A Multicomponent THF Hydroxylase Initiates Tetrahydrofuran Degradation in Cupriavidus metallidurans ZM02.

Tetrahydrofuran (THF) has been recognized as a water contaminant because of its human carcinogenicity, extensive use, and widespread distribution. Previously reported multicomponent monooxygenases (MOs) involved in THF degradation were highly conserved, and all of them were from Gram-positive bacteria. In this study, a novel THF-degrading gene cluster (dmpKLMNOP) encoding THF hydroxylase was identified on the chromosome of a newly isolated Gram-negative THF-degrading bacterium, Cupriavidus metallidurans ZM02, and functionally characterized. Transcriptome sequencing and RT-qPCR demonstrated that the expression of dmpKLMNOP was upregulated during the growth of strain ZM02 on THF or phenol. The deletion of oxygenase alpha or beta subunit or the reductase component disrupted the degradation of THF but did not affect the utilization of its hydroxylated product 2-hydroxytetrahydrofuran. Cupriavidus pinatubonensis JMP134 heterologously expressing dmpKLMNOP from strain ZM02 could grow on THF, indicating that the THF hydroxylase DmpZM02KLMNOP is responsible for the initial degradation of THF. Furthermore, the THF and phenol oxidation activities of crude enzyme extracts were detected, and the highest THF and phenol catalytic activities were 1.38 ± 0.24 μmol min-1 mg-1 and 1.77 ± 0.37 μmol min-1 mg-1, respectively, with the addition of NADPH and Fe2+. The characterization of THF hydroxylase associated with THF degradation enriches our understanding of THF-degrading gene diversity and provides a novel potential enzyme for the bioremediation of THF-containing pollutants. IMPORTANCE Multicomponent MOs catalyzing the initial hydroxylation of THF are vital rate-limiting enzymes in the THF degradation pathway. Previous studies of THF degradation gene clusters have focused on Gram-positive bacteria, and the molecular mechanism of THF degradation in Gram-negative bacteria has rarely been reported. In this study, a novel THF hydroxylase encoded by dmpKLMNOP in strain ZM02 was identified to be involved in both THF and phenol degradation. Our findings provide new insights into the THF-degrading gene cluster and enzymes in Gram-negative bacteria.