A new thermophilic extradiol dioxygenase promises biodegradation of catecholic pollutants
Xiaowen Zhang, Zihao Huang, Dan Wang, Yan Zhang, Bekir Engin Eser, Zhenyu Gu, Rongrong Dai, Renjun Gao, Zheng Guo
Abstract
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.