advantages for current applications and provides a powerful tool for generation of more efficient biocatalysts for specific applications since it is widely acknowledged that thermostable proteins present an enhanced mutational robustness and evolvability.