Characterization of chlorophenol 4-monooxygenase (TftD) and NADH:flavin adenine dinucleotide oxidoreductase (TftC) of Burkholderia cepacia AC1100.
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.
Gisi
Michelle R
MR
School of Molecular Biosciences, Washington State University, Pullman 99164-4324, USA.
Xun
Luying
L
D001426:Bacterial Proteins / Q000378:metabolism*; D016956:Burkholderia cepacia / Q000201:enzymology*; D002733:Chlorophenols / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000378:metabolism*; D005822:Genetic Vectors; D006899:Mixed Function Oxygenases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012256:Riboflavin / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002733:Chlorophenols; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; C058805:1,5-dihydro-FAD; C009534:2,4,5-trichlorophenol; D005486:Flavin Mononucleotide; D006899:Mixed Function Oxygenases; C099366:chlorophenol 4-monooxygenase; D009247:NADH, NADPH Oxidoreductases; D012256:Riboflavin