Affiliation:
1. School of Molecular Biosciences, Washington State University, Pullman, Washington
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
.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
89 articles.
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