A new thermophilic extradiol dioxygenase promises biodegradation of catecholic pollutants

Extradiol dioxygenases (EDOs) catalyze the meta cleavage of catechol into 2-hydroxymuconaldehyde, a critical step in the degradation of aromatic compounds in the environment. In the present work, a novel thermophilic extradiol dioxygenase from Thermomonospora curvata DSM43183 was cloned, expressed, and characterized by phylogenetic and biochemical analyses. This enzyme exhibited excellent thermo-tolerance, displaying optimal activity at 50 °C, remaining >40% activity at 70 °C. Structural modeling and molecular docking demonstrated that both active center and pocket-construction loops locate at the C-terminal domain. Site-specific mutants D285A, H205V, F301V based on a rational design were obtained to widen the entrance of substrates; resulting in significantly improved catalytic performance for all the 3 mutants. Compared to the wild-type, the mutant D285A showed remarkably improved activities with respect to the 3,4-dihydroxyphenylacetic acid, catechol, and 3-chlorocatechol, by 17.7, 6.9, and 3.7-fold, respectively. The results thus verified the effectiveness of modeling guided design; and confirmed that the C-terminal loop structure indeed plays a decisive role in determining catalytic ring-opening efficiency and substrate specificity of the enzyme. This study provided a novel thermostable dioxygenase with a broad substrate promiscuity for detoxifying environmental pollutants and provided a new thinking for further enzyme engineering of EDOs.