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J. Biol. Chem., Vol. 279, Issue 8, 6696-6700, February 20, 2004

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A Monooxygenase Catalyzes Sequential Dechlorinations of 2,4,6-Trichlorophenol by Oxidative and Hydrolytic Reactions^*

Luying Xun and Chris M. Webster

From the School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4234

Ralstonia eutropha JMP134 2,4,6-trichlorophenol (2,4,6-TCP) 4-monooxygenase catalyzes sequential dechlorinations of 2,4,6-TCP to 6-chlorohydroxyquinol. Although 2,6-dichlorohydroxyquinol is a logical metabolic intermediate, the enzyme hardly uses it as a substrate, implying it may not be a true intermediate. Evidence is provided to support the proposition that the monooxygenase oxidized 2,4,6-TCP to 2,6-dichloroquinone that remained with the enzyme and got hydrolyzed to 2-chlorohydroxyquinone, which was chemically reduced by ascorbate and NADH to 6-chlorohydroxyquinol. When the monooxygenase oxidized 2,6-dichlorophenol, the product was 2,6-dichloroquinol, which was not further converted to 6-chlorohydroxyquinol, implying that the enzyme only converts 2,6-dichloroquinone to 6-chlorohydroxyquinol. Stoichiometric analysis indicated the consumption of one O₂ molecule per 2,4,6-TCP converted to 6-chlorohydroxyquinol, ruling out the possibility of two oxidative reactions. Experiments with ¹⁸O-labeling gave direct evidence for the incorporation of oxygen from both O₂ and H₂O into the produced 6-chlorohydroxyquinol. A monooxygenase that catalyzes hydroxylation by both oxidative and hydrolytic reactions has not been reported to date. The ability of the enzyme to perform two types of reactions is not due to the presence of a second functional domain but rather is due to catalytic promiscuity, as a homologous monooxygenase converts 2,4,6-TCP to only 2,6-dichloroquinol. Employing both conventional catalysis and catalytic promiscuity of a single enzyme in two consecutive steps of a metabolic pathway has been unknown previously.

Received for publication, November 4, 2003 , and in revised form, December 2, 2003.

^* This work was supported by United States National Science Foundation Grant MCB-0323167. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: School of Molecular Biosciences, Washington State University, Abelson Hall 301, Pullman, WA 99164-4234. Tel.: 509-335-2787; Fax: 509-335-1907; E-mail: xun{at}mail.wsu.edu.

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