Characterization of Chlorophenol 4-Monooxygenase (TftD) and NADH:Flavin Adenine Dinucleotide Oxidoreductase (TftC) of Burkholderia cepacia AC1100

Abstract

ABSTRACT Burkholderia cepacia AC1100 uses 2,4,5-trichlorophenoxyacetic acid, an environmental pollutant, as a sole carbon and energy source. Chlorophenol 4-monooxygenase is a key enzyme in the degradation of 2,4,5-trichlorophenoxyacetic acid, and it was originally characterized as a two-component enzyme (TftC and TftD). Sequence analysis suggests that they are separate enzymes. The two proteins were separately produced in Escherichia coli , purified, and characterized. TftC was an NADH:flavin adenine dinucleotide (FAD) oxidoreductase. A C-terminally His-tagged fusion TftC used NADH to reduce either FAD or flavin mononucleotide (FMN) but did not use NADPH or riboflavin as a substrate. Kinetic and binding property analysis showed that FAD was a better substrate than FMN. TftD was a reduced FAD (FADH 2 )-utilizing monooxygenase, and FADH 2 was supplied by TftC. It converted 2,4,5-trichlorophenol to 2,5-dichloro- p -quinol and then to 5-chlorohydroxyquinol but converted 2,4,6-trichlorophenol only to 2,6-dichloro- p -quinol as the final product. TftD interacted with FADH 2 and retarded its rapid oxidation by O 2 . A spectrum of possible TftD-bound FAD-peroxide was identified, indicating that the peroxide is likely the active oxygen species attacking the aromatic substrates. The reclassification of the two enzymes further supports the new discovery of FADH 2 -utilizing enzymes, which have homologues in the domains Bacteria and Archaea .