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Journal of Bacteriology, May 2003, p. 2786-2792, Vol. 185, No. 9
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.9.2786-2792.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Michelle R. Gisi{dagger} and Luying Xun*

School of Molecular Biosciences, Washington State University, Pullman, Washington

Received 23 December 2002/ Accepted 17 February 2003

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 (FADH2)-utilizing monooxygenase, and FADH2 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 FADH2 and retarded its rapid oxidation by O2. 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 FADH2-utilizing enzymes, which have homologues in the domains Bacteria and Archaea.

* Corresponding author. Mailing address: School of Molecular Biosciences, Abelson Hall 301, Washington State University, Pullman, WA 99164-4324. Phone: (509) 335-2787. Fax: (509) 335-1907. E-mail: xun{at}mail.wsu.edu.

{dagger} Present address: Battelle Dugway Operations, Special Programs Division, Dugway, UT 84022.

Journal of Bacteriology, May 2003, p. 2786-2792, Vol. 185, No. 9
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.9.2786-2792.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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